kazakh national agrarian university doi:616.98:615.37:636 ... · gost 13805-76 dry enzymatic...
TRANSCRIPT
1
Kazakh National agrarian university
DOI:616.98:615.37:636.2 On the rights of manuscript
ZHOLDASBEKOVA ASSEL YERKIMBEKOVNA
Immunoprophylaxis against salmonellosis of cattle
6D120100 – Veterinary medicine
A dissertation submitted for the degree
of Doctor of Philosophy (Ph.D
The domrstic scientific adviser
Doctor of veterinary science, prof.
Biyashev B.K.
Foreign scientific adviser
Doctor of Philosophy (Ph.D),
Professor of the Latvian Agricultural
University (Latvia) Valdovska A.
Republic of Kazakhstan
Almaty, 2018
2
CONTENTS
NORMATIVE LINKS………………………………………….. 3
DEFINITIONS…………………………………………………… 4
DESIGNATIONS AND ABBREVIATIONS…………………... 5
INTRODUCTION………………………………………………. 6
1 REVIEW OF LITERATURE………………………………….. 10
1.1 Prevalence of cattle salmonellosis………………………………… 10
1.2 Control measures of bovine salmonellosis………………………... 17
2 PERSONAL RESEARCH……………………………………… 25
2.1 Materials and methods of the research…………………………… 25
3 RESULTS OF THE STUDY………………………………….. 28
3.1 Prevalence of bovine salmonellosis ……………………………… 28
3.2 Biological properties of salmonella cultures……………………… 30
4 Development of genetically characterized strains of Salmonella… 38
4.1 Method of producing an attenuated strain of Salmonella……….. 42
4.2 Characteristics of the biological properties of the attenuated strain
Salmonella dublin 31…………………………………………….
45
4.3 Investigation of immunogenic properties of S. dublin strain 31… 52
4.3.1 Studying the immunizing properties of the attenuated S. dublin 31
strain on mice ……………………………....................................
53
4.3.2 Studying the immunizing properties of the attenuated S. dublin 31
strain on calves……………………………………………………
56
5 Development of technological regulations for the production of
live vaccines against bovine salmonellosis……………………….
58
5.1 Method for manufacturing live vaccines against salmonellosis in
animals……………………………………………………………
59
6 Post-vaccination reaction after immunization with live vaccine
from the strain S. dublin 31……………………………………….
62
6.1 Reactogenicity and bacterial carriage…………………………….. 62
6.2 Serological indicators…………………………………………….. 64
7 Production tests…………………………………………………… 70
DISCUSSION OF THE RESULTS…………………………… 76
CONCLUSION…………………………………………………… 89
PRACTICAL OFFERS………………………………………….. 90
REFERENCES………………………………………………….. 91
ANNEXES ………………………………………………………. 102
3
NORMATIVE REFERENCES
References to the following standards were used in this thesis:
GOST 1530-65 Paperparchment;
GOST 3164-78 Medical vaseline oil. Technical conditions;
GOST 4233-77 Sodiumchloride. Technicalconditions;
GOST 5962 –67 Rectifiedethylalcohol 96%;
GOST 6038-79 Sugars;
GOST 6709-72 Distilledwater. Technicalconditions.
GOST 12923-82 Medicalalignin. Technical conditions;
GOST 13805-76 Dry enzymatic peptone for bacteriological purposes.
Technical conditions;
GOST 16280-88 Foodagar. Technica conditions;
GOST 17206-84 Microbiological agar. Technica lconditions;
GOST 20227-91 Laboratory glassware. Graduatedpipettes;
GOST 20292-74 Flasks with a capacity of 100, 200, 1000 cm3;
GOST 20730-89 Meat-peptonebroth;
GOST 22967 Injectionsyringes 2 cm3, 5 cm3;
GOST 25336-82 Е Laboratory utensils and equipment, glass.
Technicalconditions;
GOST 28085-89 Determination of sterility;
GOST 4.452-86 Biolumbiological microscope
ТC 480-11-10 –73 Pencils for the glass;
IRTC 42-102-63 Different scissors
4
DEFINITIONS
The following terms are used in this research report with the corresponding
definitions:
Morphological properties - the size, shape, nature of the relationship.
Tinctorial properties - the ability to stain with various dyes. A particularly
important feature is the ratio to the ink according to Gram, which depends on the
structure and chemical composition of the bacterial cell wall.
Cultural properties - features of bacterial growth on liquidmedia(film formation,
sediment, cloudiness) and solid media (shape, size, consistency, edges, surface, colony
transparency, pigment formation and other properties).
Biochemical properties - the ability to ferment various carbohydrates, proteolytic
activity, the formation of indole, hydrogen sulphide, the presence of urease.
Pathogenicity - the ability of microorganisms to cause an infectious process in
macroorganisms of a certain type.
Culture - a collection of bacteria that have grown on a solid or liquid nutrient
medium.
A strain-genetically homogeneous population of microorganisms with specific,
stable morphological and biological properties defined for a given species
Virulence - is a measure of pathogenicity.
Adhesion - the ability of bacteria to adsorb (attach) to epithelial cells of the small
intestine with the help of villi (pili, pili, pilus).
Endotoxins - toxins, which are structural components of gram-negative microbial
cells, enter the environment after their death and destruction, are thermostable, less
poisonous in antigenic properties than exotoxins, represent lipopolysaccharide
complexes that are not inactivated by formalin.
Attenuation- is an artificial persistent weakening of the virulence of pathogenic
microorganisms that retain the ability to induce immunity. Used in the manufacture of
live vaccines.
Vaccine strain - attenuated strain having stable immunobiological properties.
Residual virulence - is the degree of relative stability of pathogenicity.
Immunization - the creation of specific immunity to certain infectious diseases.
5
NOTATIONS AND ABBREVIATIONS
LLP - limited liability partnership
CF - collective farming
I/p - Intraperitoneal route of administration
LD50- Average lethal dose
MA - Ministry of Agriculture
MES RK - Ministry of Education and Science of the Republic of Kazakhstan
RK - Republic of Kazakhstan
OIE - International Epizootic Bureau
WHO- World Health Organization
KazNAU - Kazakh National Agrarian University
CFU -Colonium-forming unit
pH - Hydrogen index
LD50- Average lethal dose
ELISA - enzyme-linked immunosorbent assay
DNA - deoxyribonucleic acid
dNTPs (dNTPs) - deoxynucleotide triphosphates
SDS - sodium dodecyl sulfate
ELISA-enzyme immunoassay
m is the error of the arithmetic mean
μl - microliter
MPA - meat-peptone agar
MEB - meat-peptone broth
PA - agglutinationreaction
P istheconfidencefactor
6
INTRODUCTION
Relevance of the topic Modern agrarian policy of our country is aimed at fulfilling
the main task - satisfying the ever-growing needs of the people in food products. In
order to successfully solve these problems, it is necessary to ensure further growth in the
production of livestock products. The preservation of newborn animals and the
cultivation of a healthy, well-developed and adapted to new conditions of young animals
is the basis for increasing the yield of livestock products [1].
The development of livestock farms is impossible without the creation of long-
lasting protection from infectious diseases, including salmonellosis.
Salmonellosis is the most widespread zooantroponosis in the world and according
to WHO (1999) poses a significant problem in all countries of the world every year. The
damage caused by this disease is not only direct effect on the poultry, but also the fact
that infected birds having contacted with salmonella carriers from outside have become
permanent sources of contamination of the environment. Carrying among chickens is
widespread (5-22,2%), ducks (10-15%), geese (5-20%). On average, carriers have been
detected among healthy birds in the range from 0.25 to 7.0%, among diseased and
forcedly killed from 2.9 to 30% [2].
The problem of salmonellosis in animals is becoming increasingly important. This
is due to a wide circulation in general, including in nature, the polydeterminateness of
the virulence factors of the pathogens, the variety of ways of entry into the body of
animals and humans. The damage caused by this disease is not only dead animals but
also bacterial carriers,which become permanent sources of contamination of the
environment. Products of animal origin (meat, milk, eggs), obtained from salmonella
carriers in case of insufficient heat treatment can cause foodborne toxic infections in
humans and detection and the control of foodborne diseases is a very actual daily
practice of veterinary and medical workers [3].
Epizootic and epidemiological tensionsaround intestinal infections caused by
enteroinfections in recent years has increased in connection with changes in methods of
cattle breeding and fattening, as well as the rules of zootechnical and veterinary care of
animals. Vaccination of animals and birds against salmonellosis of animals became
optional and not considered in the plan of antiepizootic measures of the Veterinary
Committee of the Ministry of Agriculture of the RK [4].
In the current socio-economic conditions, the specific features of combating
diseases common to humans and animals are largely related to the development of the
private sector in livestock production, uncontrolled migration of livestock, including
from disadvantaged regions. This makes it difficult to take into account and carry out
vaccination of animals, creates difficulties in the implementation of state veterinary and
sanitary-epidemiological surveillance. Exceptional resistance of pathogens of
enteroinfections and their cyclic increase in activity cause periodic sharp increases in
morbidity. The increase in the scale and intensity of development of territories where
7
active natural foci are located leads to a wide spread of these diseases among the
population[5].
Prevention of zooantroponoses primarily based on the timely detection of the risk
of infection of people with an infection. Epizootic and epidemiological features of
infection, effective means of prevention and the possibility of their use determine the
choice of key activities. In some cases, this may be regime-restrictive measures, in
others - veterinary and sanitary, sanitary and anti-epidemic measures, use of specific
prevention tools, etc[6].
This increases a demand in the study of the epizootic situation on this topic,
insights in the main factors of the infectious process, as well as the improvement of
therapeutic and specific prevention and development of veterinary and sanitary
measures.
The issues of prevention of salmonellosis most often come down to vaccination, as
the most traditional and universal method [7,8].
In case of animal salmonellosis, various killed vaccinesarethe most studied. Some
of them are used now. However, practice has shown that killed vaccines do not supple a
sufficiently intense immunity in young animals, especially in those farms where disease
outbreaks are caused by enteropathogenic Salmonella.
Over the past decade, studies on the attenuated strains of salmonella and on the
immunological justification for the use of live vaccineshave been conductedin
Kazakhstan [9].
The live vaccines against salmonella from calves, pigs, horses and sheep developed
by them have been tested and applied in the republic's farms for many years.
In this regard, the improvement of specific prevention of bovine salmonellosis
through the development and introduction of live vaccines from genetically
characterized strains of Salmonella is an urgent issue.
All of the above determine the choice of the topic of research.
Purpose and objectives of research. The purpose of the work is to develop a
technology for the production of live vaccine against bovine salmonella.
To achieve this goal, the following tasks were identified:
1. To study the prevalence of salmonellosis in cattle in Almaty, Zhambul,
Kyzylorda regions;
2. To study the biological properties of salmonella cultures.
3. To study the biological properties of the attenuated strain of S.dublin 31 in vitro
and in vivo.
4. To develop a technological regulation for the production of live vaccines against
bovine salmonella.
5. Approbation of live vaccine against bovine salmonellosis in production
conditions and development of normative and technical documentation for the
manufacture and control of vaccines.
8
Scientific novelty. The results of the conducted studies testify the etiological role
of various serovars of salmonella in the development of diseases of calves in those
affected by salmonellosis in a number of farms in Kazakhstan. An attenuated strain of
S.dublin 31 with stability of biological properties, moderate reactogenicity, weak
residual virulence, high immunogenic activity, epizootically safe and having a genetic
label, allowing to differentiate it from epizootic prototypes was studied.
The studies have shown that the live vaccine from the attenuated strain S.dublin 31
is more immunogenic than the vaccines currently used against salmonella in calves.
A live vaccine against bovine salmonellosis from S. dublin 31 strain creates high-
tension immunity when administered once and is quite suitable for specific prevention of
bovine salmonellosis and is compatible with other vaccines. The presence of genetic
markers makes it possible to differentiate the vaccine strain from the field in the
laboratory if there is a suspicion of salmonellosis or when salmonella is isolated in
products of animal origin.
Practical value of the work. A live vaccine from an attenuated strain of S. dublin
31 for the prophylaxis of bovine salmonella.
The use of live vaccine against bovine salmonellosis in production conditions in a
number of farms in the country made it possible to significantly reduce the incidence in
animals, the mortality in calves and improve the epizootic situation in the farms.
The normative and technical documentation (the Technical condition, the
Temporary instruction, the Manual on the application of "Live vaccine against
salmonella in cattle), approved at the NTS of the Institute of Problems of Animation of
KazNAU from 13.10.17.
The recommendation on "Salmonellosis of cattle and struggle measures” was
approved by the NTS Institute of Problems of Animation of KazNAU from 13.10.17.
Implementation of the research results. Materials of the thesis were reported and
discussed at: According to the materials of the thesis 16 published works were
published, including: 1 - in journals with impact factor, Journal of Pharmaceutical
Sciences and Research. India, Vol. 10 (1), 2017, pages 162-163. Scopus; 5 in the
journals recommended by CCSON MES RK: Izdenister, non-diesel engine; Proceedings
of NAS RK. Series of Agricultural Sciences; "The White Journal", a scientific and
practical journal, the West Kazakhstan Agricultural and Technical University named
after Zhangir Khan;
-The Bulletin of Science of the Kazakh Agrotechnical University named after S.
Seifullin, which reflects the main results of experimental research, 5 - in the materials of
international conferences:
- Collection of materials of the international scientific and practical conference of
young scientists "Contribution of young scientists to the industrial and innovative
development of the agro-industrial complex", Almaty, 2016; Material XXX
International Scientific and Practical Internet Conference "Problems and prospects for
the development of science at the beginning of the third millennium in Europe and
9
Asia." Ukraine, 2016; Proceedings of the XXXVI International Scientific and Practical
Conference "Modern Problems of the Humanities and Natural Sciences", Moscow,
2017; 2 - in the international journal: Modern science, International scientific journal,
Moscow 2018. 2 - Methodological instructions: "Fight salmonellosis in animals and it's
prevention"; "Fight against Salmonellosis in Animals and their Prevention". Technical
conditions for the preparation "Live vaccine against salmonellosis of cattle"; Time
instruction on the preparation and control of the preparation "Live vaccine against
salmonella of cattle".
The main highlights of the study put on the defense:
1. The vaccine strain S.dublin 31 and its biological properties.
2. Development and introduction of live vaccine against salmonellosis of cattle.
Publication of the research results. 16 scientific papers, including one NTD were
published on the topic of the thesis
The volume and structure of the thesis.Thesis work was carried out according to
the standard pattern. It consists of content, normative references, definitions, notations
and abbreviations, introduction, literature review, body part, conclusion, bibliography
and appendix. The volume of the work is 130 pages, the text is illustrated by 15 tables,
20 pictures.
10
1. Literature review
The research part of the work includes a literary search, the collection of
information and statistical materials published in domestic and foreign scientific
publications, in the official collections of the International Program for the OIE and
WHO on the control and surveillance of infections and toxic infections in Europe, the
Centers for Disease Control in the United States and other published sources .
Our literary search is devoted to highlighting the species composition of
enteroinfective pathogens most often released in animals, birds and humans, reflecting
the interconnection of epizootic and epidemiological conditions.
In total, over 100 publications of domestic and foreign authors have been
processed. The collected material is generalized, systematized. We have carried out a
patent search for the problem posed for resolution up to 10 years.
1.1 Prevalence of cattle salmonellosis
Salmonellosis is one of the most widespread zooanthroponosis in most countries
of the world, including various regions of the Republic of Kazakhstan. Despite current
methods of diagnostics, treatment and prevention, salmonellosis still causes great
economic damage to livestock and poses a serious threat to human health [10].
The burden of foodborne diseases is substantial: every year almost 1 in 10 people
fall ill and 33 million of healthy life years are lost. Foodborne diseases can be severe,
especially for young children. Diarrhoeal diseases are the most common illnesses
resulting from unsafe food, 550 million people falling ill each year, including 220
million children under the age of 5 years. Salmonella is 1 of the 4 key global causes of
diarrhoeal diseases.
Salmonella is a gram negative rods genus belonging to the Enterobacteriaceae
family. Within 2 species, Salmonella bongori and Samonella enterica, over 2500
different serotypes or serovars have been identified to date. Salmonella is a ubiquitous
and hardy bacteria that can survive several weeks in a dry environment and several
months in water [11].
While all serotypes can cause disease in humans, a few are host-specific and can
reside in only one or a few animal species: for example, Salmonella enterica serotype
Dublin in cattle and Salmonella enterica serotype Choleraesuis in pigs. When these
particular serotypes cause disease in humans, it is often invasive and can be life-
threatening. Most serotypes, however, are present in a wide range of hosts. Typically,
such serotypes cause gastroenteritis, which is often uncomplicated and does not need
treatment, but disease can be severe in the young, the elderly, and patients with
weakened immunity. This group features Salmonella enteric serotype Enteritidis
and Salmonella enterica serotype Typhimurium, the two most important serotypes of
Salmonella transmitted from animals to humans in most parts of the world [12].
11
Annually, the Ministry of Health registers about 3000 cases of salmonellosis in
Israel. The peaks of morbidity fall on the hottest months, because this bacterium loves
heat very much. According to the Ministry of Health, in July-August 2017, 1327 people
became ill with salmonellosis, 1,044 people fell ill in the same period of last year, 825 in
2015, 1007 in 2014, and 943 in 2013. That is, there is indeed growth, but without serious
analysis is difficult to determine, it is associated with the deterioration of the sanitary
and epidemiological situation in the country or with some other factors. Despite this, it
can still be said that even with such figures, the annual incidence rate is still within the
usual background [13].
In addition, world statistics show: over the past 15 years, the incidence of
salmonellosis has increased worldwide. In recent months, an increased level of
salmonella infection has been observed in countries such as Great Britain, Holland,
Italy, Hungary, Norway, Sweden, etc [14].
According to experts, this is due not only to the fact that a single economic space
allows unhindered to "disperse" food products contaminated at one enterprise by a fan in
all EU countries, but also because the resistance of the bacterium to antibiotics has
increased, and that before it killed, now only makes it stronger [15].
Global monitoring of foodborne infections for 12 years, showed that 47% of all
outbreaks were caused by salmonella, and of the -34% of it , due to consumption of
chicken meat [16].
It is estimated that in the United States, 1.4 million people are susceptible to this
infection every year. Material costs are estimated at $ 2.6 billion a year, including
medical expenses, loss of productivity, losses to food producers and public catering
enterprises, and research costs [17].
The epidemic situation of salmonellosis in the Russian Federation is unsuccessful.
On average for the period 2009-2012 - about 50,000 cases were recorded per year.
Mortality is 0.01-0.02 per 100 thousand of the population, among children - 0.03.
At the same time, poultry products are considered as the main way of transmission of
infection to humans [18].
Economic damage, with the registration of about 50,000 cases of human salmonella
infection per year is 1.55 billion rubles (only medical expenses) [19].
"According to the results of 4 months of this year in Astana, there is an increase in
the incidence of salmonellosis 4 times: from 2.5 (20 cases) to 10.3 per 100 thousand
people (86 cases). The incidence of salmonellosis exceeds the average republican
indicator by 4 times (RK - 17.7) and by the OCI group by 2 times (RK - 17.7). In the
context of the regions, according to the incidence of salmonellosis and acute intestinal
infections, Astana ranks first, "the capital department for the protection of consumers'
rights said [20].
So, for intestinal infections, the risk group is children under the age of 14 - 76%,
the figure among this age group exceeds the republican by 2 times. According to the
incidence of salmonellosis, 82% are adult towns people. The microbial landscape of
12
salmonella infection is represented in 98% of Salmonella eneteridis, which confirms the
food transfer factor. In the epidemiological investigation of foci of intestinal infection,
the effect of the food path was established in 97%, in 3% of the contact-household
pathway [21].
The high incidence of salmonellosis directly depends on the quality of poultry
products supplied to the city.
"To ensure that the situation is not uncontrolled and does not lead to other
unfavorable epidemics, we believe that in accordance with the Code" On the health of
the people and the health care system, "an industrial control should be considered as an
alternative," the department notes [22].
The highest incidence of salmonellosis is in economically developed countries.
Analysis of literature data and information bulletins of national centers on salmonellosis
of individual countries and WHO, allows to assume that in recent decades, there has
been a distinct tendency of increase in the incidence of these infections, accompanied by
an increase in the number of cases of detection of salmonella in animals, food, feed and
other environmental objects in most countries of the world.
The increase in the incidence of salmonellae in animals and humans, the increase in
the number of Salmonella serovars isolated from them, and the increased incidence of
contamination of food products of animal origin and objects of the external
environmentby these bacteria, have put forward this infection in a list of important
zooanthroponosis [23,24].
It is important to note that due to various circumstances salmonellosis retains a
significant part in the structure of infectious pathology. Thus, according to researchers,
in the structure of the incidence of all zoonoses of agricultural animals salmonellosis
makes 15-45% [24,25].
In recent years, the incidence of salmonellosis in farm animals has been increased 2
times in Kazakhstan.
Every year the problem of salmonellosis acquires a growing national economic
importance due to its widespread distribution among animals and people, an increase in
Salmonella contamination of the environment, and a marked increase in the incidence
rate [26].
A number of scientists analyzing the current situation note that the difficulties of
combating salmonella infection with modern antimicrobial agents are due to the
variability and quick adaptability of microbes, as well as the possibility of repeated
infection, especially in the patients with immune deficiency [27,28].
The growth of subclinical and latent forms of the disease, the infection of fodder
and the environment, the different routes of entry into the animal and human organism,
the selection and circulation of strains bearing R- factors that are formed under the
influence of antibiotics and chemopreparations contribute to the spread of salmonellosis
in animals [29].
13
The problem of salmonellosis is relevant not only in connection with its prevalence,
but also due to the course of the disease in the form of latent and “removed” clinical
forms and the possibility of transition to a long-term bacterial carriage. According to
B.A. Matviyenko latent infections of salmonella- carriers are very common in animals
[30].
Bacterial - carrying by animals is considered as one of the clinical forms of
salmonellosis. A significant amount of work has been devoted to this problem of both in
our country and abroad.
Some researchers argue that the peculiarity of salmonellosis in recent years is the
high frequency of bacterial transport, which is detectable only when examining various
groups of animals and people for a variety of reasons.
Matviyenko B.A. [5], Biyashev K.B. [6] consider that infected animals remain
carriers and excrete salmonella for a long time and serve as a source of infection for
healthy animals and humans.
A number of researchers believe that infected animals stay for a lifetime as carriers
of salmonella.
P.P. Rakhmanin, A.V. Kulikovsky indicate that the problem of human
salmonellosis is becoming increasingly important. According to WHO experts it is a
global pattern: this is partly due to the fact that majority of the farm animals acquiring
the causative agent with food and water become asymptomatic carriers of various
salmonella serovars pathogenic to humans. Infection of people occurs through the
products obtained from these animals [31].
According to WHO experts, the absence of symptoms of infection in farm animals
and the technical difficulties associated with the detection of these bacterial carriers
make them permanent sources of contamination of the environment and products of
animal origin.
Analysis of outbreaks showed that the frequency of Salmonella isolation from
poultry, including chickens, had increased. In many cases, the chickens are infected with
S. enteritidis and other serovars, which do not cause clinical signs of the disease and
death of birds, which makes it difficult to assess the well-being of the farms for this
infection [32].
B.A. Matvienko, P.P. Popova, M.M. Rementsova, A.A. Kim believe that the spread
of salmonella is largely promoted by high stability, plasticity and survival in the external
environment. The study of the samples of previously infected soil sections showed that
the causative agent of salmonella survives for more than 120 days, while preserving all
cultural and morphological and biochemical properties [5,33,34].
A number of researchers consider that adult individuals of cattle who have
recovered from the clinical form of salmonellosis excrete the pathogen of salmonellosis
continuously and in large numbers [35].
Some cows and calves do not excrete salmonella with feces when infected. They
should be considered as latent bacterial carriers.
14
Some researchers consider that the main way of natural infection of calves is direct
contact of sick animals with healthy ones. In addition, infection by indirect contact is
also acknowledged, when consuming contaminated milk [36].
Infection of animals occurs most often through infected feed and water, during
transportation and on pastures in places of watering. The main way of distribution of
salmonellosis among animals is a direct transmission of infection from the patient to the
healthy. Salmonella is excreted from sick animals with various secretions, contaminating
objects of the external environment that serve as factors for the spread of infection. The
greatest danger is represented by the aborted animals, which stay for a long time as
salmonella carriers. It was reported that animals of one species can be a source of
salmonellosis for others [37].
Ways of distribution of salmonellosis are diverse: direct transmission from animal
to animal (intraspecies or interspecies), from animal to human and vice versa,
transmission of the pathogen through animal feed. A significant role in the spread of
salmonella infection is played by newly imported “carrier animals”, as well as wild
animals, birds, and even cold-blooded animals.
Most researchers argue that infection with salmonellosis most often occurs
alimentary by eating food contaminated with Salmonella [38].
Thus, salmonella are excreted from the body of patients and serve as an ultimate
cause of infection of animals of all ages.
A number of researchers studied the natural focality of salmonellosis in different
zones of Kyrgyzstan and Kazakhstan, and found salmonella in seven species of wild
warm-blooded animals. The total infection rate was 11.8%, and the species composition:
72.3% - S. typhimurium and 27.7% S.enteritidis. It indicates the probability of carrying
out the causative agent of infection in nature by carrier animals and on the reverse
transformation of salmonella infection from natural foci into livestock and poultry farms
[39].
Kotova A.L. noted that acute salmonellosis epizootic is observed among cold-
blooded animals (frogs, snakes, lizards, etc.). They established that salmonellae among
them reach up to 30-50% [40].
Wild mice can serve as a reservoir of the pathogen for cattle. This conclusion was
made due to the isolation of S. dublin from the feces, liver, spleen, and kidneys of mice
in a farm unfavorable by salmonellosis. It is believed that S. dublin originally from cows
infiltrated into the population of mice and subsequently began to infect calves
periodically.
Salmonella bacteriosis in mice and rats is very significant in places of the slaughter
of animals, as well as in food and livestock breeding facilities and varies widely [41].
Most researchers prove that in adult animals, a prolonged and sometimes lifelong
bacterial carriage is observed after a clinical recovery, especially when infected by
S.dublin. Moreover, the isolation of S. dublin depends on the season and the stage of the
infectious disease. The greatest number of pathogens is released at the beginning and in
15
the midst of an infectious disease. Six to twelve weeks after the disappearance of the
clinical signs of the disease, it is possible to isolate the maximum number of constant
bacterial carriers [42].
Akhmedov A.M. argues that the main cause of salmonellosis among the calves is
salmonella carrier cows [43].
A high level of salmonellosis infection was recorded in 140 species of wild birds,
which indicates a long-term preservation of the causative agent of salmonella in the
population of wild birds and their definite role in reserving and dispersing salmonella in
nature over considerable distances [44].
Researchers note that in our country and abroad the epidemiological situation for
salmonella remains tense. At present, a large amount of data is accumulated, indicating
that all representatives of the genus Salmonella are potentially pathogenic to humans. It
is established that all kinds of salmonella that cause diseases in agricultural animals and
birds are able to persist in the human body and cause toxic infections [45].
In addition, the epizootic situation of salmonella infection is complicated by the
fact that most serovars of this pathogen do not cause clinical symptoms of the disease in
animals that carry this infection dangerous for human.
The greatest danger is represented by the products obtained from the animals with
subclinical and latent forms of the disease, which, if insufficiently processed, can cause
foodborne toxic infections in humans [46,47].
Therefore, the fight against food-borne diseases is a very urgent daily task for
veterinary and medical workers.
Most researchers believe that salmonellae form a thermostable endotoxin which is
released when the bacterial cell is destroyed. Thermostable endotoxin causes severe
intoxication in sick animals and food toxicosis in humans [48,49,50].
Salmonella-carriage in the slaughter of clinically healthy animals is recorded in
0.1-7.7%, and in those who were forcedly killed in 7.2-12.85% cases, which maintains
epidemiological tensionsaround salmonellosis at a high level.
Data shows that salmonella causes food-borne toxic infections in humans through
consuming milk and dairy products, eggs, meat from salmonella- carriers animals and
birds or patients with salmonellosis. In this case, the bacteria colonize the
gastrointestinal tract, enter the lymphatic system and blood. In the body, salmonella
perishes and forms endotoxin. Of those who have recovered, about 2-3% remain the
carriers [51].
In addition, the maintenance and spread of the infection are facilitated by many
mammals (including rats and mice), as well as wild birds, reptiles, insects. Humans can
also get infected from domestic animals (dogs, cats, turtles, pigeons, etc.) [52].
Analysis of the statistical data of recent years indicates an increase in the incidence
of abortions in cows caused by S. dublin and S. typhimurium. Infections caused by S.
dublin and S. typhimurium are zooanthroponotic. Infection of humans occurs from
infected cows, most often during abortions, and as a result of the use of milk [53].
16
The frequency of seeding of salmonella in animals corresponds to the frequency of
detection of this serovar in humans. This proves that the epidemiological situation often
reflects the epizootic situation and vice versa [54].
In humans, it is recorded as anthroponous salmonellosis (typhoid fever,
paratyphoid (A, B, C) and zooanthroponous, associated with infected products of animal
origin (food poisoning).
A close attention is paid to the asymptomatic carriage of salmonella in animals,
which is becoming increasingly important in human infectious pathology, in the
majority of developed countries[55].
The most common route of human infection is an alimentary route (60-70%),
followed by the direct contact - up to 30% [56].
The main transmission factors are food products of animal origin (meat and dairy).
The serious danger is represented by the food products from meat of forcedly
slaughtered animals with an unrecognized disease and food products consumed without
additional heat treatment. Cases of food poisoning related to the consumption of eggs or
food made of raw eggs were observed for a number of years [57].
The analysis of national and foreign literature showed that the dominant serovars of
salmonella isolated from humans are S. dublin, S. enteritidis, S. typhimurium, S.
thompson and S. anatum [58].
Salmonellosis is a group of infectious diseases of animals and humans caused by
bacteria of the genus Salmonella, characterized by significant polymorphism of the
clinical course [59].
The causative agents of salmonellosis are microorganisms belonging to the genus
Salmonella of the family Enterobacteriaceae. Salmonella is small sticks with round ends,
1 to 3 microns in length and 0.5-0.8 microns in width, usually mobile due to the
presence of peritrichically located flagella (S. pullorum-gallinarum immobile). They are
well dyed with aniline dyes, they are negative at Gram staining, and do not form spores
and capsules. Bacteria grow abundantly on common nutrient media, forming small
colonies of 2-4 mm in diameter. A total of 2300 species of Salmonella are known, and
Kaufman suggests that there are at least 10,000 species in the nature. A characteristic
feature of these microorganisms is their ability to form toxins [60].
Salmonella persists in the environment for a long time. And in some products
(milk, meat products) salmonella can not only be preserved, but also multiply, without
changing the state of products. Pickling and smoking have a very weak effect on them,
and freezing even increases the survival time [61].
The main causative agents of calf salmonellosis are - S. dublin, S. typhimurium,
and S. enteritidis.
During the study of the role of salmonella-bearing animals on the farms
unfavorable by salmonellosis of cattle and pigs the following serotypes were identified:
S. dublin-73%, S. typhimurium-18%, S. enteritidis-7%, and S. choleraesuis-2%, from
17
animals of unfavorable pig farms-S. choleraesuis-68%, S. typhisuis-14%, S. dublin-
11%, and S. typhimurium-7% [62].
Long-term studies conducted by P.P. Popova on the territory of Central Kazakhstan
aimed at establishing the level of salmonella contamination and etiologic structure of
salmonellosis in various animals, showed, that out of the total number of isolated
Salmonella cultures, the majority was obtained from the cattle and pigs and are
represented by serological variants of four groups: D-35.6%; B-32.3%; C-22.8% and E-
9.3%. Circulation of 10 salmonella serovars was established in cattle, most of them were
isolated as a percentage of S. dublin, S. typhimurium and S. enteritidis [22,63].
The analysis of outbreaks of salmonellosis in Kazakhstan made it possible to
establish that the serovars S. typhimurium and S. dublin were often the cause of the
disease of piglets and suckling pigs [64].
According to the data of the majority of researchers, the causative agents of calves'
salmonellosis are most often S. dublin, S. enteritidis, S. rostok and S. typhimurium, less
often other salmonella serovars.
Analysis of literature data indicates that the most common serovars of bovine
salmonella are S.dublin and S. typhimurium. The increase of S. typhimurium which
causes disease and carriage of salmonella in cattle should also be considered [65].
Thus, salmonellosis is a typical zooanthroponosis that represents an important
veterinary and biological problem, characterized by a high incidence of disease and
carriage both in wild and farm animals, significant resistance and dissemination of the
pathogen in the external environment. The participation of invertebrates, vertebrates and
environmental objects in the circulation of the pathogens of salmonellosis indicates a
natural focal character of salmonellosis with the facultative-transmissive mechanism of
transmission of its pathogen. All this explains the relevance of the study of epizootic and
epidemiological situation of this infection, the main driving forces of the infectious
process, as well as the improvement of specific prevention and veterinary and sanitary
measures [66].
1.2 Control measures of bovine salmonellosis
The success of the fight against salmonellosis is based on the systematic
implementation of measures aimed at eliminating the causes, contributing and
predisposing to the emergence. In this regard, a special attention should be paid to
prevention, which combines a set of common measures, veterinary sanitary as well as
special measures. It is also necessary to systematically study and analyze the epizootic
situation, to clarify the epizootic features of the disease course in a particular region
[67].
According to available information, prevention of salmonellosis should be based on
the use of a set of measures that are developed in a specific situation. This complex
should include the following measures: organizational-economic, special anti-epizootic
and epizootological forecast. Organizational and economic measures begin with the
18
grouping of a herd of cattle from salmonellosis farms that are safe from salmonella. The
newly imported animals are to be kept in quarantine for 30 days. Bacteriological studies
on salmonella can be carried out if necessary. Feeds entering the farms are subjected to
bacteriological examination, since they can be contaminated with salmonella. Corpses of
wild rodents living in cattle-breeding farms and buildings are also subjected to
bacteriological research [68,69].
Farm workers must undergo an annual preventive examination and examination for
salmonella in the Sanitary and Epidemiological Station (SES) [70].
The complex of general measures also includes preventive disinfection with
mandatory testing of its quality, deratization and disinfestation, utilization of corpses
and aborted fetuses, organization of active animal petting, control of indoor
microclimate and animal feeding; ultraviolet irradiation of calves in the winter, the use
of gernerphages, probiotics, antibacterial substances [71].
The fight against salmonellosis should be systematic, aimed at the systematic
destruction of Salmonella in the external environment, the identification and removal of
sources of infectious agents from the herd [71].
Daily disinfection with disinfection solutions at least twice a day should be carried
out in the facilities where sick animals are kept are treated. Animals are isolated and the
facilitiesare re-disinfected when new cases are identified,.
Treatment for salmonellosis should be complex, aimed at destroying the pathogen
in the body, eliminating intoxication and restoring the function of digestion and
respiration [72].
Nowadays, a wide experience has been gained in the use of specific biological
products, antibiotics, sulfonamides, nitrofurans and other antimicrobial agents against
salmonellosis. In all treatment protocols the best effect is obtained when activities to
increase the overall resistance of the animal organism (full feeding, improvement of
zoogeogenic conditions, etc.)are carried out. Preliminary titration of drugs with the
determination of the sensitivity is an obligatory condition for the use of drugs [73].
In addition to specific preventive measures urgent effective treatment is necessary
when salmonella infection occurs.
A number of researchers widely use various antibiotics against salmonellosis along
with preventive and treatment purposes. The most effective treatment for animals
suffering from salmonellosis are levomycetin, sintomycin, biomycin, tetracycline,
dibiomycin, streptomycin and other antibiotics, either alone or in various combinations
[74].
Treatment of patients with calf salmonellosis should begin as early as possible,
while internal organs do not have destructive, irreversible morphological changes yet. It
is necessary to use a complex method of treatment aimed at suppressing Salmonella in
the body, removing intoxication and restoring the disturbed functions of the digestive
and respiratory organs [75].
19
The issue of a significant reduction of salmonellosis in animals is resolved on the
basis of extensive veterinary sanitary and zootechnical measures, where an important
role is played by specific serotypes and vaccinoprophylaxis. Immunoprophylaxis shifts
the correlation of forces between a macroorganism and a microorganism in favor of
humans and is the main mechanism for breaking the epizootic chain [76].
Among the measures aimed at increasing the general resistance of the animal
organism and preservation of the number of cattle and newborn calves, application of
therapeutic and prophylactic purpose of hyperimmune sera is included [77].
Therapeutic and prophylactic serums are means of emergency prevention of
treatment, their advantage over the medicines used for active prevention is that they
contain the antibodies capable of neutralizing the action of pathogens and toxic products
of their vital activity. In this case, the use of immune sera for prevention purposes allows
the creation of immunity in the shortest timepossible, and in the case of a developed
disease, such drugs are specifically indispensable [78].
According to observations of many scientific and practical workers the use sera,
gives good results in the overall complex of measures to combat calf salmonella.
In the complex of measures to combat infectious diseases of agricultural animals,
active immunization is considered as one of the most effective measures to solve the
problems of significant reduction ofanimal salmonellosis. In this regard, vaccine
prophylaxis takes a special place in the prevention and control of salmonellosis [79].
Literary data on the vaccination against salmonella infections indicate the
development and use of various types of vaccines: killed, live, chemical.
Inactivated (killed) vaccines are immunoprecipitates that contain microorganisms
treated in such a way that they have lost the ability to reproduce in the body of
vaccinated animals [80].
Killed vaccines are prepared from virulent strains of microorganisms, inactivated
by one of the methods that causes minimal damage to structural proteins. The term
"inactivation" in this aspect means the loss of the biological agent's ability to reproduce
while preserving the immunogenic and antigenic properties. The problem of inactivated
vaccines is that when the pathogen is inactivated, a greater or lesser part of its
immunogenic structure is lost under the influence of physicochemical effects on the
microbial cell. Therefore, it is especially important that the initial material for the killed
vaccines contains the antigen at the highest concentrations possible. Hence causing a
need for large doses and multiple vaccinations. The lack of the possibility of
reproduction and replication of the antigen in the organism of vaccinated animals leads
to limited circulation of it, as well as insufficient involvement of the immune cells,
which ultimately leads to a low immunogenicity index of the inactivated vaccine. The
immunogenicity of inactivated vaccines is enhanced by the selection of immunogenic
strains of the pathogen and the addition of adjuvants, which enhance the stimulation of
the immune system in the vaccinated animals, but do not act an antigen [81,82].
20
The killed vaccines also have a complex composition. They contain not only
antigenic substances, but also inactivating substances, adjuvants, as well as a number of
concomitant compounds possessing antigenic, allergenic and at some point even toxic
effects.The unintended side effects that they cause, especially in the form of allergic
reactions, often affect negatively both the vaccinated animals and the cost of their
maintenance, and is one of the obstacles in completion of the vaccine process [83].
B.A. Matvienko notes that many years of experience with the use of killed vaccines
indicate their insufficient immunogenic efficacy, especially in conditions of livestock
complexes. Their immunogenic activity is directly related to the number of microbial
cells and their decay products (endotoxins). They weakly stimulate the immune system
against salmonella due to a certain degradation of antigenic properties under the
influence of physico-chemical factors on the microbial cells and limited circulation of
the antigen. A significant shortcoming of the killed vaccines is the need for multiple
vaccinations [5].
Long-term studies have shown that inactivated vaccines form the immunity of
insufficient voltage and duration. In this connection, the search for improvements in
various areas has been constantly conducted and is still being carried out: improving the
methods of preparation of corpuscular vaccines (broth and agar vaccines, inactivated by
washing with various physical and chemical agents); selection of vaccine-rich - antigen-
bearing strains; use of adjuvants that increase the immunogenic properties of vaccines
[84,85].
Currently, in the CIS countries, the standard vaccine against salmonella of calves is
the concentrated form of a leukocyte vaccine (VGNKI). The vaccine is prepared from a
virulent strain of S.dublin [86].
As practice shows, vaccination at young age is not always effective, which is
explained by physiological immaturity of the organism and abnormal microclimate of
the facilities (low temperature and high humidity). Vaccination at two to five days of
age is a strong stress factor, contributing to the occurrence of acute digestive disorders.
In recent years, live vaccines based on attenuated strains have been successfully
used in agricultural animals and birds to prevent salmonellosis in our country and abroad
[87,88].
In recent years, live vaccines based on attenuated strains have been successfully
used in agricultural animals and birds to prevent salmonellosis in our country and
abroad.
Cellular immune responses are crucial, since salmonellae are capable of
intracellular parasitization. The level of antibodies does not reflect the intensity of
immunity. In this regard, live vaccines are the most promising for the prevention of
salmonella in farm animals [89].
Live vaccines are biological preparations made from attenuated strains of
salmonella, having sufficiently high immunogenicity and weak residual virulence,which
21
are safe for the immunized organism and have genetic markers that make it possible to
distinguish them from virulent prototypes.
The introduction of live attenuated microbes that survive for some time in the host
organism, causes a longer antigenic effect on the cells of immunocompetent organs,
which is accompanied by the development of a pronounced protective effect [90].
Few scientific discoveries have had such an impact on world health as the
discovery of vaccines. The phenomenon that individuals who recovered from some
infectious diseases were resistant to subsequent re-infection was observed by Edward
Jenner and Louis Pasteur and provided the impetus for the early development of
vaccines. Thanks to the advances in immunology and molecular biology the field of
Vaccinology has undergone considerable development during the last century mainly
because of new techniques: attenuation and inactivation of pathogens, cell-culture of
viruses, genetic engineering and acellular component identification [91].
In recent years considerable progress has occurred in areas such as combination
vaccines, new adjuvants, proteomics, reverse vaccinology and vaccines for
noninfectious diseases. These various revolutions have resulted in the appearance of
many different types of vaccines such as whole cell inactivated vaccines, bacterins
(Pasteurella multocida, Salmonella), live attenuated vaccines (tuberculosis and
Salmonella Typhi infections), toxoids (tetanus and diphtheria toxoids, Salmonella
toxoid), acellular vaccines or subcellular vaccines or subunit vaccines (pertussis,
Salmonella infections), polysaccharide vaccines (Haemophilus influenzae type B, Vi
capsular vaccine for Salmonella Typhi infection), recombinant protein vaccines
(hepatitis B, antigens expressed in yeast cells and Salmonella), anti- synthetic peptide
vaccines (hepatitis B, foot and mouth disease), DNA and mRNA vaccines, live vectored
vaccines such as vaccinia- VRG, an oral rabies vaccine, pox and adenoviruses exploited
as vectors).
Although, there is no systematic surveillance in operation in India and other south
East Asian countries, salmonellosis, an important zoonotic disease, is an endemic
problem in the region. More than 2500 serovars of genus Salmonella have been
identified, contributing to massive global losses in human and animal productivity as a
result of diarrhoea. A few strains, particularly host-adapted ones also cause heavy
mortality in young, immunocompromised and stressed populations. Year after year,
millions of people suffer with salmonellosis and about one third of the foodborne
disease outbreaks in humans are caused by Salmonellae alone [92].
Transmission of salmonellosis is often associated with animal and plant products
and more than 235 Salmonella serovars were found to be prevalent in India alone. Of the
many vaccines tried for control of salmonellosis, killed vaccines are serovar specific and
produce only short lived immunity. Live vaccines may turn infective in
immunocompromised individuals, in elderly and in infants as well as in healthy people
because of the zoonotic potential of Salmonella. Despite these limitations many different
types of vaccines, broadly classified as killed vaccines or bacterins, subunit vaccines and
22
live vaccines have been developed to control salmonellosis. Advantages and
disadvantages of each type of vaccine are summarized in Table 1.
Table 1. Advantages and Disadvantages of Live and Inactivated Vaccines.
Criteria Live Vaccine Inactivated Vaccine
Oral dosing Good immunity No or poor immunity
Duration of immunity Long Short
Requirement of adjuvant No Yes
Cross protection from related
strains
Present Rare
Safety on inoculation Varies Often safer
Horizontal spread of the vaccine
strain
Possible Not applicable
Vertical spread of vaccine strain Possible Not possible
Potential contamination Possible Remote chance
Stability and maintenance Poor and difficult Good and easy
CMI induction Good Poor
Secretary IgA and local mucosal
immunity
Good No
Reversion of vaccine strain to
pathogenic
Possible No
Persistence in the vaccinee Yes No
Interference from normal flora
of vaccinee
Possible No
Cost of the vaccine Less More
Requirement for
immunomodulators
No Yes
Vaccine marker Genetic markers Serological markers
Potential for vector vaccine
development
Good Poor
Potential for use in multivalent
combination
Less Good
Changes in growth conditions
during production have impact
on immunogenicity
Less More
There is no ideal vaccine available for control of salmonellosis. Such a vaccine
must be cheap, minimally reactive, induces mucosal immunity and has self-boostering
quality. It should be a single dose oral vaccine, preferably live and invasive but still safe
to induce durable immunity but not causing any disease in progeny of vaccinated
23
animals either on vertical or horizontal transmission. However, an ideal vaccine should
afford a life-long protection. Except for broilers and pig which are reared for a short
duration of 2-3 months, no Salmonella vaccine affords protection even for 1 year. An
ideal vaccine must be enabling differentiation of vaccinated from infected animals
(DIVA vaccine) [93].
A good vaccine candidate must easily be distinguished from wild type Salmonella
in a basal bacteriology laboratory by antigenic or genetic or phenotypic markers. Some
of the identifiable phenotypic characters such as susceptibility to low or high
temperature and requirement of some specific ingredients for growth (auxotrophic) have
been incorporated into modern vaccine candidates along with their compatibility with
growth promoting antibiotics, probiotics and prebiotics. However such phenotypic
markers must be non-transferable to the wild type homologous or heterologous strains
[94].
A vaccine should not deteriorate on storage if killed and should be stable and non-
reverting to pathogenic if live. It should not be interfering with colonization of normal
mucosal flora necessary for pathogen exclusion mechanism in healthy individuals,
should not cause development of tolerance on overuse, and must not be interfering with
other vaccines to be used in tandem [95].
German scientists point out the advantage of living vaccines for the prevention of
salmonella infections in animals, and a large amount of research has been done to obtain
live vaccines against salmonella infections [96].
Trmendous amount of work was conducted by Professors of the Department of
Microbiology and Virology of the Alma-Ata Zoo Veterinary Institute, B.A. Matvienko,
K.B. Biyashov and other employees, as well as the Whole Union State Scientific and
Control Institute of Veterinary Preparations (B. Yu. Shuster) and the Whole Union
Research Institute of Epidemiology and Microbiology named after. N.F. Gamalei (V.G.
Petrovskaya, V.M. Bondarenko) on the problems of specific prophylaxis of
salmonellosis in animals [5,6].
B.A. Matviyenko [5] received an attenuated strain of S. dublin 17, which differed
from the typical culture by agglutinability, sensitivity to bacteriophage and virulence,
but retained antigenic, immunogenic properties and typical biochemical activityby
passaging paratyphoid bacteria on poisonous snakes. The experimental vaccine from the
attenuated strain 17 with positive results was tested on calves in farms of disadvantaged
by paratyphoid. Despite this, strain 17 had not reached wide production use.
Later on, B.A. Matviyenko [5] received vaccine strains against the major
salmonellosis of domestic animalsusing chemicals and antibiotics. However, the lack of
genetic characteriszation of the vaccine strain, which makes it possible to distinguish it
from an epizootic prototype, as well as an increased residual virulence for white mice,
did not allow this strain to find wide industrial application in practice.
24
Great work was carried out by B. Yu. Shuster with co-authors on the preparation of
streptomycin-dependent mutants of S. dublin No. 6 and S. choleraesuis No. 9, the results
of the application were positive [97].
In the future, as a result of creative cooperation between the Alma-Ata Zoo-
veterinary Institute and the Whole Union Research Institute of Epidemiology and
Microbiology, N.F. Gamals, living and genetically characterized vaccines against
salmonellosis of animals were obtained and developed.
In recent years K.B.Biyashev, B.K.Biyashev and A.Zh. Makbuz attenuated strains
of Salmonella S. dublin-12, S. cholera suis B-17, S. typhimurium 09, S. typhimurium
58/2, S. abortusequi B-47, S. abortusovis 10in Kazakhstan. Currently, young animals
and adult farm animals and birds are vaccinated with live vaccines prepared from the
vaccine strainsmentioned above. The vaccines are approved in the Ministry of
Agriculture of the Republic of Kazakhstan and registered in the State Standard of the
Republic of Kazakhstan [6].
Thus, to date, considerable experience has been gathered on the use of live and
killed vaccines used abroad and in our country against bovine salmonellosis, with an
immunogenicity coefficient of live vaccines being higher than inactivated.
High immunizing activity of live vaccines is explained by the presence in them of a
number of antigenic complexes, preservation of the main metabolic pathways,
reproduction of the vaccine culture in the body, involvement of large tissue surfaces in
the immunogenesis process, the onset of rapid, intense and prolonged immunity with a
single administration of vaccines.
According to the literature, specific prevention is one of the main means in the fight
against salmonellosis in cattle.
Analysis of literature data indicates conflicting information about the specific
prevention of bovine salmonellosis using live and inactivated vaccines. However, it
should be noted that the literature data of research results of many scientists indicate the
significant advantages of live vaccines, because they fully preserve the antigenic set of
the pathogen and provide a longer- lasting immunity.
25
2. PERSONAL RESEARCH
2.1 Materials and methods of the research
The work was carried out in the period from 2015 to 2017 in the laboratory of
antibacterial biotechnology of the Kazakh National Agrarian University as well as in a
number of Kazakhstani farms.
The issues of epizootology of bovine salmonellosis were studied directly at the
farms. Annual reports of regional and district veterinary laboratories and veterinary
reports of the veterinary department of the regional territorial inspection of the Ministry
of Agriculture were applied.
Analysis of statistical data has shown that this infection has a significant spread
among the animals and birds of the republic, causing great economic damage not only to
livestock, poultry enterprises, but also to private farms.
Attention was drawn to the prevalence of the disease among adult cows and
newborn calves.
The clinical course of the disease was studied both in spontaneously sick animals
under unfavorable farm conditions and in experimentally infected animals.
Under natural conditions, we observed that salmonellosis in animals was in
intestinal (enteric) and septic forms. The main clinical signs of the disease were the
followng: diarrhea, turning into profuse, weakness, loss of appetite, depression,
dehydration.
Pathoanatomical studies were conducted in accordance with GOST 7269-91 «Meat.
Methods of sampling and organoleptic methods of freshness test» [98].
For intravital bacteriological diagnostics, samples of faeces in patients with
diarrhea of calves that had not been treated with antibacterial drugs. Samples of feces
were taken from sick calves to sterile test tubes directly from the rectum using a boiled
rubber catheter.
Samples from dead calves for the first ten days were examined for postmortem
bacteriological diagnostics. For the study, the liver, spleen, lungs, mesenteric lymph
nodes, a thin intestinal tract, heart, tubular bone were taken.
Nutrient media was prepared according to GOST 29112-91 «Solid culture media
(for veterinary purposes). Characteristics» [99].
The following nutrient media were used for the study of primary cultures, such as:
meatpeptone broth (MAP), meatpeptone agar (MPA), mediums of Endo, Kitt-Tarozzi,
Mink, Kaufmann, Levin [99].
In dysfunctional farms («Anisan», «Khabit», «Turtan-Ata») on mass intestinal
diseases of animals, 140 samples of pathological material obtained from calves with
clinical signs of diarrhea were subjected to bacteriological examination.
For postmortem bacteriological diagnostics, 160 samples from fallen calves were
examined during the first ten days. For the study, the liver, spleen, lungs, mesenteric
lymph nodes, a thin intestinal tract, heart, tubular bone were taken.
26
For intravital bacteriological diagnostics, 50 fecal samples were examined in
healthy calves that had not been treated with antibacterial drugs. Samples of feces were
taken from diseased and healthy calves to sterile test tubes directly from the rectum
using a boiled rubber catheter. Of all farms were taken ACTs to collect pathological
materials (Annex 3)
The diagnosis of salmonellosis was performed according to conventional methods.
MU 4.2.2723-10 Laboratory diagnostics of salmonellosis, detection of salmonella
in food and environmental objects (Methodical instructions of the Foundation of the
Central Research Institute of Epidemiology of Russian consumer supervision [100].
Meat-peptone agar (IPA), meat-peptone broth (MPB) and Endo agar were applied
for bacteriological testing using standard methods. In individual experiments Ploskirev
Agar and bismuth-sulfite agar were used as a selective medium. The selection of
cultures was carried out on the basis of the features of growth on media and microscopy
of preparations from individual colonies.
In virulent cultures, morphological, cultural, biochemical, antigenic properties and
pathogenicity were studied.
The pathogenicity of the isolated cultures was studied in experiments on laboratory
animals and calves.
The main objective of our studies was to investigate the biological properties of the
attenuated strain of S. dublin 31, immunological responses to vaccination, and to test an
experimental live vaccine against bovine salmonellosis in production conditions.
The attenuated strain S. dublin 31 was studied in comparison with the virulent S.
dublin 315/52 culture.
We studied 179 cultures isolated from the dead and diseased calves with clinical
signs of salmonellosis from farms of disadvantaged by this infection.
Morphology and cultural properties of attenuated and virulent strains of Salmonella
have been studied with multiple crossings on solid, semi-liquid and liquid nutrient
media. The main attention was paid to the possibility of dissociation of cultures, which
was controlled by the nature of growth on nutrient media and the stability of native and
warm suspensions of passages.
The biochemical properties of attenuated and virulent strains of salmonella have
been studied many times, by inoculating carbohydrate media with the Andrede indicator
and on selectively differential media by Endo and Ploskirev [99].
If cultures do not ferment lactose, do not split urea, but ferment glucose and form
hydrogen sulphide, they are suspicious of belonging to the genus Salmonella and are
subjected to further study.
The fermentation of lactose (and sucrose) in Olkenitsky medium and fermentation
of lactose in the Kligler and Russell medium is judged by the appearance of a yellow
color in the sloping part of the agar, and the fermentation of glucose by the same
staining in the column [100].
27
Gas formation was established by the presence of gas bubbles and agar rupture,
and the formation of hydrogen sulphide by the blackening of the medium. In the
environment of Olkenitsky with the growth of a culture hydrolyzing urea, the medium
will acquire a diffuse bright red-crimson color.
In the isolated cultures, their enzymatic characteristics were studied, which made
it possible to determine the generic accessory of the isolated bacteria.
For these purposes, tests were applied to determine the ability to form indole, the
presence of growth on media with citrates, the decomposition of salicin and malonate
sodium, the presence of lysine decarboxylase, phenylalanine deaminase, the ability to
decompose urea, the formation of acetyl-methyl carbinol in the Foges-Proskauer
reaction. A sample was also made with methyl red and the mobility was determined.
The antigenic properties of attenuated and virulent strains of salmonellae have been
repeatedly tested with poly- and monoreceptor (O- and H-) agglutinating sera in the
unfolded agglutination reaction and on the glass with sera of the serological group D (1,
9, 12, g, p) and the serological group B [100] .
Virulent properties of isolated Salmonella cultures were determined by setting up a
biological test on white mice weighing 14-16 g. Inbred white mice were infected with
isolated bacterial culture, intraperitoneally, at a dose of 0.3-0.5 ml; in an inoculated dose
of the culture, 3-105 to 106 colony forming units were contained in the control of the
original cultures according to the turbidity standard.
The residual virulence of the strain S. dublin 31 was tested on white mice and
calves, taking into account their reaction to the introduction of S. dublin 12 strain,
seeding and dissemination of the vaccinal culture at various doses and methods of
infection.
The constancy of the biological properties of the attenuated strain S. dublin 31 was
studied in long-term storage, repeated crossings on artificial nutrient media, after freeze-
drying and after passaging on sensitive animals.
Immunogenic activity was studied in experiments on white mice and calves.
Evaluation of the immunogenic activity of the attenuated strain was determined by
the Kerber method in the modification of I.P. Ashmarin and A.A. Vorob'ev [101].
The statistical processing of the results was carried out according to the method
described by R.F. Sosov and A.A. Glushkov (1974). The level of reliability was
determined using the Student-Fisher test. The data were considered reliable at P = 0.05
[102].
28
3. RESULTS OF THE STUDY
3.1 Prevalence of bovine salmonellosis
Analysis of statistical data, conducted by us in the veterinary departments of
Almaty, Kyzylorda and Aktobe regional- territorial inspectorates of the Ministry of
Agriculture of the Republic of Kazakhstan showed salmonellosis of cattle is widely
spread among livestock, causing great economic damage to all categories of farms.
In order to study the prevalence of bovine salmonellosis, studies were conducted in
different farms of Almaty, Aktobe and Kyzylorda regions. In most of the surveyed
farms diseases have been observed for several years. For instans, in the Almaty region,
Enbekshikazakh district, Karakemer village farm «Habit» salmonellosis was registered
for 5 years, and in the Kyzylorda region, Zhanakorgan district farm «Turtan-Ata»
registered for 3 years, Aktobe region the village of Belogorka farm «Anisan»
salmonellosis was registered for 4 years.
Analysis of statistical data, conducted by us in the department of veterinary
medicine of Almaty, Kyzylorda, Aktyubinsk regional territorial inspectorates of the
Ministry of Agriculture of the Republic of Kazakhstan for salmonellosis of cattle
showed that this disease has a significant spread among the cattle population, causing
great economic damage to all categories of farms.
Epizootological studies of Habit, Anisan, and Turtan-Ata farms established in
farms show that salmonella infection in calves is most often observed at the age of 10 to
60 days and is manifested by lethargy, unsteady gait, profuse diarrhea, dehydration of
the body, conjunctivitis, rhinitis and an increase in body temperature to 40.6-42 ° C.
At the opening of the fallen calves with signs of intestinal diseases, a characteristic
picture was noted - the contents of the intestine with gas bubbles, spotted haemorrhages
are noticeable on the mucosa of the thin and thick intestine, ulcers are detected. The
spleen is enlarged grey-yellow in colour, in the kidney are visible point haemorrhages,
the capsule is easily removed. When cutting the affected lobe of the lungs, a
mucopurulent mass is released. Bronchial, mediastinal and mesenteric lymph nodes are
enlarged, with haemorrhages.
Veterinary specialists regularly sent pathological materials for bacteriological
studies. In the farms under study over the last 5 years, salmonellosis has been confirmed
in 14 cases, which is 62%.
Analyzing the statistical reports and taking into account the opinions of the
veterinary specialists of the region, we note that the main cause of the emergence of
salmonella in young animals is the non-observance of zoogenic conditions of keeping
and feeding the breeding stock and the birth of a non-viable offspring. A complex
epizootic situation in the incidence of the gastrointestinal tract, in particular in new-born
calves, salmonella is observed in farms engaged in dairy cattle, where most of the milk
is of commercial value, is sent for sale. Along with this, there is a violation of content
technology and a low culture of animal husbandry. It should also be noted that adverse
conditions (high relative humidity, fluctuations in indoor temperature, inadequate
29
feeding) reduce the natural resistance of young animals. An infectious process of
salmonella carriers is aggravated, the number of patients is sharply increasing.
Under natural conditions, we observed that salmonella in calves flowed in intestinal
(enteric) and septic forms. The main clinical signs of the disease were: diarrhea turning
into profuse form, weakness, loss of appetite, depression, dehydration.
Pathological changes in the dead calves had a picture of catarrhal and catarrhal-
hemorrhagic gastroenteritis, ulcers, multiple ulcers on the stomach mucosa, large and
small intestine, under the capsule of the spleen. Regional mesenteric lymph nodes
enlarged, edematous.
In the farms with mass intestinal diseases of animals, 140 samples of pathological
material were obtained from calves with clinical signs of diarrhea and were subjected to
bacteriological examination.
For postmortem bacteriological diagnostics, 160 samples from dead calves were
examined during the first ten days. Liver, spleen, lungs, mesenteric lymph nodes, a thin
intestinal tract, heart, tubular bone were taken.
For bacteriological diagnostics, 50 fecal samples from healthy calves that had not
been treated with antibacterial drugswere examined. Samples of feces were taken from
diseased and healthy calves to sterile test tubes directly from the rectum using a boiled
rubber catheter.
Primary selection of cultures was carried out on the basis of features of growth on
media and microscopy of preparations from individual colonies. Morphological,
cultural, biochemical properties were tested according to the generally accepted
schemes.
Identification of the isolated cultures was carried out according to Berdzhi's
determinant.
Table 2. Results of bacteriological studies of organs from sick and dead calves, as
well as from faeces of healthy calves.
Kind of animal Where sampled Number of
samples
Number of
isolated
salmonella
cultures
Calves Sick 140 45
Calves Dead 160 116
Calves Healthy 50 18
In total - 350 179
30
Figure 1 – Percentage bacteriological studies of organs from sick and dead calves,
as well as from faeces of healthy calves.
As a result of the studies of organs of diseased and dead calves, as well as faeces
of healthy calves, 179 Salmonella cultures were isolated and identified, including from
diseased calves - 45, from dead - 116 and from healthy calves – 18 (Table 2, Figure 1)
[103] (Annexes 1,2,3).
3.2 Biological properties of salmonella cultures
Salmonellosis belongs to the group of intestinal infections, but the fight against
them and their prevention is much more complicated than with other gastrointestinal
infections. This is due to a wide circulation of numerous serovars of salmonella in
nature, polyethiologic, a variety of ways of introduction into the body of animals and
humans.
In farm («Khabit», «Anisan», «Turtan-Ata»)conditions, we studied the clinical
picture of diseased calves, and also noted some signs of salmonellosis in adult cattle.
In the «Khabit» farm, the disease of calves with salmonellosis was observed at the
age of 10 to 60 days. Typical clinical signs were: high temperature (40-42 ºС), diarrhea
(mucus, blood), respiratory tract infection (discharge from the nose, cough, frequent,
painful), arthritis (swelling, lameness), impaired coordination of movements, sluggish
reacts to the environment. In the absence of treatment for 5-10 days, calve's diseases
mostly died.
31
At autopsy of the fallen calves the following pathoanatomical picture was noted:
the spleen was enlarged, gray-red, its edges were rounded, the capsule was tense, small
hemorrhages were present, the parenchyma of the spleen was cherry brown, bloody and
easily scraped; The mucous membrane of the stomach is swollen, hyperemic with
hemorrhages; the thin part of the intestine is swollen with gases, the mucous parts are
catarrally inflamed, there are pinpoint hemorrhages; the lymph nodes of the mesentery
are enlarged, juicy, hyperemic, on a section of their hemorrhage; the liver is enlarged,
flabby, with a clay shade, on the incision is dryish, small necrotic nodules are found; the
kidneys are pink or gray-yellow, the blood vessels are injected, spot pinpoint
hemorrhages are spotted.
The biological properties of the isolated cultures were studied by their cultural,
biochemical and antigenic properties.
The cultural properties of the isolated cultures were studied on meat-peptone agar
and on semi-liquid media. After a 16-18 hour cultivation on sloping agar, a rather mild
growth in the form of a gentle plaque was observed in most test tubes, while in others - a
uniform growth, with a bluish tinge was seen. Colonies were round S-shaped. An early
clouding with a small precipitate was observed in liquid media. Salmonella in smears
were located singly, randomly, in the form of short sticks. All cultures had good
mobility. They were negatively stained by Gramm.
Fermentation of carbohydrates was determined on media containing sugars and
polyhydric alcohols on semi-liquid agar with the Andrede indicator. The results were
observed for 10 days.
The formation of hydrogen sulphide and indole by the cultures was determined by
means of a strip of filter paper with an aqueous solution of acetic acid lead and 12%
solution of oxalic acid. The plates were kept in a thermostat at 37 ° C. Counting was
performed after two days.
The results of studying the biochemical properties of the Salmonella cultures
studied on media containing sugar and polyhydric alcohols on semi-liquid agar with the
Andrede indicator showed that the range of growth temperature is 37-39 ºС, the
optimum temperature is 37 ºС. The optimum pH is 6.8-7.5. As a carbon source, strains
use glucose, maltose, arabinose, xylose, rhamnose, mannitol, sorbitol. Form hydrogen
sulphide. They possess lysine - and ornithine decarboxylase activity, do not possess any
contagious activity. Do not form an indole.
The studies of the morphological, tinctorial, cultural and biochemical properties of
179 cultures isolated from diseased and dead calves, as well as from the faeces of
healthy calves showed that they were typical for the Salmonella genus.
For the serological tests of isolated cultures, general and monoreceptor
agglutinating sera (O and H) were used. It was established that all strains of salmonella
were agglutinated to the same degree in four crosses by common and monoreceptor sera.
32
Table 3 - Antigenic structure and serovariants of salmonella, isolated from calves
of unfavorable farms.
Farms Number
of cultures
Multi
valen
t
the reaction of
agglutination with
sera to antigens
Gro
ups
Serovars
О – antigen
1,4,5,9,12
Н-
antigen
i,q,p,m
«Khabit»
«Khabit»
«Khabit»
«Khabit»
«Khabit»
1-30
31-39
40-55
60-65
66-80
+
+
+
+
+
+ 0 0 + +
+ 0 0 + +
+ 0 0 + +
+ 0 0 + +
+ + + 0 +
0 + + 0
0 + 0 +
0 + + 0
0 + + 0
+ 0 0 0
Д1
Д1
Д1
Д1
В
S.dublin
S.enteritidis
S.dublin
S.dublin
S.typhimurium
«Аnisan»
«Аnisan»
«Аnisan»
«Аnisan»
81-90
91-100
101-113
114-118
+
+
+
+
+ 0 0 + +
+ 0 0 + +
+ + + 0 +
+ 0 0 + +
0 + + 0
0 + 0 +
+ 0 0 0
0 + + 0
Д1
Д1
В
Д
S.dublin
S.enteritidis
S.typhimurium
S.dublin
«Тurtan-Аtа»
«Тurtan-Аtа »
«Тurtan-Аtа »
«Тurtan-Аtа »
«Тurtan-Аtа »
119 -130
131-134
135-147
148-161
162-179
+
+
+
+
+
+ 0 0 + +
+ 0 0 + +
+ + + 0 +
+ 0 0 + +
+ 0 0 + +
0 + + 0
0 + + 0
+ 0 0 0
0 + + 0
0 + + 0
Д1
Д1
В
Д1
Д1
S.dublin
S.enteritidis
S.typhimurium
S.dublin
S.dublin
Note – «+»- agglutination
In the identification of 179 Salmonella cultures isolated from diseased and dead
calves, as well as from the faeces of healthy calves, it was found that 121 (68.0%)
belonged to Salmonellа dublin (group D) , S. typhimurium (group B) - 38 (21.0%) and
20 (11.0%) - Salmonella enteritidis (group B )(Table 3, Figure 3).
Table 4 - Saerovariants of salmonella isolated from calves
Serovars Calves
from sick from dead from feces
Salmonellа dublin 31 78 12
S.typhimurium 10 24 4
Salmonellа enteritidis 4 14 2
Total cultures studied 45 116 18
33
Figure 3 - Saerovariants of salmonella isolated from calves
The purpose of our research was to determine the pathogenicity of salmonella
isolated from animals and birds for the selection of production strains of enteroinfection
pathogens that will be used to manufacture innovative biologics against
enterobacteriocinosis in animals and birds.
Previously, the pathogenicity of all the isolated cultures was checked on white mice
injected intraperitoneally at doses of 103, 104, 105, 106 and 109 colony-forming units.
The results of the experiment indicated that the experimental animals died completely
when infected with a dose of 105 CFU or higher.
As a result, strains of salmonella isolated from fallen calves: S.dublin,
S.typhimurium, S.enteritidis (3 strains from each salmonella serovar) were selected
based on the study of morphological, biochemical and antigenic properties and the
degree of pathogenicity of the isolated cultures.
The virulence of S.dublin, S.typhimurium, S.enteritidis cultures was studied in
experiments on white mice.
White mice were infected intraperitoneally with salmonella strains at various doses
of colony forming units (CFU). The results were evaluated by the survival of the
experimental animals (Table 5,6,7).
34
Table 5 - Virulence of S.dublin strains isolated from diseased calves in experiments
on white mice.
№ Culture name
No of
animals
Infecti
on
dose
(CFU)
Inoculat
ion
method
Result
Died Alive % of
staying
alive
1 S.dublin -14 20 103 I/p 14 6 30
-//- 20 104 I/p 19 1 5
-//- 20 105 I/p 20 - -
-//- 20 106 I/p 20 - -
2 S.dublin -76 20 103 I/p 18 2 10
-//- 20 104 I/p 20 - -
-//- 20 105 I/p 20 - -
-//- 20 106 I/p 20 - -
3 S.dublin -66 20 103 I/p - 20 100
-//- 20 104 I/p - - 100
-//- 20 105 I/p 4 16 80
-//- 20 106 I/p 9 11 55
Note. Observation period 15 days
Figure 4 - Percentages virulence of S.dublin strains isolated from diseased calves in
experiments on white mice.
35
Table 6 - Virulence of strains of S.enteritidis isolated from calves in experiments
on white mice.
№ Culture name
No of
animal
s
Infectio
n dose
(CFU)
Inoculat
ion
method
Result
Died Alive % of
staying
alive
1 S. enteritidis -36 20 103 I/p 2 18 90
-//- 20 104 I/p 2 18 90
-//- 20 105 I/p 7 13 65
-//- 20 106 I/p 12 8 40
2 S. enteritidis -91 20 103 I/p - 20 100
-//- 20 104 I/p - 20 100
-//- 20 105 I/p - 20 100
-//- 20 106 I/p 3 17 85
3 S. enteritidis -54 20 103 I/p 10 10 50
-//- 20 104 I/p 15 5 25
-//- 20 105 I/p 18 2 20
-//- 20 106 I/p 20 - -
Note. Observation period 15 days
Figure 5 - Percentages virulence of strains of S.enteritidis isolated from calves in
experiments on white mice.
36
Table 7 - Virulence of strains of S. typhimurium isolated from calves in
experiments on white mice
№ Culture name
No of
animal
s
Infectio
n dose
(CFU)
Inoculat
ion
method
Result
Died Alive % of
staying
alive
1 S. typhimurium -9 20 103 -//- 16 4 20
-//- 20 104 -//- 18 2 10
-//- 20 105 -//- 20 - -
-//- 20 106 -//- 20 - -
2 S. typhimurium -
86
20 103 -//- 1 19 95
-//- 20 104 -//- 3 17 85
-//- 20 105 -//- 5 15 75
-//- 20 106 -//- 9 11 55
3 S. typhimurium -
69
20 103 -//- 10 10 50
-//- 20 104 -//- 20 - -
-//- 20 105 -//- 20 - -
-//- 20 106 -//- 20 - -
Note. Observation period 15 days
Figure 6 - Percentages virulence of strains of S.typhimurium isolated from calves in
experiments on white mice.
37
As can be seen on the tables 4,5,6 the results of the experiments showed that
the cultures tested had a sufficiently high virulence, especially strains: S.dublin 76
and S. typhimurium 69, isolated from the dead calves, causing 100% death of the
experimental animals at a dose of ≥104 CFU.
An autopsy was carried out in all experiments. Infectious cultures were
constantly isolated.
The virulence of S.dublin 76, S. typhimurium 69, S.enteritidis 54 strains were
tested on calves. All animals were 1 months old. Reference virulent strains of S.
typhimurium 371, S. dublin 315/52, S.enteritidis 51, taken from VGNKI (Moscow) were
used as control.
The experimental calves were infected intraperitoneally by daily agar culture in
appropriate doses. Experimental animals mostly died on the 6th -12th day after the
infection with obvious signs of salmonellosis.
The results of the experiment are shown in Table 8.
An autopsy was carried out in all experiments. Infectious cultures were constantly
isolated.
The results of the conducted studies testify to the etiological role of the studied
salmonella in the disease of calves.
The main goal of our research was to obtain attenuated strains of salmonella, to
study their biological properties, to use it as a vaccine strain for the production of a live
vaccine against bovine salmonellosis.
Table 8- Test of virulence of S.dublin 76, S. typhimurium 69, S. enteritidis 54
strains on calves.
Culture name
No of
anim
als
Infectio
n dose
(CFU)
Inoculation
method
Result
Died Alive % of
staying
alive
S.dublin 76
10
10
10
10
109
2*109
4*109
6*109
Intraperitoneally
-//-
-//-
-//-
10
10
10
10
-
-
-
-
-
-
-
-
Died on
day 7-10
S.dublin 315/52
(control strain)
10
10
10
10
109
2*109
4*109
6*109
Intraperitoneally
-//-
-//-
-//-
9
10
10
10
1
-
-
-
10
-
-
-
Died on
day8-12
38
S. typhimurium
69
10
10
10
10
109
2*109
4*109
6*109
Intraperitoneally
-//-
-//-
-//-
10
10
10
10
-
-
-
-
-
-
-
-
Died on
day6-9
S.typhimurium
371
(control strain)
10
10
10
10
109
2*109
4*109
6*109
Intraperitoneally
-//-
-//-
-//-
9
10
10
10
1
-
-
-
10
-
-
-
Died on
day7-9
S. enteritidis 54
10
10
10
10
109
2*109
4*109
6*109
Intraperitoneally
-//-
-//-
-//-
4
6
10
10
6
4
-
-
60
40
-
-
Died on
day9-10
S. enteritidis 51
(control strain)
10
10
10
10
109
2*109
4*109
6*109
Intraperitoneally
-//-
-//-
-//-
9
10
10
10
1
-
-
-
10
-
-
-
Died on
day8-10
Note. Observation period 20days
Our studies showed that the strains studied preserved the typical morphological,
tinctorial, cultural, biochemical, antigenic and pathogenic properties characteristic of the
corresponding salmonella serovars.
The studied S. dublin 76 strain was selected as the initial strain for use in the
development and design of a live vaccine (using the attenuation method) against bovine
salmonellosis.
The task of our further research was to obtain an attenuated strain of salmonella, to
study its biological properties, to use it as a vaccine strain for the production of a live
vaccine against bovine salmonella [104] (Annexes 4).
4. Development of genetically characterized strains of Salmonella
Principles of obtaining attenuated strains of enterobacteria.
Live vaccines are biological preparations from hereditarily altered forms (mutants)
of pathogens of infectious diseases suspended or dried in appropriate protective
39
environments. Mutants of pathogens with genetic characteristicsare defined as forms
that underwent genotypic changes, as a result of which they irreversibly lost the ability
to cause pathological changes in the susceptible organism. At the same time, they
retained their genetic constitution determinants determining their ability to cause
specific immunological changes and restructuring. In accordance with the transformed
genome these mutants also changed their phenotypic area.
To obtain hereditarily altered mutants of pathogens that are suitable as live
vaccines, researchers used various pathways.
From early literature sources it was established that the most common way of
obtaining avirulent strains is the method of passaging virulent cultures on artificial
nutrient media and in non-susceptible animals.
So, in Romania, Istratic co-authors proposed a vaccine strain for enteric
vaccination, an avirulent variant of the Flexner strain obtained by selection from a
virulent strain passaged multiple times (32 passages) on bile medium.
V.D.Hecker and co-workers obtained vaccine strains of Flexner, 516 and Zonne–20
by selecting from the population of virulent strains after multiple passaging of the latter
on artificial nutrient media.
B. Matvienko (1958) by passaging the virulent culture of S.dublin through
poisonous snakes vaccine strain S.dublin-17 was obtained. This strain lost
agglutinability with Hertner's serum, sensitivity to phage, high virulence, but retained
antigenic, immunogenic properties and typical biochemical activity.
Later, a number of researchers studied the possibility of obtaining attenuated strains
of typhoid bacteria under the influence of physical, chemical and biological factors.
B. Elbert, G. Vilenchik, D. Emushko (1957) by freezing and thawing, growing at
high temperatures, storage of distilled water and under the influence of lithium salts
embodiment typhoid received coccoid bacteria that has lost the ability to ferment
glucose and agglutinated specific serum. At the same time, to coccoid serum
agglutination caused embodiment as a variant to the culture, and the starting virulent
strain [104].
Extensive research on the preparation of attenuated strains of coliform conducted
Romanian scientists.They were obtained avirulent strains of S.typhi, S.typhimurium,
S.enteritidis by exposing the microbial surface active agents, anesthetic agents, coloring
agents, antibiotics.
Working on the issue ofmaking live vaccines against salmonella infections, B.A.
Matvienko presented vaccine strains of S. choleraesuis TC-177, S. abortusovis Trp
1/10, S. typhimurium Tr-Br -1, S abortusegui E-841 under the influence of a chemical
mutagen (trypaflavin). According to the scheme of sequential passaging, there was an
adaptation of a pathogen to increasing concentrations of tripaflavin [5]. Vaccine strains
with positive results were tested on laboratory animals, sheep, calves and pigs in
production conditions.
40
Successes of bacterial genetics and molecular biology have recently made it
possible to proceed to targeted methods of obtaining vaccine strains with reduced
virulence. Auxotrophic mutantsrequiring addition of growth factors, hybrid - strains
obtained as a result of interspecific crossings and ribosomal mutants (in particular,
streptomycin-dependent forms of bacteria) became mostly widespread.
The idea of obtaining hybrids and using them as vaccine strains is due to the
genetic studies of S. Faloometal , which showed that some chromosomal areas of the
Flexler's Escherichia and Shigella are structurally similar, that crosses are admissible,
although the frequency of recombinations between them is much lower than that
between the same strains . It is shown that in these cases hybrid forms are not perceived
by all, but only by certain genetic markers. The hybrid obtained in this way by a group
of American authors lost the ability to cause symptoms of the disease in guinea pigs and
monkeys, but retained the ability to cause kerato – conjunctival infection in guinea pigs,
penetrate the epithelial cells of the intestinal mucosa and reach laminapropria, but lost
the ability for intensive propagation in this region.
In the last decade streptomycine - dependent (str-d) forms of bacteria have become
the most widespread for the production of live bovine vaccines.
For the first time, streptomycin-dependent mutants were obtained by Yugoslav
researchers D. Melom and co-workers. The authors report that in their morphological,
serological, immunological and toxic properties, as well as in relation to phages, str-
dstrains are identical to the original strains. They differ from the latter only in their
inability to grow on nutrient media in the absence of streptomycin, as well as in the body
of experimental animals.
The search for live vaccines based on the principle proposed by the Yugoslav
authors was conducted in Russia.
V. Sergeev, V. Shuster and others isolated streptomycin-dependent mutants Shigell
and Flexner and studied their biological properties. It was shown that this mutation did
not lead to a change in the antigenic structure of the microbes.
The authors report that in their morphological, serological, immunological and toxic
properties, and also with respect to phages, str-d strains are identical to the original
strains. They differ from the latter only in their inability to grow on nutrient media in the
absence of streptomycin, as well as in the body of experimental animals.
The above-mentioned experimental studies on obtaining attenuated strains of
enterobacteria, suitable as live vaccines, indicate significant progress in this direction,
achieved mainly during the last decade.
However it should be noted that one of the main reasons for the slow introduction
of live intestinal vaccines into practice, made from avirulent strains obtained by the
above mentioned methods, is associated with the possibility of reversion of the vaccine
strain cells into the original virulent state. In particularit is applied to avirulent strains
obtained by passaging on nutrient media, in non-susceptible animals, by adapting
41
virulent cultures to increasing concentrations of the chemical compound (analogues of
nitrogenous bases, oxidants, reducing agents and acridine crushers).
This is explained by the fact that for obtaining vaccine strains, it is not only
necessary to influence a virulent culture with a particular mutagen, but also directional
effect, i.e. the one that would ensure the shifts in its genetic apparatus. With the methods
of obtaining avirulent strains, the specific mechanism of action of the selected mutagen
is not clear on the completely determined discreteness of the genetic apparatus of the
individual, i.e. there are no genetic marks, which make it possible to distinguish them
from natural prototypes.
The data on a sufficiently high reversion into the prototrophic (virulent) state of
auxotrophic mutants, in particular those dependent on purine are established. They also
found that streptomycine - dependent mutants of salmonella in 20% of cases are
reversed into a virulent state.
Thus, the creation of live vaccines based on attenuated strains with a single
mutation is inappropriate because of the possibility of reversion into a virulent state.
According to international requirements and standards, attenuated strains used for
the production of live vaccines must have a minimum of two characterized mutations,
have a stable biological properties, have a weak residual virulence and provide
immunity to infection of most animals by a single immunization.
In contrast to the method for obtaining avirulent suppressor streptomycin-
dependent mutants, we developed and tested a new method of bacterial attenuation that
provides two-marker potential vaccine strains of salmonellae with an optimal level of
attenuation to the corresponding host.
The method was developed as a result of creative collaboration between the
laboratories of the genetics and laboratory of virulence of bacteria NIIEM named after
N.L. Gamaleya of the Academy of Medical Sciences of Russian Federation and the
laboratory of antibacterial biotechnology (authors – V.G. Petrovskaya, Y.M. Romanova,
K.B. Biyashev, B.K. Biyashev, A.Zh.Makbuz). The method of obtaining avirulent
bacterial strain was as follows: the initial strain highly virulent for mice (LD50 = 8 cells)
under the influence of mutagens reduced virulent properties, i.e. attenuation occured.
Attenuation is associated with a violation of the translation of genes that encode the
synthesis of important pathogenicity factors of the bacterium or genes the products of
which are important for life of the bacterium. The study of virulence of the resulting
transductants on white mice intraperitoneally revealed a decrease in clones that acquired
simultaneously 2 mutations that impart resistance to the two mutagens, and each
separately. The presence of two mutations with known mechanisms of actionin the
avirulent strain, each of which can significantly reduce virulent properties, serves as a
convincing genetic evidence of the stability and safety of the avirulent vaccine strain.
The theoretical frequency of the reverse mutation simultaneously for both markers
is approximately 10-14, although in practice this is not possible. Live vaccines prepared
42
from attenuated strains were harmless, immunogenic, labeled and genetically safe in the
study on various biological objects.
We have developed a method that allows the addition of a third mutation in the
two-marker strain, which informs the high sensitivity of the bacterium to surface active
substances. This mutation, referred to as Hst (high sensibility), does not affect the
attenuation and immunogenicity of the strain, but it limits the time of bacterial
experience in the host's intestine and in the external environment. Potential vaccine
strains possessing Hst mutation are not capable of prolonged exposure to the
environment, and therefore they can be considered as "environmentally friendly" live
vaccines characterized by limited ability to form an infectious chain (i.e., incapable of
epidemic or epizootic distribution).
In conclusion, it should be noted that the detection and study of genetic control of
the virulence factors of salmonella, responsible for the development of the infectious
process and the toxic effect on the body is of paramount importance for deciphering
many vailed aspects of the infection pathogenesis and developing effective drugs for
specific prevention of salmonella infections. Successes in the study of the genetics of
virulence of enterobacteria already allows the construction of safe, immunogenic,
effective, labeled, potentially vaccine strains.
4.1. Method of producing an attenuated strain of Salmonella
The aim of our research was to obtain a strain of salmonella that possesses stability
of biological properties, moderate reactogenicity and residual virulence, harmlessness,
high immunogenicity, epizootic safety and having genetic markers to distinguish it from
a wild type strain.
Salmonella dublin strain 31 was obtained by genetic method from the wild type
virulent strain Salmonella dublin 76. The initial strain Salmonella dublin 76, virulent for
mice (LD50 = 100 bacteria), was plated on the plates with the Hottinger agar at a
concentration of 1010CFU (colony forming units). The agar plates contained 100 μg / ml
of Nal mutagen and 50 μg / ml of Rif mutagen. Mutants that had acquired resistance to
400-500 μg / ml Nal and 100-200 μg / ml Rif were selected.The obtained mutants with
the desired phenotype were 3 times passaged on a selective medium with mutagens.
Virulence of isolated clones was studied in experiments on mice by intraperitoneal
infection. Mutants with phenotype NalR 500 and RifR 100 were characterized by
decrease in virulence by 6-7 in contrast to mutants resistant to Nal and high
concentrations of Rif (phenotype Nal R 700 and RifR 200). An attenuated clone # 31
(LD50 = 107 bacteria) was selected, which was a donor strain in transduction
experiments using bacteriophage P22. It was shown that the transfer of each mutation,
which caused mutant No. 31phenotype, to the initial virulent strain # 76 leads to a
decrease in its virulence. Among the transductants that simultaneously acquired
mutations that impart resistance to Nal R and RifR, a strain of Salmonella dublin 31 was
selected.
43
The obtained strain Salmonella dublin 31 is deposited in the Collection of
Microorganisms of the Republican State Enterprise "Scientific Research Institute of
Biological Safety Problems" of the Ministry of Education and Science of the Republic of
Kazakhstan (RSE NIIPBB KN MES RK).
Collection number M-42-15 / D
The strain Salmonella dublin 31 is characterized by the following features.
Morphological signs.
The cells of the strain are straight, rod-shaped (1.5-1.7) * (2.4-5.6) μm, mobile,
gram-negative, do not form spores.
Cultural properties.
The bacteria of the strain, when grown on Hottinger agar and the Difco nutrient
broth after 24 hours, form smooth, round, shiny, translucent colonies of gray color with
a flat edge of 2 mm, in Endo medium after 24 hours - round colorless translucent
colonies with a flat edge of 2 mm. When cultivated in liquid media - Difco broth
bacteria form a uniform turbidityin 18h.
Physiological and biochemical signs.
The range of growth temperatures is 37 - 39 ° С, the optimum temperature is 37 °
С. The optimum pH is 6.8-7.5. The source of carbon is glucose, maltose, galactose,
mannitol, sorbitol. Possesses lysine and ornithine decarboxylase activity, does not
possess urease activity. Does not form hydrogen sulphide and indole.
Antigenic structure.
Typical for birds’Salmonella 0-1,9,12; Hg, p.
It is sensitive to bacteriophage P22,
Resistance to mutagens.
It is resistant to Nal R 100 μg / ml and RifR 100 μg / ml.
Residual virulence.
At intraperitoneal infection of white mice Lg LD50 = 7.0 ± 0.3 (according to the
method of Reed and Mench with the mean error calculated by the Pizzi formula).
Stability of residual virulence.
A 10-fold passage of S. dublin 31 strain through the body of white mice revealed
the retention of the initial level of residual virulence and the stability of resistance
markers to NaR and RifR. All subculturesisolated from the body of the mouse after each
passage have approximately the same indices of residual virulence Lg LD50 = 0.7 ± 0.3.
Stability of attenuation and markers of resistance to NalR and RifR in S. dublin 31
strain was determined by treatment of the strain with a mutagen nitrosoguanidine. The
study of these properties in 10 clones of the strain selected after such treatment revealed
their preservation at the same level as in the untreated cultures (Lg LD50-7.0 ± 0.3 the
minimal suppressive is-NalR 100 μg / ml and RifR 100 μg / ml).
Immunogenicity for white mongrel mice.
44
The strain has a pronounced immunogenicity for mice, protecting 100% of the
animals when immunized with a dose of 105 cfu, 95% in white mice with a dose of 104
cfu.
Genetic analysis of the strain safety as a live vaccine.
A genetic study of the Salmonella dublin strain 31 performed using a transducing
bacteriophage P22, showed that its phenotype is determined by the presence of 2
independent mutations, in the RifR gene and in a gene that determines resistance to
NaR. It has been established that mutations controlling the resistance to RifR and NalR
lead to damage of ribosomal proteins (S13, S16) and thus influence the correctness of
the reading of genetic information. As a result, the virulent properties decrease, i.e.
attenuation occurs. Thus, attenuation is associated with a violation of the translation of
genes encoding the synthesis of important pathogenicity factors of the bacterium or
genes products of which are important in the life of the bacterium.
A study of the virulence of the resulting transductants by intraperitoneal infection
of white mice revealed a decrease in its clones, which acquire both 2 mutations that
confer resistance to Nal R and RifR, and each separately. The presence in the S. dublin
31 strain of two mutations, each of which can reduce virulent properties, is a convincing
proof of the stability and safety of the vaccine strain Salmonella dublin 31.
We have developed a method that allows the addition of a third mutation in the
two-marker strain, which informs the high sensitivity of the bacterium to surface active
substances. This mutation, referred to as Hst (high sensibility), does not affect the
attenuation and immunogenicity of the strain, but it limits the time of bacterial
experience in the host's intestine and in the external environment. Potential vaccine
strains possessing Hst mutation are not capable of prolonged exposure to the
environment, and therefore they can be considered as "environmentally friendly" live
vaccines characterized by limited ability to form an infectious chain (i.e., incapable of
epidemic or epizootic distribution).
Differentiation of the vaccine strain from wild type cultures.
The strain Salmonella dublin 31 is differentiated from wild type cultures due to
resistance to Nal R, RifR and high sensitivity to surface active substances. The presence
of genetic markers for the three mutagens allows to differentiate vaccine strains from the
field strarins in 16-20 hours in case when salmonellosis is suspected or when salmonella
are isolated in products of animal origin.
Thus, the strain Salmonella dublin 31 meets all the requirements for vaccine
strains: it has a stable biological properties, moderate reactogenicity and residual
virulence, high immunogenicity for mice and chickens, is epizootically safe for use and
has three genetic markers for distinguishing it from a wild type strain. The presence in of
three mutations Salmonella dublin strain 31,with known mechanisms of action serves as
a convincing genetic evidence of the stability and safety of the attenuated strain
Salmonella dublin 31. The theoretical frequency of reverse mutation simultaneously for
all markers is approximately 10-21, which is practically impossible.
45
The purpose of the strain Salmonella dublin 31 is the production of vaccines
against salmonellosis of cattle [105].
4.2 Characteristics of the biological properties of the attenuated strain Salmonella
dublin 31.
Our studies had showed that the attenuated strain Salmonella dublin 31, preserved
typical morphological, tinctorial, cultural, biochemical and antigenic properties
matching the corresponding serovar.
We drew attention to the possibility of dissociation of the vaccine strain S.dublin
and the virulent culture of S. dublin 315/52.
Figure 7 - Tinctorial properties of the S. dublin strain 31. The photo was taken with
the Levenhuk t 800 Microscope.
46
Figure 8 - The process of seeding culture of Salmonella dublin 31on medium MPA.
Estimation of the degree of dissociation of salmonella was carried out by multiple
scatters on petri dishes with MPA and taking into account the agglutination reaction in
physiological sodium chloride solution. After boiling for an hour, the above strains did
not precipitate. All this gives grounds to believe that all strains (vaccine and virulent) are
in a stable S-form (Figure 9).
Figure 9 –The growth of Salmonella dublin 31 culture at MPA (S-form).
47
Close attention was paid to the possibility of dissociation of the vaccine strain S.
dublin 31. An evaluation of the dissociation degree of salmonella was carried out by
multiple scatters on plates of Petri with MPA, the agglutination reaction was taken into
account, and virulent strains formed stable suspensions in physiological saline solution
at 37 ° C, Over the course of an hour the above strains did not precipitate. All this gives
grounds to believe that all strains (vaccine and virulent) are in a stable S-form (Figure 9)
[106] (Annexes 5).
One of the important requirements for attenuated vaccine strains is the retention of
residual virulence, on which the high immunizing ability of the live vaccine depends. In
this connection, throughout our experiments attention was drawn to the virulence
consistency.
Figure 10 - The process of intraperitoneal injection of white mice.
The residual virulence of the vaccine strain S.dublin 31 was tested in comparison
with the virulent culture of S. dublin 315/52 on white mice (weighing 14-16 g) and
calves (aged 8-10 days) in several repetitions, taking into account their survival,
dissemination process and timing of elimination of the culture of the vaccine strain.
White mice were infected with the daily culture of the attenuated S. dublin 31
strain subcutaneously at doses of 104, 105, 106, 107, 108CFU and intraperitoneally at a
dose of 106 107 and 108CFU (Figure 10).
48
It should be noted that these doses of the S.dublin 31 strain correspond to 50 to
10,000 fatal doses of the virulent culture of S.dublin 315/52 (LD50 102 CFU). The results
of the experiment are shown in Table 9.
Table 9 - Tests of the virulence of an attenuated strain S.dublin 31 in an
experiment on white mice.
Name of the
strain
Infection Results
No of
animals Infection
dose
(CFU)
Inoculation
method Died Alive
%
Of
staying
alive
S.dublin 31 30 104 Subcutaneously - 30 100
30 105 -//- - 30 100
30 106 -//- - 30 100
30 107 -//- - 30 100
30 108 -//- 3 27 90
S.dublin
315/52
(virulent)
30 103 Subcutaneously 17 13 43
30 104 -//- 25 5 16
30 105 -//- 30 - 0
S.dublin 31 30 106 Intraperitoneally - 30 100
30 107 -//- 2 28 93
30 108 -//- 3 27 90
S.dublin
315/52
(virul.)
30 103 Intraperitoneally 23 7 23
30 104 - / / - 27 3 10
30 105 - / / - 30 - 0
Note: The observant+-ion period was 20 days after infection
Table 8 shows that all mice infected with S. dublin 31 culture survive in 90-100%
of cases during the 20 days of observation, whereas control mice infected with the
virulent culture of S.dublin 315/52 at a dose of 103, 104 and 105CFU died from 57 to
100% of cases.
In the experiments on calves the residual virulence of S.dublin strain 31 was
studied by intraperitoneal administration. A total of 70 calves were used in the
experiment. The virulence of the strain was controlled by the survival rate of both the
general and local response. The results of the experiment are given in Table 9.
Table 10- Test of attenuatedstrain S.dublin 31 virulence in the experiment on calves.
Name of the
strain
Infection Results
No of Inoculation Infection Died Alive % of
49
animals method dose(CFU) staying
alive
S. dublin 31 10 Intraperitoneally 4*109 - 10 100
10 Intraperitoneally 6*109 - 10 100
10 Intraperitoneally 8*109 - 10 100
10 Intraperitoneally
1010
1
9
90 (died on
day14 post
infec.)
S.dublin
315/52
10 Intraperitoneally 2*109 4 6 60
10 Intraperitoneally 4*109 9 1 10
10 Intraperitoneally 6*109 10 - -
Note: The observation period was 20 days after infection
As can be seen from Table 10, intraperitoneal infection with the vaccine strain
S.dublin 31 did not cause significant disease in animals, only one calf infected with
large-dose (1010CFU) died on the day 14 without manifesting clinical signs of the
disease.
Control calves infected with the virulent culture of S.dublin 315/52 died with
symptoms of acute salmonellosis.
The data presented indicate that the attenuated S. dublin strain 31 has a weak
residual virulence.
Along with this, the degree of dissemination and the timing of elimination of the
vaccine strain from the animal organism was studied. At subcutaneous infection of white
mice with a vaccine strain at a dose of 106 cfu, the isolates were cultured from organs
and blood during 15 days, from inguinal lymph nodes during 30 days.
Table 11- Timing of elimination of S.dublin 31 strain with subcutaneous
immunization of white mice (dose106CFU).
White
mice
Terms of
slaughter
Crops from
Spleen The
liver
Blood
of
heart
Bone
marrow
inguinal
lymph
node
Bile Lungs
1 On the
3rd day
++++ ++++ ++++ ++-- ++++ ++++ ++++
2 ++++ ++++ +++- ++-- ++++ ++++ ++++
3 ++++ ++++ +++- ++-- ++++ ++++ ++++
4 ++++ ++++ ++++ ++-- ++++ ++++ ++++
5 ++++ ++++ ++++ ++-- ++++ ++++ ++++
1 On the
7th day
++++ ++++ ++-- +-- ++++ ++++ ++++
50
2 ++++ ++++ ++-- ++-- ++++ ++++ +++-
3 ++++ ++++ +++- ++-- ++++ ++++ +++-
4 ++++ ++++ +++- ++-- ++++ ++++ +++-
5 ++++ ++++ +++- +--- ++++ +++- ++--
1 For the
14th day
++-- ++-- ---- ---- ++-- +--- +---
2 ++-- +--- ---- ---- ++-- ++-- ++--
3 ++-- +--- ---- ---- ++-- +--- +---
4 ++-- +--- ---- ---- +--- +--- ++--
5 +--- ++-- ---- ---- +--- ---- ----
1 For the
21st day
---- ---- ---- ---- ---- ---- ----
2 ---- ---- ---- ---- ---- ---- ----
3 ---- ---- ---- ---- ---- ---- ----
4 ---- ---- ---- ---- ---- ---- ----
5 ---- ---- ---- ---- ---- ---- ----
Note: ++++ - abundant growth
+++ - - over 20 colonies
++ - - over 10 colonies
+ --- - unit colonies
---- - no growth
In calves subcutaneously infected with a vaccine strain at a dose of 2 * 109 cfu,
generalized vaccine infection was noted in the first three days. After 7 days the culture
was well isolated from the lymph nodes and spleen, weak culturing from the liver and
bone marrow; after 14 days, the culture in the form of single colonies was isolated from
spleen, pre-lobed, mediastinal and mesenteric lymph nodes (Table 11 ).
Thus, in experiments on laboratory animals and calves, the inability of the
attenuated strain S.dublin 31 to cause a typical infectious process was established.
The persistence of the biological properties of the vaccine strain has been studied
during long-term storage (for 4-5 years) and repeated crossings on semi-liquid and solid
nutrient media, after freeze-drying of the S.dublin strain 31, and also after 10-fold
passage on white mice and three times through the bodies of calves.
Passage of S.dublin strain 31 was carried out on white mice by intraperitoneal fatal
infection of mice at a dose of 3 * 107CFU. Passage of the strain S.dublin 31 was carried
out on white mice by intraperitoneal fatal infection of mice at a dose of 107CFU (Table
12).
51
Table 12 - Residual virulence of the vaccine strain S. typhimurium 10 - 52
passaged in the experiment on white mice.
Passage
The
number of
white
mice
Dose
(CFU)
Method of
administration
Results
Dead Surviv
ed
Survival
Rate
1 20 107 intraperitoneally - 20 100
2 20 107 ---«---«--- - 20 100
3 20 107 ---«---«--- - 20 100
4 20 107 ---«---«--- - 20 100
5 20 107 ---«---«--- - 20 100
6 20 107 ---«---«--- - 20 100
7 20 107 ---«---«--- - 19 95
8 20 107 ---«---«--- - 20 100
9 20 107 ---«---«--- - 19 95
1 20 107 intraperitoneally - 20 100
2 20 107 ---«---«--- - 20 100
3 20 107 ---«---«--- - 20 100
4 20 107 ---«---«--- - 20 100
5 20 107 ---«---«--- - 20 100
6 20 107 ---«---«--- - 19 95
7 20 107 ---«---«--- - 20 100
8 20 107 ---«---«--- - 18 90
9 20 107 ---«---«--- - 20 100
10 20 107 ---«---«--- - 20 100
Virulent
culture
Salmonell
a dublin
371
20
20
105
105
Subcutaneously 20
20
-
-
-
-
Note: The observation period is 15 days.
Passage of S.dublin strain 31 was carried out on 8-10 day-old calves by
intraperitoneal infection in a dose of 2 * 1010 CFU. Calves responded to infectionwith
significant oppression, fever, digestive disorders, but did not die.
On the 3rd and 5th day, the experimental white mice and calves were killed for
bacteriological examination.
52
A total of 10 passages were made on white mice and calves.
All passivated strains were controlled by the nature of growth and agglutinability.
In addition, they were tested for virulence, by subcutaneous infection of white mice at
doses of 107 and 108CFU, the experimental animals remained alive.
The conducted studies testify to the constancy of the properties of vaccine strain
S.dublin 31 when grown on artificial nutrient media and when passaging on susceptible
animals.
The absence of reversion of the vaccine strain is indicated by numerous
immunological experiments in laboratory animals and calves, as well as immunization
with an experimental vaccine from the same strain of more than 2,000 heads of cattle in
farms infected with salmonellosis.
The study of S.dublin strain 31 culture properties after freeze-drying showed good
survival, which complies with the standard and preservation duration.
Thus, the obtained results on the study of the biological properties of the attenuated
S.dublin 31 strain obtained by the genetic method indicate that the strain S.dublin 31 is
in stable S form, has stable, typical for S.dublin 31 morphological, cultural, biochemical
and antigenic properties, weak residual virulence, is well established in the body, does
not reverse during prolonged passage on susceptible animals. The presence of three
genetic markers in S.dublin 31 strain is a convincing proof of the stability and safety of
an attenuated strain, and also allows it to be differentiated from a natural prototype
[107].
4.3. Investigation of immunogenic properties of S. dublin strain 31.
After studying the cultural-biochemical, antigenic properties and residual virulence
of the attenuated strain S. dublin 31, we proceeded to study immunogenic properties in
experiments on laboratory animals and calves, in comparison with a concentrated mold-
wax vaccine of biomedical production.
Veterinarians have a great advantage over health professionals, they can determine
the true value of a vaccine or serum by testing it on the same animals on which it will be
used.
The level of protection is determined by experimentally infecting the vaccinated
and control groups of animals with a contagious dose of infectious material. It is quite
obvious that in order to study reproducible results, this dose in each experiment must be
strictly determined. It is expressed in units of the amount of the infectious organism
which, after administration to each group of experimental animals, causes a 50% death
in a certain time. This dose is called a 50% lethal dose or LD50. The reason for choosing
a value that characterizes the effect of only 50% of the animals in this group is that when
using infectious material in an amount causing 100% death, there would be no other way
to determine if the test dose is more than sufficient to cause the death of all animals.
The method for determining LD50 is as follows: several groups of animals are
taken and representatives of each of them are injected with a suspension of the pathogen
53
in one of the serial dilutions, which are usually prepared with a tenfold increase. It can
be expected that in a group in which a suspension with less dilution is introduced (i.e.,
containing more virulent culture), all animals will die, and no animal will perish in the
group to which the suspension is administered with the largest dilution. Based on the
percentage of death of animals in groups between these extreme values, according to the
method of Reed and Mench, LD50can be calculated (then breeding, in which exactly half
of the animals died in the group).
For a continuous analysis of the experimental vaccine, it is sufficient to have one
pre-established infectious dose.
The successes of fighting intestinal diseases are closely related to the effectiveness
of specific prevention of intestinal infections in animals and birds, which are the main
source of infection. In the materials of the WHO (1991) on the problem of intestinal
infections in humans and animals, it is emphasized that the use of effective vaccines in
livestock and poultry can reduce the incidence of intestinal diseases by 100,000 times.
Therefore, in the CIS and abroad over the past 20 years, intensive research has been
conducted to obtain intestinal vaccines. The large experimental material convincingly
demonstrates that live vaccines possessed the most reliable protection against intestinal
infections. In contrast to immunity caused by killed vaccines, the immunity that occurs
when live vaccines are administered occurs quickly, often even after a single injection of
the vaccine and is marked by high intensity and duration.
4.3.1. Studying the immunizing properties of the attenuated S. dublin 31 strain on
mice.
Having studied the cultural - biochemical, antigenic properties and residual
virulence of the attenuated strain S. dublin 31, we proceeded to study immunogenic
properties in experiments on laboratory animals and calves, in comparison with
concentrated formate - alum vaccine of biomedical production.
Immunogenic activity of S. dublin strain 31 was studied in white mice, during
which various doses were tested, local and general reaction and immunity after the
control infection were taken into account. Controls were animals vaccinated with a
concentrated formulose-vaccine and non-immunized animals.
The virulent S. dublin strain 315/52 was titered on white mice and calves prior to
the experiments. From the materials of titration it was established that the virulent strain
of S. dublin 315/52 causes the death of mice at a dose of 104 CFU, and calves in a dose
of 2-109 CFU at intraperitoneal administration.
The immunizing activity of the vaccine strain was first studied in 150 white mice
(90 experimental and 60 control mice) weighing 14-16 grams. The mice were
54
immunized subcutaneously with a lyophilized culture of the S. dublin 31 vaccine strain
at doses of 104, 105, and 106 CFU.
Mice immunized once with concentrated formulose vaccine in a dose of 0.1 ml (5-
108 CFU and unvaccinated) were controls. Twenty days after vaccination, the
experimental and control mice were infected with a virulent strain of S. dublin 315/52 at
a dose of 106 CFU, and controls (unvaccinated) ten times less than 105 CFU. The
materials of the experiment are shown in Table 12.
Table 12 shows that with a single subcutaneous immunization with the culture of S.
dublin 31 strain at 104, 105 and 106 CFU doses, high-voltage immunity is created in
mice. 100% of the experimental mice remained alive.
Mice immunized with a concentrated formulose vaccine (at a dose exceeding 10
times the live vaccine), with subsequent subcutaneous infection with a virulent culture,
died more than in 70% cases. All of control mice died in 10 days (Table 13) [108].
55
Table 13 - Testing the virulence of the attenuated strain S.dublin31 in an experiment on white mice.
Name of the
vaccines
Vaccination Infection with a virulents strain S. dublin 315/52
No of
mice
Inoculation
method
Inoculation
dose (CFU)
No of
mice
Inoculation
method
Inocul
ation
dose
(CFU)
results
died aliv
e
%
Of
alive
S. dublin 31 30 subcutaneously 104 30 intraperitoneally 106 - 30
100
30 subcutaneously 105 30 intraperitoneally 106 - 30
100
30 subcutaneously 106 30 intraperitoneally 106 - 30
100
Concentrated
formulose
vaccine 30 subcutaneously
0,1 ml
(5-108)
30 intraperitoneally 106 21 9 30
Controls (non -
vaccinated) 30 - - 30 intraperitoneally 106
30
- -
Note: Observation period 18 days post infection
56
4.3.2 Studying the immunizing properties of the attenuated S. dublin 31 strain on
calves.
The obtained materials of experimental studies on laboratory animals allowed us to
continue studying the immunizing properties of the attenuated S. dublin 31 strain on
calves. Harmlessness, reactogenicity and immunizing doses were determined. The
possibility of both early reactions (general and local) that occurred after vaccination and
later, long-term vaccination results, which could be due to the development of the
vaccine process were taken into account.
In total, there were 45 calves of 12-14 days old in the experiment.
In the first group, 20 calves were vaccinated with a vaccine from S. dublin strain 31
in a volume of 2 ml with 107CFU in 1 ml.
In the second group, 20 calves were inoculated twice with a concentrated formulose
vaccine in doses of 5ml and 10ml.
The third group - (5 calves) control, were not exposed to vaccination.
The vaccine was administered to the experimental calves in the region of the upper
third of the neck.
Immunized calves were monitored for up to 3 months.
The test calves were under observation and the body temperature was measured
daily. In calves vaccinated by strain 12 there was a state of some depression, a slight
increase in body temperature by 0.5 ° C, the appetite was preservedon the first day. At
the injection site, limited edema appeared, which resolved on day 2-3.
Control intraperitoneal infection of all experimental and control (10) calves was
carried out by flushing out the daily agaric virulent culture of S. dublin 315/52 at a dose
of 1010CFUafter twenty days from the inoculation. The intensity of immunity was
checked by the degree of survival, general and local response and gastroenteric
disorders.
As a result of infection in the calves of all groups, an increase in body temperature
from 40 to 41.5 ° C was observed on the following day, which in the group of
experimental calves persisted for 2-6 days. 3 calves showed lethargy, decreased appetite,
increased pulse and respiration. After 4 days the signs of the disease disappeared and the
appetite recovered. There were no other clinical abnormalities. All the experimental
calves remained alive (Table 14).
In calves of the second group (inoculated with a concentrated formulose vaccine),
the high temperature was held for 7-10 days. The increase in temperature was
accompanied by significant depression, decreased appetite, palpitations and breathing, as
well as intermittent diarrhea. In 4 calves, there remained depression, dyspnea, frequent,
arrhythmic pulse, cough, weight loss and death on 9-10 days.
57
Table 14 - Testing the virulence of the attenuated strain S.dublin 31 in an experiment on white calves.
Test vaccines
No of
animals
Vaccination Infection with a virulents strainS. dublin
315/52
Inoculation
method
Inoculation
dose
Inoculation
method
Inoculation
dose (CFU)
Result
Died Alive
Vaccination from
an attenuated
strain of S.dublin
31
20 subcutaneously
107 intraperitoneally 1010 - 20
Concentrated
vaccine against
salmonellosis in
calves
20 subcutaneously 2 сm 3 intraperitoneally 1010 2 17
Control
(unvaccinated)
5 - - intraperitoneally 1010 5 -
Note - infection with virulent culture 20 days after vaccination;
- the observation period is 15 days after infection
58
In the control calves, on the second day after infection, severe inhibition was
observed, an increase in temperature to 41.6 ° C and 41.8 ° C, which persisted until the
animals died: the calves died on the 10th -12th day after the challenge. An autopsy of
the dead calves revealed typical changes of an acute salmonellosis: the presence of
densified areas in the lungs, the blood supply to the liver, a sharp swelling of the lymph
nodes (especially the pre-lobed, mediastinal, mesenteric), the spleen, and the small
intestinal mucosa with hemorrhages. Hemorrhages were also found under the capsule of
the kidneys and at the atria. From the blood (heart), liver, gall bladder, spleen and
mesenteric lymph nodes, the infecting culture was abundantly isolated. In a
bacteriological study, the infecting culture from organs, lymph nodes and bone marrow
was abundantly isolated [108].
The "Salmonella dublin 31 strain, used for the production of live vaccine against
bovine salmonella", was obtained for the strain Salmonella dublin 31 for No.32222 of
03/31/2017 [109] (Anexes 11).
5. Development of technological regulations for the production of live vaccines
against bovine salmonellosis.
Epizootic and epidemiological tensions around intestinal infections caused by
enteric pathogens have increased in recent years due to the changes in methods of cattle
breeding and fattening, as well as the rules of zootechnical and veterinary care of
animals. Vaccination of animals and birds against intestinal diseases became optional,
and it is not mandatory in the plan of anti-epizootic measures of the Veterinary
Committee of the Ministry of Agriculture. In the current socio-economic conditions, the
specific features of combating diseases common to humans and animals are largely
related to the development of the private sector in livestock production, uncontrolled
migration of livestock, including from those from disadvantaged regions. This makes it
difficult to consider and carry out vaccination of animals and creates difficulties in the
implementation of state veterinary and sanitary-epidemiological surveillance.
Exceptional resistance of pathogens of enteroinfections and the cyclic increase in their
activity, cause periodic and sharp increases in morbidity. The increase in the scale and
intensity of development of territories where active natural foci are located leads to a
wide spread of these diseases among the population. Prevention of zoonoses primarily
based on the timely detection of the risk of humaninfection.
Epizootic and epidemiological features of infection, effective means of prevention
and the possibility of their use determine the choice of key activities. In some cases, this
may be regime-restrictive measures, in others - veterinary and sanitary, sanitary and
anti-epidemic measures, use of specific prevention tools etc.
Specific prophylaxis in the form of vaccination and the use of serums is an
important part of the overall complex of antiepizootic measures.
59
Given the high protective properties, the vaccine strain S. dublin 31 studied has all
the grounds for a wide study of it as a vaccine for specific immunoprophylaxis of bovine
salmonella.
5.1. Method for manufacturing live vaccines against salmonellosis in animals.
To date, considerable experience has been gained on the use of killed and live
vaccines. Scientists recommend to use live vaccines against salmonellosis for
prophylactic purposes, the immunogenicity coefficient of which is higher than that of
the killed ones.
Inactivated (killed) vaccines are immunopreparations that contain microorganisms
that have been treated in such a way that they have lost the ability to reproduce in the
body of vaccinated animals. The problem of inactivated vaccines is that when the
pathogen is inactivated, a greater or lesser part of its immunogenic structure is lost under
the influence of physicochemical effects on the microbial cell. Therefore, it is important
that the starting material for the killed vaccines contains the antigen at the highest
possible concentrations. Hence there is the need for large doses and multiple
vaccinations. The lack of the possibility of reproduction and replication of the antigen in
the organism of vaccinated animals leads to limited circulation, as well as insufficient
involvement of immune cells, which ultimately results in a low immunogenicity index
of the inactivated vaccine. The immunogenicity of inactivated vaccines is enhanced by
the selection of immunogenic strains of the pathogen and by the addition of adjuvants
that enhance the stimulation of the immune system in the vaccinated animals, but do not
act as an antigen.
Despite all the measures, inactivated vaccines do not provide as much tense and
prolonged protection as live vaccines.
High immunizing activity of live vaccines is explained by preservation of the main
metabolic pathways and the whole complex of antigenic properties of the microbial cell,
reproduction of the vaccinal culture in the body and wide circulation, stimulation of the
immune system on large tissue surfaces. In the prevailing number of live vaccines
attenuated (weakened) strains of the pathogen are used. In essence, these mutants of
infectious agents that have vaccine properties have sufficiently high immunogenicity,
weak residual virulence and are safe for the immunized organism. Strains should have
genetic markers that distinguish them from virulent prototypes.
Many researchers believe that salmonellosis is determined by cellular immune
responses, since salmonella are capable of intracellular parasitization. The level of
antibodies does not reflect the intensity of immunity. In this regard, live vaccines are the
most promising for the prevention of salmonellosis in farm animals.
The scheme of the technological process for the production of live vaccines
includes:
- preparation of nutrient medium for cultivation;
- preparation of inoculum;
60
- cultivation of the vaccine strain;
- determination of bacterial mass concentration;
- lyophilization of the preparation;
- drug control;
- Packaging of the preparation and labeling of the vials with and packaging.
Preparation of nutrient medium. To prepare the vaccine from the vaccine strain the
main Hottinger's digest containing 10% hepatic extract and 0.4% peptone is used, then
distilled water is added so that the amine nitrogen content is not less than 200-250 mg
/%. The mixture is brought to a boil. Boiling is continued for 30 minutes. Then, pH of
the medium is adjusted to 7.7-7.8 by adding 20% sodium hydroxide, 0.3% chemically
pure sodium chloride. The medium is boiled for 1-1.5 hours after boiling, it is filtered
through a cotton-gauze filter and pumped to the reactor.
To control the sterility of the medium, samples should be taken and passaged on
mediums: MPA, MPB, MPPB, under vaseline layer, Endo.
Preparation of inoculum. To prepare the vaccine from the vaccine strain, a separate
ampoule with a lyophilized culture should be used. An ampoule with a dry vaccine
strain is opened and 2 ml of sterile physiological saline is added to it. The resulting
suspension is plated in bottles with Hottinger broth (capacity of the bottle is 100 ml).
The vaccine strain is grown for 14-16 hours at a temperature of -37-380C (first
generation culture).
The culture of the first generation of the vaccine strain (after checking for purity) is
inoculated into a bottle containing 10 liters of Hottinger broth (second generation
culture). Cultivation is carried out for 18-20 hours at T-37-380 C.
Cultivation of the vaccine strain to obtain bacterial mass. The culture of the second
generation of the vaccine strain (after checking for purity) is inoculated in AKM-III or
into a reactor (apparatus for cultivation of microorganisms) with a sterile nutrient
medium.
The vaccine strain grown and tested for cleanliness are put into reactors with an
amount of 8-10% of the volume of the nutrient medium.
The vaccine strain is grown at a temperature of 37 ° C, with an addition of 0.1% (in
terms of dry matter) of glucose in the form of 20-40% sterile solution, for 18-20 hours
with constant stirring and aeration with sterile air.
Cultures grown in the reactor are tested for growth purity by microscopy of smears
and culturing on nutrient media. Accumulation of microbial cells in an 18-20-hour
culture corresponds to 10-15 billion in 1 ml. At the same time, the grown culture is
cooled down by passing cold water under the reactor.
Determine the concentration of bacterial mass in each reactor. For this purpose,
saline is added to 1 ml of the culture taken from the reactor with a pipette to a
concentration of 1 billion colony-forming units (CFU) according to the bacterial
turbidity standard. Bacterial mass in each reactor is diluted with sterile saline solution to
a concentration of 4 billion CFU in 1 ml.
61
After this period of cultivation of the vaccine strain, a sterile washable liquid
(drying medium) is introduced into the apparatus and flushing is performed by rotating
the apparatus and its petals. The drying medium contains 1.5-2% of gelatin, 10% of
sucrose, and the pH of the medium is 7.8-8.0.
After flushing, the microbial mass is stored at T -2-100 C for 2-3 days. The
resulting microbial mass is checked for purity and typical growth by culturing in tubes
with MPA, MPB, MPBB, Endo.
Concentration of bacterial mass. The resulting microbial mass is adjusted by
adding a drying medium to a concentration of 1010 CFU per cm3 according to the optical
turbidity standard of Tarasevich.
Before the packaging the culture is checked for cleanliness and absence of
extraneous contamination by microscopy of smears stained by Gram and made passages
on MPA, MPB, MPPB, Endo. The passages are kept at T-37-380 C for 10 days.
The technological process of vaccine lyophilization includes the following steps:
Packaging of the vacine. The microbial suspension of the vaccine strain in a
concentration of 1010CFU in 1 cm3was filled in accordance with the optical turbidity
standard by 3 ml in vials, with continuous stirring. The vials are closed with a sterile
three-layer gauze napkin and prepared for freezing and drying.
Drying the preparation. Vials with the vaccine are placed in cassettes and placed in
refrigerated chambers for freezing at temperatures from minus 45 to 00 C. Freezing is
carried out for 10-16 hours, counting from the moment of reaching a temperature of
minus -500 C in the refrigerating chamber.
The frozen preparation is subjected to drying (sublimation) in TG-50 units
equipped with devices for measuring vacuum, condenser temperature, shelf temperature,
and temperature in the preparation.
After loading and creating a vacuum, the shelves are heated, and after 6-10 hours
the temperature of the shelves must be + 300 ° C.
Sublimation of ice in the preparation (from minus 45 to 00 C) lasts 40-45h.
The first period of drying from 00 C to + 300 C - 10-15 h.
The duration of the entire drying process is 70-72 hours.
The day of the end of lyophilization is considered the date of preparation.
Vials with dried vaccinesare covered with sterile rubber stoppers under vacuum in
a chamber of a sublimation unit and rolled with metal caps.
The method of controlling the vacine from the vaccine strain comprises the
following steps:
The vaccine is checked for sterility, harmlessness and activity. To check the
sterility,0.2 ml of the vaccine from 5 bottles are taken for passaging on MPB, MPA,
MPBP under vaseline layer. Passages on all media are kept in a thermostat at 37 ° C for
3 days. Plates with vaccine bacteria should remain sterile.
The vaccine is tested for harmlessness on 5 white mice weighing 16-18 g and 5
guinea pigs weighing 300-350 g. The vaccine is administered intraperitoneally to white
62
mice at 0.3 ml, guinea pigs - 2 ml each. Laboratory animals should remain alive and not
show signs of disease during the 10-day observation period.
Control of immunogenic activity is performed on 25 guinea pigs weighing 350-400
g. Vaccine is administered under the skin into the abdomen in a dose of 0.5 ml. After 16
days, the vaccinated guinea pigs, along with the control guinea pigs (5 ml per each
virulent strain), are infected with pre-titrated lethal doses of virulent cultures,
respectively, for each type of live vaccine (obtained from VGNKI, Moscow) grown for
16-18 hours. Experiment animals should remain alive in the case of all control animals.
This indicates that the proposed vaccine has a high immunogenic activity.
After the inspection, the bottles are labeled with the name of the product, the serial
number and the date of manufacturing.
Certain amount of the vaccine obtained as a result of one-time mixing in one
container and having the same concentration of living microbial cells simultaneously
fused into bottles, dried under the same regime and received its number, the number of
state control issued by one quality document (passport) with the indication of the name
of the manufacturer, the name of the product, the serial number, the state control
number, the date of manufacturing (month, year), test results on indicators of quality,
shelf life, storage conditions, specifications, number and date of issue of the document
on the quality, the conclusion and signature of the person issuing the document is
considered as one serie of a vaccine.
The preparation from live cultures is a dry, fine-porous mass of white or grayish-
yellow color, containing 50-60% of living microbial cells, easily soluble in
physiological solution, distilled or boiled water. The vaccine is suitable for use within
12-18 months from the date of manufacture, provided that it is stored at a temperature of
+4 - +10 ° C.
The technology described above was used to prepare a live vaccine of strain S.
dublin 31 against bovine salmonellosis [110].
6. Post-vaccination reaction after immunization with live vaccine from the strain S.
dublin 31.
The defense reaction can be used to test the experimental vaccine before extensive
testing in production conditions. In this sense, this reaction is of particular importance,
since allows to determine the main quality of the vaccine - the ability to create immune
protection, which can not always be judged on the basis of the results of other
immunological reactions.
6.1 Reactogenicity and bacterial carriage.
The study of the expression of local and general reactions is of particular interest
due to the fact that the duration and intensity of the antigenic stimuli are largely
63
determined and, when compared with other indicators give a more complete picture of
the vaccination process.
The reactogenicity of the vaccine strain S. dublin 31 was determined on white
mice, guinea pigs and calves.
When immunizing doses of S.dublin strain 31 (104CFU, 105CFU, 106CFU, and
107CFU) administered subcutaneously to white mice no clinically pronounced reaction
was noted.
In experiments on guinea pigs, subcutaneous vaccination with vaccine strain
S.dublin 31 (immunizing dose 3 * 108CFU), caused the development of slight edema
(0.5x1 cm)on the second day, which increased by days 3-5 with severe limitation and
then, in some guinea pigs, after 7-10 days benign abscesses without necrosis were
formed. In guinea pigs vaccinated with the strain S.dublin 31 and then infected with
virulent culture, small abscesses without necrosis were also formed.
General reaction in guinea pigs vaccinated with live vaccine was manifested in the
form of short-term depression without a noticeable loss in weight.
In pigs immunized with a concentrated formulose vaccine, after infection with
virulent culture significant swelling and necrosis of the skin was formed. Along with
this, the general reaction in the form of considerable oppression and weight loss was
more pronounced.
Local reaction in calves was manifested by inflammatory phenomena: hyperemia,
infiltration, edema formation of 3x4 - 4x5 cm. After 3-5 days, edema had decreased,
became less painful and then resorption occured.
The general reaction was characterized by mild depression during the first half of
the day, temperature remained normal or increased by 0.5-1 degrees, the appetite in
calves usually remained unchanged. All these phenomena disappeared in 2-3 days
without any complications.
The same reaction was observed in calves when vaccinated with elevated doses (4
* 108CFU). In pregnant cows and heifers, the vaccine strain only developed a local
reaction.
To identify the bacterial carriage, as well as to study some questions of
immunomorphology, 3 calves were killed, 3, 7 and 14 days after vaccination with the
strain S.dublin 31, at a dose of 2-109CFU(a lyophilized dried culture diluted in
physiological saline).
Bacteriological study of the material from the killed calves was carried out
according to a conventional method (culturing from the liver, spleen, bone marrow,
prenopathic, mediastinal, mesenteric and inguinal lymph nodes).
In calves killed 3 and 7 days after vaccination, the presence of compaction and
swelling in the area of administration of live vaccine was noted. The basis of a skin and
hypodermic layer were filled with an infiltrate. In a calf slaughtered after 14 days,
subcutaneous tissue was hyperemic without tissue infiltration.
64
The results of a bacteriological study showed that the culture of the S.dublin strain
31 wasabundantly isolated from all organs, 3 and 7 days after vaccination. In calves,
slaughtered 14 days after vaccination, the culture was isolated only as single colonies
from the spleen, pre-lobed, mediastinal and mesenteric lymph nodes.
Bacteriological study of immunized and then slaughtered mice, showed that the
culture of the vaccine strain is abundantly isolated from the liver, spleen and inguinal
lymph nodes for 7 and 15 days, less abundantly from blood and bone marrow. After 25-
30 days the strain was only isolated from the inguinal lymph nodes.
The foregoing allowed us to conclude that an attenuated strain of S. dublin 31 in
white mice, guinea pigs and calves causes a benign local and general reaction. Local
reaction was characterized by leukocyte infiltration, general-short-term depression and
rise in temperature (calves).
The vaccine strain was well isolated from the parenchymal organs and lymph nodes
during the first week, later the isolation decreased, and in white mice the vaccine strain
remained permanently in the lymph nodes (25-30 days).
The danger of vaccinal bacterial carriagewas eliminated because the attenuated
strain of S.dublin 31 does not cause infection when parenterally infected with massive
doses (white mice and calves) and was not accompanied by the development of an
infectious process. The safety of S.dublin strain 31 was confirmed by the constancy of
biological properties. The attenuated strain of S.dublin 31 meets the requirements for
live vaccines: stability of biological properties, animal safety and well-expressed
immunizing activity.
6.2. Serological indicators.
Humoral factors of body protection in vaccinated animals were determined by the
dynamics of specific antisalmonella antibodies, total protein content, quantitative and
qualitative content of immunoglobulins.
The serological evaluation of postvaccinal immunity was determined by the
increase in antibodies in the agglutination reaction (AR).
The agglutination reaction was set in polystyrene plates with lunettes and, in a
volume of 1 cm3 in diluted serum, from 1:25 to 12800. To dilute the serum, 0.98 cm3
was poured into the first well, and 0.5 cm3 of phenolized 0.5 %) saline solution. Then,
0.02 cm3 of the test serum was added to the first well, mixed thoroughly and transferred
0.5 cm3 to the second, from the second the same amount to the third, and so on. From
the last well, 0.5 cm3 of diluted serum was removed. After the preparation of the sera
dilution, 0.05 cm3 of antigen with 1010 colony forming units in 1 (one) ml was added to
each well.
To control the absence of spontaneous agglutination of the antigen to 0.05 cm3 of
physiological solution, 0.05 cm3 of the antigen was added. The plate with wells was
gently shaken and placed in a thermostat for 8-10 hours at 370C, then it was additionally
65
kept at room temperature for 12-14 hours, the reaction was taken into account visually
and evaluated in "crosses".
The results of AR in the test tubes were taken into account according to the 5-point
system:
4 crosses - complete bleaching of the liquid, agglutinate settles on the bottom of the
tube as an umbrella, which, when shaken lightly, breaks into flakes and lumps, and the
liquid remains clear, 100% agglutination.
3 crosses - incomplete fluid clarification and well-defined "umbrella", 75%
agglutination.
2 crosses - liquid clarification and "umbrella" are moderately expressed, in the
center of the umbrella a dense antigen deposit in the form of a "fad", 50% agglutination.
1 cross - a barely noticeable "umbrella" around a solid antigen deposit, the clarity
of the fluid is negligible, flakes or lumps are barely noticeable when shaken, 25%
agglutination.
Minus - the bleachings of the liquid and the formation of an umbrella are not
observed, the antigen settles in the form of a "fad" and, with slight shaking, forms a
homogeneous suspension.
When evaluating the reaction, agglutination in two crosses and above is estimated
as positive, one cross - as doubtful, the absence of agglutination - a negative result.
We conducted studies of the titers of agglutinins in blood serum and colostrumof
cows, as well as in calves' blood after the colostrum administration.
The titers of agglutinins were taken into account at various times after
immunization of cows and calves.
For comparison, agglutinins were determined in cows and calves vaccinated with a
concentrated formulose vaccine of biomedical production.
In order to clarify the role of colostral protection on the formation of postvaccinal
immunity, a group of calves born from the immunized cows was selected, which were
also vaccinated with the vaccine from the strain S.dublin 31. '
Cows were vaccinated with live vaccine once for 50-60 days before calving in a
dose of 2-109 CFU; calves were inoculated at a dose of 109CFUat the 12-14 days of age.
Concentrated formulose vaccine was administered twice: to cows 50 and 60 days before
calving in a dose of 10 and 15 ml, calves 1.5 and 2 ml (according to the instructions).
Agglutinins in the serum of cows were determined before vaccination, 10, 20, 30,
40 days after vaccination, before calving and at 1, 3, 5, 15 and 30 days after calving. In
the colostrum, the agglutinins were determined on days 1, 3, 5, 10, 20 and 30 days after
calving.
The calves' serum was examined before the colostrum was drunk and after 1, 3, 5,
10 days of life and on days 5, 15 and 25 after vaccination. A total of 1650 samples of
blood of cows, 840 samples of colostrum and 1440 of samples calves' blood were
examined.
Blood serum was obtained according to the generally accepted method.
66
Serum of colostrum was obtained by adding of 1 ml of 1 % pepsin solution to 10
ml of colostrum, followed by keeping in the thermostat for 3-5 hours.
The serum was then aspirated and centrifuged for 15-20 minutes (2,000 rpm) to
separate it from clumps of casein. The agglutination reaction was put in a volume of 1
ml according to the generally accepted procedure. A 5 billion suspension of daily agar
culture of the virulent strain S.dublin 315/52 was used as an antigen. The test sera were
diluted in physiological saline 1: 50-1: 800.
The indices of agglutinins in the blood serum of cows indicate that after the
vaccination attenuated S. dublin 31 strain shows significant shifts.
Thus, on day 10 after vaccination the agglutinin titers reached 1: 800 in 25, 1: 400-
29 and 1: 200 in 10 cows (out of 80 examined).
On day 30 - 1: 800 in 13, 1: 400-32 and 1: 200 in 9 cows. Before calving and in the
first days after calving a slight decrease in the titer of agglutinins (1: 100-1: 200) was
noted.
On the 5th day after calving, their growth was observed: 1: 800 in 19, 1: 400-35
and 1: 200 in 6 cows. The same indices with small fluctuations persisted in the study at
30 days after calving (Figure 11).
Figure 11 - Titers of agglutinins in blood serum of cows vaccinated with attenuated
strain S.dublin 31 (out of 80 examined)
Agglutinins in the serum of colostrum of 70 vaccinated cows reached 1: 800 in 17,
1: 400 in 30 and 1: 200 in 15 cows after 1 day post - vaccination.
67
On the 3rd day - 1: 800 in 4, 1: 400 in 19 and 1: 200 in 21 cows. Then there was a
gradual decrease in them and on the 10th day agglutinins were detected only in dilutions
of 1: 100 in 10 and 1:50 in 12 cows (Figure 12).
Figure 12 - Titers of agglutinins in colostrum of cows vaccinated with an attenuated
strain S.dublin 31 (70 vaccinated cows)
Figure 13 - Titers of agglutinins in blood serum of cows vaccinated with concentrated
formulose vaccine (out of 40 examined).
68
In the blood serum of cows (40 heads) inoculated a concentrated formulose vaccine
the titer of agglutinins reached 1: 400 in 10, 1: 200 in 15 cows after 20 days.
After 30 days-1: 800 in 5, 1: 400 in 10 and 1: 200 in 11 cows (Figure 13).
In colostrum one day after calving, agglutination titers were 1: 800 in 2, 1: 400 in 9
and 1: 200 in 12 cows (out of 35 examined); aftre 3 days-1: 400 in 1, 1: 200 in 6 and 1:
100 in 11 cows (Figure 14).
Figure 14 - Titers of agglutinins in colostrum of cows vaccinated with with
concentrated formulose vaccine (out of 35 examined).
In the blood serum and colostrum of cows not subjected to vaccination, during the
experiment the agglutinins were not detected or were in low titers.
To determine the duration of agglutinin retention in calves that received colostrum
from immunized and non-immunized cows, serum was examined within 10 days after
birth.
It was found that the agglutinins in the blood serum of calves from cows vaccinated
with live vaccine from S. dublin strain 31 (70 calves) reached the highest titer one day
after colostrum feeding: 1: 200 in 30 calves, 1: 400 in 27 and 1: 800 in 6. These
parameters were kept with minor fluctuations up to 3 days, then there was a decrease by
10 days, agglutinins in the serum of calves were not detected.
69
Figure 15 - The titres of agglutinins in the blood serum of 35 calves obtained from
cows imunized with a concentrated formulose vaccine.
In the blood serum of 35 calves obtained from cows imunized with a concentrated
formulose vaccine, the titres of agglutinins reached 1: 400 in 1 day after calving; 1: 200
in 10 and 1: 100 in 8 calves; on the 3rd day - 1: 200-4, 1: 100 - 10 calves, and on the
10th day only in the 1:50 dilution in two calves (Figure 15).
The control calves did not show agglutinins or in a small number of calves they
were detected at 1:50 (nonspecific reaction).
Thus, the attenuated strain of S.dublin 31 causes a moderate accumulation of
agglutinins in the serum and cow colostrum (1: 200, 1: 400, 1: 800).
In the blood of calves, after feeding with colostrum from vaccinated cows,
agglutinins reached the highest titer in a day (1: 200, 1: 400) and remained with slight
fluctuations for 3-5 days.
In calves at the age of 12-14 days vaccinated with live vaccine, agglutinins were
detected in moderate titres on days 10-25 after vaccination (1: 200, 1: 400).
Differences in the agglutinin counts in vaccinated calves obtained from immunized
and non-immunized cows were not observed.
The lack of parallelism between the high immunity intensity created by live
vaccine from S. dublin strain 12 and moderate indices of agglutinins in calves is in
accordance with the literature data.
70
7. Production tests
Specific prophylaxis of salmonellosis and other infectious diseases of young calves
is aimed at raising the level of specific antibodies in colostrum and in the organism of
the nascent offspring.
A more promising way to protect newborn calves from salmonellosis is to create a
high resistance to infection through colostral immunity. Immunization of pregnant cows
and heifers provides accumulation of specific antibodies in colostrum and transfer to
their offspring.
In the beginning, a live vaccine in the form of a suspension in saline was tested on
individual groups of calves. Subsequently, we proceeded to immunize the entire
emerging animal population with a dry live vaccine from the attenuated strain
Salmonelladublin 31.
Preparation and control of dry live vaccine was carried out in the laboratory of
antibacterial biotechnology of the NPJSC Kazakh national agrarian university, including
freeze drying.
Preparation and control of dry live vaccine. From the matrix- attenuated strain
Salmonella dublin 31, which was stored in the dried form, the cultures were made on
meat-peptic agar pH 7.2-7.4 in a test tube, and also planted onto petri dishes to exclude
contamination of the culture and its dissociation. The daily agar culture was tested for
growth purity and agglutinability with total and monoreceptor sera.
After that, the slurry was prepared in physiological saline solution and the
necessary amount was plated into a Tartakovsky flask with MPA, pH 7.2-7.4.
After 20 hours of incubation in a thermostat, the culture was washed with
physiological saline until a thick suspension was obtained. The concentration of bacteria
was checked by diluting 1 ml of a suspension to 109CFU with an optical salmonella
standard. The main flush with Tartakovsky flasks, containing 10 ml of 10-109 CFU (10
doses for calves), packaged in ampoules of 1ml and the same amount of skim milk was
added.
Figure 16 - Dry live vaccine from an attenuated strain of Salmonella dublin 31.
71
Figure 17 - Drying the culture of the attenuated strain on the freeze-dried INAY-6.
The whole described process of vaccine preparation was carried out in the
laboratory of antibacterial biotechnology of the Kazakh National Agrarian University,
including freeze drying.
Drying was carried out under the following conditions: freezing in vacuum at -40 °
for 20 hours, then drying at 25 ° for 24 hours.
The dried culture was an amorphous mass that dissolved readily in saline or water,
turning into a uniform suspension (Figure 16,17).
After drying, the culture was tested for the purity and content of living bacteria, by
plating onto Petri dishes.
The control on the number of bacteria was carried out by dilution and subsequent
plating on Petri dishes (from 2-3 ampoules) in terms of 1000 cells according to the
optical standard. Counting was conducted according to the number of colonies grown,
50-70% of the number of plated bacteria by the optical standard (ie 500-700 colonies).
The cultures of strain 12 retained typical morphological, cultural, biochemical and
agglutinabile properties after drying.
Control for the stability of residual virulence was carried out on 10 white mice (14-
16 g.). White mice were injected subcutaneously with 107CFU (0.2 ml of 5-107
suspension from the dried culture). Within 15 days the mice remained alive.
Control for activity was also tested on white mice. 10 mice were injected
subcutaneously with 106 CFU (0.2 ml of 106suspension from the dried culture). After 15
72
days, 10 vaccinated and 5 control mice were infected subcutaneously with a virulent
culture of Salmonella dublin 315/52 at a dose of 106CFU. Control mice died within 10
days, all vaccinated mice remained alive.
We conducted research and production experience in three farms, which were
disadvantaged for salmonellosis of cattle: in the peasant farming Khabit of the Almaty
region, the Anisan farm of the Aktobe region, and the Turtan-Ata farm of the Kyzylorda
region.
For the production tests, we divided the calves into two groups, a dry live vaccine
was introduced from the attenuated S.dublin strain 31 into the experimental group, and a
concentrated vaccine was introduced into the control group.
To the experimental group vaccination was carried out with the coverage of all
young animals (regardless of fatness and development) subcutaneously, once, in the
region of the 1/3 of the neck, in a dose of 1 ml (109CFU).
In the control group, they were inoculated with a molded vaccine twice in the 10-20
day period at a dose of 1.0 cm3 and at 8-10 days after the first vaccination again with a
dose of 2.0 cm3.
Vaccinated animals were under clinical observations. A few hours after the
vaccination, the calves experienced a short-term depression, and the appetite remained
on the same level. Local reaction was accompanied by the formation of edema (size
3x4-4x5 cm), which resolved on 4-6 days. Along with this, the vaccine was tested on
cows in the last stage of pregnancyonce, in a dose of 2 ml (2-109CFU) subcutaneously,
in the region of 1/3 of the neck. Vaccinated cows demonstrated only local reactions.
Calves from vaccinated cows were born viable and did not develop salmonellosis.
After 21 days we infected with a virulent culture Salmonella dublin 315/52 in a
dose of 1010 intraperitoneally.
As a result of studies in the experimental group, the survival rate of the young
animals was 98%, in the control group 75% (Table 15 ).
Later in the vaccine was successfully tested in a number of Kazakhstani farms that
were not healthy for salmonellosis of cattle.
These livestock farms were infectedwith salmonellosisfor several years. Newborn
calves on farms were systematically immunized with a concentrated formulose vaccine
according to the instructions. Despite this, there were quite frequent cases of calves'
infection with salmonella. Autopsy revealed that salmonellosis was caused by
Salmonella dublin strain, which was repeatedlyconfirmed by bacteriological
laboratories.
Initially a live vaccine in the form of a suspension in saline was tested on individual
groups of calves. Subsequently, we proceeded to immunize the entire emerging
population with a dry live vaccine from the attenuated strain Salmonella dublin 31.
73
Table 15 - Production tests of live dry vaccines from an attenuated strain S.dublin 31.
Farms Group No of
animals
Infection
dose
(CFU)
Inoculation
method
virulent
culture
Infection
dose
(CFU)
Inoculation
method
Result
Dead Survived % of
staying
alive
Khabit
(Almaty
region)
Experimental 20 1 ml
(109)
subcutaneously S.dublin
315/52
1010 Intraperitoneally - 20 100 %
Control 20 1 cm3 subcutaneously – // – 1010 Intraperitoneally 5 15 75%
Turtan –Аtа
(Kyzylorda
region)
Experimental 20 1 ml
(109)
subcutaneously – // – 1010 Intraperitoneally - 20 100%
Control 20 1 cm3 subcutaneously – // – 1010 Intraperitoneally 4 16 80%
Аnisan
(Аktobe
region)
Experimental 20 1 ml
(109)
subcutaneously – // – 1010 Intraperitoneally 1 19 95%
Control 20 1 cm3 subcutaneously – // – 1010 Intraperitoneally 6 14 70%
74
Figure 18 – Percentage of the production test of a live dry vaccine from an
attenuated strain S.dublin 31.
In total, during 2015-2017, a total of more than 2,000 heads of cattle were
vaccinated, including 960 cows 20-25 days before calving and 1,040 calves. During this
period, cases of calf disease and their death from salmonellosis have not been recorded.
Observation of vaccinated calves and cows showed that the vaccine against bovine
salmonellosis from the attenuated strain S. dublin 31 does not cause complications.
The epizootological data of the farms where vaccine trials were conductedin
comparison with previous years indicate the efficacy and safety of the experimental live
vaccine and indicates the possibility of its widespread use as one of the measures to
combat bovine salmonellosis.
Economic efficiency as a result of immunization of cattle with live vaccine from S.
dublin strain 31 can be achieved due to decrease in the incidence and mortality of calves,
labor costs and amounted to 60 tenge per one spent tenge.
The effectiveness of immunization of cattle in disadvantaged for salmonellosis
farms was studied by conducting an epizootic analysis before and after its use and taking
into account a decrease in the incidence of morbidity and calf death.
Thus, to date, significant experience has accumulated on the use of live vaccines. It
was established that in the experiments on the evaluation of the efficacy of live and
killed vaccines used in our country against salmonella of animals, the immunogenicity
75
coefficient for live vaccines was higher than for the killed ones. In addition, the use of
live vaccines will make it possible to improve the economy from salmonella in animals,
increase the yield of young animals, improve the epizootic situation of farms, sharply
reduce foodborne toxic infections in people with salmonella ethiology. The economic
effect of carrying out routine preventive vaccination of animals against salmonellosis
with live vaccines is 2-3 times higher than when immunized with animals killed by
vaccines [111, 112]
We have applied for a patent for the invention of the Republic of Kazakhstan on the
subject "the method of preventing bovine salmonellosis with a vaccine from the strain
Salmonella dublin 31" , registration number 2018/0296-1, from 14.05.2018 (Anexes 12).
.
76
DISCUSSION OF THE RESULTS
Modern agrarian policy in our country is aimed at fulfilling the main task -
satisfying the ever growing needs of the people in food products. To successfully solve
these problems, it is necessary to ensure further growth in the production of livestock
products. The preservation of newborn animals and the cultivation of a healthy, well-
developed and adapted to new conditions of young animals is the basis for increasing the
yield of livestock products.
The development of livestock farms is impossible without the creation of lasting
protection from infectious diseases, including salmonellosis.
Salmonellosis is the most widespread zooantroponosis in the world and according
to WHO (1999) sets a significant problem in all countries of the world every year. The
damage caused by this disease is not only direct effect on the poultry, but also the fact
that infected birds having contacted with salmonella carriers from outside become
permanent sources of contamination of the environment. Carrying among chickens is
widespread (5-22,2%), ducks (10-15%), geese (5-20%). On average, carriers were
detected among healthy birds in the range from 0.25 to 7.0%, among diseased and
forcedly killed from 2.9 to 30% [1].
The problem of salmonellosis in animals is becoming increasingly important. This
is due to a wide circulation in general, including in nature, the polydeterminateness of
the virulence factors of the pathogens, the variety of ways of entry into the body of
animals and humans. The damage caused by this disease is not only dead animals but
also bacterial carriers, which become permanent sources of contamination of the
environment. Products of animal origin (meat, milk, eggs), obtained from salmonella
carriers in case of insufficient heat treatment can cause foodborne toxic infections in
humans and detection and the control of foodborne diseases is a very actual daily
practice of veterinary and medical workers [2,3].
Epizootic and epidemiological tensions around intestinal infections caused by
enteroinfections in recent years has increased in connection with changes in methods of
cattle breeding and fattening, as well as the rules of zootechnical and veterinary care of
animals. Vaccination of animals and birds against salmonellosis of animals became
optional and not considered in the plan of antiepizootic measures of the Veterinary
Committee of the Ministry of Agriculture of the RK.
In the current socio-economic conditions, the specific features of combating
diseases common to humans and animals are largely related to the development of the
private sector in livestock production, uncontrolled migration of livestock, including
from disadvantaged regions. This makes it difficult to take into account and carry out
vaccination of animals, creates difficulties in the implementation of state veterinary and
sanitary-epidemiological surveillance. Exceptional resistance of pathogens of
enteroinfections and their cyclic increase in activity cause periodic sharp increases in
morbidity. The increase in the scale and intensity of development of territories where
77
active natural foci are located leads to a wide spread of these diseases among the
population.
Prevention of zooanthroponosis is primarily based on the timely detection of the
risk of infection of people with an infection. Epizootic and epidemiological features of
infection, effective means of prevention and the possibility of their use determine the
choice of key activities. In some cases, this may be regime-restrictive measures, in
others - veterinary and sanitary, sanitary and anti-epidemic measures, use of specific
prevention tools, etc.
All this causes the need to study the epizootic situation of these infections, the
opening of the main factors of the infectious process, as well as the improvement of
curative and specific prevention and development of veterinary and sanitary measures.
In this regard, the improvement of specific prevention of bovine salmonellosis
through the development and introduction of live vaccines, from genetically
characterized strains of Salmonella, is an urgent issue.
The purpose of our work was the development of a technology for the production
of live vaccines against bovine salmonellosis.
To achieve this goal, the following tasks were identified: To study the prevalence
of salmonellosis in cattle in various regions of Kazakhstan;To study the biological
properties of salmonella cultures; To study the biological properties of the attenuated
strain of S. dublin 31 in vitro and in vivo; To develop a technological regulation for the
production of live vaccines against bovine salmonella; Approbation of live vaccine
against bovine salmonellosis in production conditions and development of normative
and technical documentation for the manufacture and control of vaccines.
The work was carried out in the period from 2015 to 2017 in the laboratory of
antibacterial biotechnology of the Kazakh National Agrarian University, as well as in a
number of Kazakhstani farms.
The research part of the work includes a literary search, the collection of
information and statistical materials published in domestic and foreign scientific
publications, in the official collections of the International Program for the OIE and
WHO on the control and surveillance of infections and toxic infections in Europe, the
Centers for Disease Control in the United States and other published sources .
In order to study the prevalence of bovine salmonellosis, studies were conducted in
various farms in Almaty, Zhambyl, and Kyzylorda regions. In most of the surveyed
farms, diseases have been observed for several years.
The issues of epizootology of bovine salmonellosis were studied directly at the
farms. Annual reports of regional and district veterinary laboratories and veterinary
reports of the veterinary department of the regional territorial inspection of the Ministry
of Agriculture were used.
The diagnosis for salmonellosis was performed according to conventional
methods.
78
Under natural conditions, we observed that salmonellosis in animals was in
intestinal (enteric) and septic forms. The main clinical signs of the disease were:
diarrhea, turning into profuse, weakness, loss of appetite, depression, dehydration.
Pathological changes in the dead animals had a picture of catarrhal and catarrhal-
hemorrhagic gastroenteritis, ulcers, multiple ulcers on the stomach mucosa, thin and
thick intestine, under the capsule of the spleen were seen on the stomach mucosa, the
small intestine and cecum. regional mesenteric lymph nodes enlarged, edematous.
Bacteriological studies were conducted in accordance with the guidelines
"Laboratory diagnosis of human and animal salmonellosis, detection of salmonella in
feed, food and environmental objects".
In the farms with mass intestinal diseases of animals, 140 samples of pathological
material were obtained from calves with clinical signs of diarrhea and were subjected to
bacteriological examination.
For postmortem bacteriological diagnostics, 160 samples from fallen calves were
examined during the first ten days. Liver, spleen, lungs, mesenteric lymph nodes, a thin
intestinal tract, heart, tubular bone were taken.
For bacteriological diagnostics, 50 fecal samples from healthy calves that had not
been treated with antibacterial drugs were examined. Samples of feces were taken from
diseased and healthy calves to sterile test tubes directly from the rectum using a boiled
rubber catheter.
For the study of primary cultures, the following nutrient media were used: meat -
peptone broth (MBP), meat - peptone agar (MPA), Endo medium, Kitt-Tarozzi medium,
Mink, Kaufman, Levin media.
Primary selection of cultures was carried out on the basis of features of growth on
media and microscopy of preparations from individual colonies. Morphological,
cultural, biochemical properties were tested according to the generally accepted schemes
(NI Rozanov, 1952).
Identification of the isolated cultures was carried out according to Berdzhi's
determinant.
As a result of the studies of organs of diseased and fallen calves, as well as faeces
of healthy calves, 179 Salmonella cultures were isolated and identified, including from
diseased calves - 45, from dead - 116 and from healthy calves - 18.
The studies of the morphological, tinctorial, cultural and biochemical properties of
179 cultures isolated from diseased and dead calves, as well as from the faeces of
healthy calves showed that they were typical for the Salmonella genus.
In the identification of 179 Salmonella cultures isolated from diseased and fallen
calves, as well as from the faeces of healthy calves, it was found that 121 (68.0%)
belonged to S. typhimurium - 38 (21.0%) and 20 (11.0%) - Salmonella enteritidis.
The purpose of our further research was to determine the pathogenicity of
salmonella isolated from animals for the selection of production strains of
79
enteroinfection pathogens that will be used to manufacture innovative biologics against
enterobacteriocinosis in animals .
Previously, the pathogenicity of all the isolated cultures was checked on white mice
injected intraperitoneally at doses of 103, 104, 105, 106and 109 colony-forming units. The
results of the experiment indicated that the experimental animals died completely when
infected with a dose of105 CFU or higher.
As a result, strains of Salmonella isolated from fallen calves were selected on the
basis of the study of the morphological, biochemical and antigenic properties and the
degree of pathogenicity of the isolated cultures: S.dublin 76, S. typhimurium 69,
S.enteritidis 54 (3 strains from each salmonella serovar) .
The virulence of S. dublin, S. typhimurium, S.enteritidis, S. choleraesui cultures
was studied in experiments on white mice.
The results of the experiments showed that the cultures tested had a sufficiently
high virulence, especially strains: S.dublin 76 and S. Typhimurium 69, isolated from the
dead calves, causing 100% death of the experimental animals at a dose of ≥104 CFU.
An autopsy was carried out in all experiments. Infectious cultures were constantly
isolated.
The virulence of S.dublin 76, S. typhimurium 69, S.enteritidis 54 strains were
tested on calves. All animals were 1 months old. Reference virulent strains of S.
typhimurium 371, S. dublin 315/52, S.enteritidis 51, taken from VGNKI (Moscow) were
used as control.
The experimental calves were infected intraperitoneally by daily agar culture in
appropriate doses -109, 2 * 109, 4 * 109, 6 * 109 CFU. Experimental animals mostly died
on the 6th -12th day after the infection with obvious signs of salmonellosis.
Our studies showed that the strains studied preserved the typical morphological,
tinctorial, cultural, biochemical, antigenic and pathogenic properties characteristic of the
corresponding salmonella serovars.
The studied S. dublin 76 strain was selected as the initial strain for use in the
development and design of a live vaccine (using the attenuation method) against bovine
salmonellosis.
The task of our further research was to obtain an attenuated strain of salmonella, to
study its biological properties, to use it as a vaccine strain for the production of a live
vaccine against bovine salmonella.
Live vaccines are biological preparations from hereditarily altered forms (mutants)
of pathogens of infectious diseases suspended or dried in appropriate protective
environments. Mutants of pathogens with the position of genetics allow us to define
them as forms that underwent genotypic changes, as a result of which they irretrievably
lost the ability to cause pathological changes in the susceptible organism that previously
caused the disease. At the same time, they retained in their genetic constitution
determinants determining their ability to cause specific immunological changes and
80
restructuring. In accordance with the transformed genome, these mutants also changed
their phenotypic area.
The results of the conducted studies testify to the etiological role of the studied
salmonella in the disease of calves.
The main goal of our research was to obtain attenuated strains of salmonella, to
study their biological properties, to use it as a vaccine strain for the production of a live
vaccine against bovine salmonellosis.
As a result of our studies, we obtained an attenuated strain of Salmonella dublin 31,
which has genetic markers for distinguishing it from a wild type strain.
The strain Salmonella dublin 31 was studiedfor its morphological, cultural,
antigenic properties, the stability of attenuation, immunogenicity, the safety of the
vaccine strain and the differentiation of the vaccine strain from wild type cultures.
The results of the studies show that Salmonella Dublin strain31 meets all the
requirements for vaccine strains: it has stable biological properties, moderate
reactogenicity and residual virulence, high immunogenicity for mice and chickens, is
epizootically safe for use and has three genetic markers for distinguishing it from a wild
type strain. The presence in of three mutations Salmonella dublin strain 31, with known
mechanisms of action serves as a convincing genetic evidence of the stability and safety
of the attenuated strain Salmonella dublin 31. The theoretical frequency of reverse
mutation simultaneously for all markers is approximately 10-21, which is practically
impossible.
The obtained strain Salmonella dublin 31 is deposited in the Collection of
Microorganisms of the Republican State Enterprise "Scientific Research Institute of
Biological Safety Problems" of the Ministry of Education and Science of the Republic of
Kazakhstan (RSE NIIPBB KN MES RK), Collection number M-42-15 / D.
Our studies showed that the attenuated strain Salmonella dublin 31, retained the
typical morphological, tinctorial, cultural, biochemical and antigenic properties
characteristic of the corresponding serovar.
We drew attention to the possibility of dissociation of the vaccine strain S.dublin
and the virulent culture of S. dublin 315/52.
Estimation of the degree of dissociation of salmonella was carried out by multiple
scatters on petri dishes with MPA and the agglutination reaction in a physiological
saline solution. After boiling for an hour, the above strains did not precipitate. All this
gives grounds to believe that all strains (vaccine and virulent) are in a stable S-form.
One of the important requirements for attenuated vaccine strains is the retention of
residual virulence, on which the high immunizing ability of the live vaccine depends. In
this connection, throughout our experiments attention was drawn to the virulence
consistency.
The residual virulence of the vaccine strain S.dublin 31 was tested in comparison
with the virulent culture of S. dublin 315/52 on white mice (weighing 14-16 g) and
81
calves (aged 8-10 days) in several repetitions, taking into account their survival,
dissemination process and timing of elimination of the culture of the vaccine strain.
White mice were infected with the daily culture of the attenuated S. dublin 31 strain
subcutaneously at doses of 104, 105, 106, 107, 108 CFU and intraperitoneally at a dose of
106 107 and 108 CFU.
It should be noted that these doses of the S.dublin 31 strain correspond to 50 to
10,000 fatal doses of the virulent culture of S.dublin 315/52 (LD50 102 CFU).
All mice infected with S. dublin 31 culture survive in 90-100% of cases during the
20 days of observation, whereas control mice infected with the virulent culture of
S.dublin 315/52 at a dose of 103, 104 and 105 CFU died from 57 to 100% of cases.
In the experiments on calves the residual virulence of S.dublin strain 31 was
studied by intraperitoneal administration. A total of 70 calves were used in the
experiment. The virulence of the strain was controlled by the survival rate of both the
general and local response.
Intraperitoneal infection with the vaccine strain S.dublin 31 did not cause
significant disease in animals, only one calf infected with large-dose (1010 CFU) died on
the day 14 without manifesting clinical signs of the disease.
Control calves infected with the virulent culture of S.dublin 315/52 died with
symptoms of acute salmonellosis.
The data presented indicate that the attenuated S. dublin strain 31 has a weak
residual virulence.
Along with this, the degree of dissemination and the timing of elimination of the
vaccine strain from the animal organism was studied. At subcutaneous infection of white
mice with a vaccine strain at a dose of 106cfu, the isolates were cultured from organs and
blood during 15 days, from inguinal lymph nodes during 30 days.
In calves subcutaneously infected with a vaccine strain at a dose of 2 * 109cfu,
generalized vaccine infection was noted in the first three days. After 7 days the culture
was well isolated from the lymph nodes and spleen, weak culturing from the liver and
bone marrow; After 14 days, the culture in the form of single colonies was isolated from
spleen, pre-lobed, mediastinal and mesenteric lymph nodes.
Thus, in experiments on laboratory animals and calves, the inability of the
attenuated strain S.dublin 31 to cause a typical infectious process was established.
The persistence of the biological properties of the vaccine strain has been studied
during long-term storage (for 5-6 years) and repeated crossings on semi-liquid and solid
nutrient media, after freeze-drying of the S.dublin strain 31, and also after 10-fold
passage on white mice and three times through the bodies of calves.
Passage of S.dublin strain 31 was carried out on white mice by intraperitoneal fatal
infection of mice at a dose of 3 * 109 CFU.
Passage of S.dublin strain 31 was carried out on 8-10 day-old calves by
intraperitoneal infection in a dose of 2 * 1010 CFU. Calves responded to infection with
significant oppression, fever, digestive disorders, but did not die.
82
On the 3rd and 5th day, the experimental white mice and calves were killed for
bacteriological examination.
A total of 10 passages were made on white mice and calves.
All passivated strains were controlled by the nature of growth and agglutinability.
In addition, they were tested for virulence, by subcutaneous infection of white mice at
doses of 107 and 108 CFU, the experimental animals remained alive.
The conducted studies testify to the constancy of the properties of vaccine strain
S.dublin 31 when grown on artificial nutrient media and when passaging on susceptible
animals.
The absence of reversion of the vaccine strain is indicated by numerous
immunological experiments in laboratory animals and calves, as well as immunization
with an experimental vaccine from the same strain of more than 2,000 heads of cattle in
farms infected with salmonellosis.
The study of S.dublin strain 31 culture properties after freeze-drying showed good
survival, which complies with the standard and preservation duration.
Thus, the obtained results on the study of the biological properties of the attenuated
S.dublin 31 strain obtained by the genetic method indicate that the strain S.dublin 31 is
in stable S form, has stable, typical for S.dublin 31 morphological, cultural, biochemical
and antigenic properties, weak residual virulence, is well established in the body, does
not reverse during prolonged passage on susceptible animals. The presence of three
genetic markers in S.dublin 31 strain is a convincing proof of the stability and safety of
an attenuated strain, and also allows it to be differentiated from a natural prototype.
After studying the cultural-biochemical, antigenic properties and residual virulence
of the attenuated strain S. dublin 31, we proceeded to study immunogenic properties in
experiments on laboratory animals and calves, in comparison with a concentrated mold-
wax vaccine of biomedical production.
Immunogenic activity of S. dublin strain 31 was studied in white mice, during
which various doses were tested, local and general reaction and immunity after the
challenge were taken into account. Controls were animals grafted with a concentrated
formulose-vaccine vaccine and non-immunized.
Before the experiments, the virulent S. dublin315 / 52 strain was protitted on white
mice and calves. From the titration materials it was established that the virulent strain of
S. dublin315 / 52 causes death of mice in a dose of 10 CFU, and calves in a dose of 2-
109CFU with intraperitoneal administration
The immunizing activity of the vaccine strain was first studied in 150 white mice
(90 experimental and 60 control mice) weighing 14-16 grams. The mice were
immunized subcutaneously with a lyophilized culture of the S. dublin 31 vaccine strain
at doses of 104, 105, and 106 CFU. Mice immunized once with concentrated formulose
vaccine in a dose of 0.1 ml (5-10 CFU and unvaccinated) were controls. Twenty days
after vaccination, the experimental and control mice were infected with a virulent strain
of S. dublin 315/52 at a dose of 106 CFU, and controls (unvaccinated) ten times less than
83
105 CFU. The materials of the experiment are shown in Table 7. Table 7 shows that with
a single subcutaneous immunization with the culture of S. dublin 31 strain at 104, 105
and 106 CFU doses, high-voltage immunity is created in mice. 100% of the experimental
mice remained alive.
Mice immunized with a concentrated formulose vaccine (at a dose exceeding 10
times the live vaccine), with subsequent subcutaneous infection with a virulent culture,
died more than in 70% cases. All of control mice died in 10 days.
The obtained materials of experimental studies on laboratory animals allowed us to
continue studying the immunizing properties of the attenuated S. dublin 31 strain on
calves. Harmlessness, reactogenicity and immunizing doses were determined. The
possibility of both early reactions (general and local) that occurred after vaccination and
later, long-term vaccination results, which could be due to the development of the
vaccine process were taken into account.
In total, there were 45 calves of 12-14 days old in the experiment.
In the first group, 20 calves were vaccinated with a vaccine from S. dublin strain 31
in a volume of 2 ml with 107 CFU in 1 ml.
In the second group, 20 calves were inoculated twice with a concentrated formulose
vaccine in doses of 5ml and 10ml.
The third group - (5 calves) control, were not exposed to vaccination.
The vaccine was administered to the experimental calves in the region of the upper
third of the neck.
Immunized calves were monitored for up to 3 months.
The test calves were under observation and the body temperature was measured
daily. In calves vaccinated by strain 12 there was a state of some depression, a slight
increase in body temperature by 0.5 ° C, the appetite was preserved on the first day. At
the injection site, limited edema appeared, which resolved on day 2-3.
Control intraperitoneal infection of all experimental and control (10) calves was
carried out by flushing out the daily agaric virulent culture of S. dublin 315/52 at a dose
of 1010 CFU after twenty days from the inoculation. The intensity of immunity was
checked by the degree of survival, general and local response and gastroenteric
disorders.
As a result of infection in the calves of all groups, an increase in body temperature
from 40 to 41.5 ° C was observed on the following day, which in the group of
experimental calves persisted for 2-6 days. 3 calves showed lethargy, decreased appetite,
increased pulse and respiration. After 4 days the signs of the disease disappeared and the
appetite recovered. There were no other clinical abnormalities. All the experimental
calves remained alive.
In calves of the second group (inoculated with a concentrated formulose vaccine),
the high temperature was held for 7-10 days. The increase in temperature was
accompanied by significant depression, decreased appetite, palpitations and breathing, as
84
well as intermittent diarrhea. In 4 calves, there remained depression, dyspnea, frequent,
arrhythmic pulse, cough, weight loss and death on 9-10 days.
In the control calves, on the second day after infection, severe inhibition was
observed, an increase in temperature to 41.6 ° C and 41.8 ° C, which persisted until the
animals died: the calves died on the 10th -12th day after the challenge. An autopsy of
the dead calves revealed typical changes of an acute salmonellosis: the presence of
densified areas in the lungs, the blood supply to the liver, a sharp swelling of the lymph
nodes (especially the pre-lobed, mediastinal, mesenteric), the spleen, and the small
intestinal mucosa with hemorrhages. Hemorrhages were also found under the capsule of
the kidneys and at the atria. From the blood (heart), liver, gall bladder, spleen and
mesenteric lymph nodes, the infecting culture was abundantly isolated. In a
bacteriological study, the infecting culture from organs, lymph nodes and bone marrow
was abundantly isolated.
Given the high protective properties, the vaccine strain S. dublin 31 studied has all
the grounds for a wide study of it as a vaccine for specific immunoprophylaxis of bovine
salmonella.
Many researchers believe that live vaccines are the most promising for the
prevention of salmonellosis in farm animals.
We have developed a technology for manufacturing live vaccines against bovine
salmonellosis, which includes the following stages:
- preparation of nutrient medium for cultivation;
- preparation of inoculum;
- cultivation of the vaccine strain;
- determination of bacterial mass concentration;
- lyophilization of the preparation;
- drug control;
- Packaging of the preparation and labeling of the vials with and packaging.
After the shop-floor inspection, the bottles were labeled with the name of the
product, the serial number and the date of manufacture.
A series of the drug considered a certain amount of a drug obtained as a result of a
one-time mixing in one container and having the same concentration of living microbial
bodies simultaneously fused into bottles, dried under the same regime and received its
number, the number of state control issued by one quality document (passport) with the
indication in it: the name of the manufacturer, the name of the product, the serial
number, the state control number, the date of manufacture (month, year), test results on
indicators of quality, shelf life, storage conditions, refer to specifications, number and
date of issue of the document on the quality, the conclusion and signature of the person
issuing the document.
The preparation from live cultures is a dry, fine-porous mass of white or grayish-
yellow color, containing 50-60% of living microbial cells, easily soluble in
physiological solution, distilled or boiled water. The vaccine is suitable for use within
85
12-18 months from the date of manufacture, provided it is stored at a temperature of +4 -
+10 ° C.
The above technology was used to make a living vaccine of S. dublin 31 strain
against bovine salmonellosis.
The study of the expression of local and general reactions is of particular interest
due to the fact that the duration and intensity of the antigenic stimuli are largely
determined and, when compared with other indicators give a more complete picture of
the vaccination process.
The reactogenicity of the vaccine strain S. dublin 31 was determined on white
mice, guinea pigs and calves.
When immunizing doses of S.dublin strain 31 (104 CFU, 105 CFU, 106 CFU, and
107 CFU) administered subcutaneously to white mice no clinically pronounced reaction
was noted.
In experiments on guinea pigs, subcutaneous vaccination with vaccine strain
S.dublin 31 (immunizing dose 3 * 108 CFU), caused the development of slight edema
(0.5x1 cm) on the second day, which increased by days 3-5 with severe limitation and
then, in some guinea pigs, after 7-10 days benign abscesses without necrosis were
formed. In guinea pigs vaccinated with the strain S.dublin 31 and then infected with
virulent culture, small abscesses without necrosis were also formed.
General reaction in guinea pigs vaccinated with live vaccine was manifested in the
form of short-term depression without a noticeable loss in weight.
Local reaction in calves was manifested by inflammatory phenomena: hyperemia,
infiltration, edema formation of 3x4 - 4x5 cm. After 3-5 days, edema had decreased,
became less painful and then resorption occured.
The general reaction was characterized by mild depression during the first half of
the day, temperature remained normal or increased by 0.5-1 degrees, the appetite in
calves usually remained unchanged. All these phenomena disappeared in 2-3 days
without any complications.
The same reaction was observed in calves when vaccinated with elevated doses (4
* 108 CFU). In pregnant cows and heifers, the vaccine strain only developed a local
reaction.
To identify the bacterial carriage, as well as to study some questions of
immunomorphology, 3 calves were killed, 3, 7 and 14 days after vaccination with the
strain S. dublin 31, at a dose of 2-109 CFU (a lyophilized dried culture diluted in
physiological saline).
Bacteriological study of the material from the killed calves was carried out
according to a conventional method (culturing from the liver, spleen, bone marrow,
prenopathic, mediastinal, mesenteric and inguinal lymph nodes).
In calves killed 3 and 7 days after vaccination, the presence of compaction and
swelling in the area of administration of live vaccine was noted. The basis of a skin and
86
hypodermic layer were filled with an infiltrate. In a calf slaughtered after 14 days,
subcutaneous tissue was hyperemic without tissue infiltration.
The results of a bacteriological study showed that the culture of the S.dublin strain
31 was abundantly isolated from all organs, 3 and 7 days after vaccination. In calves,
slaughtered 14 days after vaccination, the culture was isolated only as single colonies
from the spleen, pre-lobed, mediastinal and mesenteric lymph nodes.
Bacteriological study of immunized and then slaughtered mice, showed that the
culture of the vaccine strain is abundantly isolated from the liver, spleen and inguinal
lymph nodes for 7 and 15 days, less abundantly from blood and bone marrow. After 25-
30 days the strain was only isolated from the inguinal lymph nodes.
The foregoing allowed us to conclude that an attenuated strain of S. dublin 31 in
white mice, guinea pigs and calves causes a benign local and general reaction. Local
reaction was characterized by leukocyte infiltration, general-short-term depression and
rise in temperature (calves).
The vaccine strain was well isolated from the parenchymal organs and lymph nodes
during the first week, later the isolation decreased, and in white mice the vaccine strain
remained permanently in the lymph nodes (25-30 days).
The danger of vaccinal bacterial carriage was eliminated because the attenuated
strain of S.dublin 31 does not cause infection when parenterally infected with massive
doses (white mice and calves) and was not accompanied by the development of an
infectious process. The safety of S.dublin strain 31 was confirmed by the constancy of
biological properties. The attenuated strain of S.dublin 31 meets the requirements for
live vaccines: stability of biological properties, animal safety and well-expressed
immunizing activity.
Humoral factors of body protection in vaccinated animals were determined by the
dynamics of specific antisalmonella antibodies, total protein content, quantitative and
qualitative content of immunoglobulins.
It was found that the agglutinins in the blood serum of calves from cows grafted
with live vaccine from the S.dublin 31 strain (70 calves), reached the greatest titer one
day after colostrum feeding: 1: 200 in 30 calves, 1: 400 in 27 and 1: 800 in 6. These
parameters were kept with minor fluctuations up to 3 days, then there was a decrease to
10 days, agglutinins in the serum of calves were not detected.
In the blood serum of 35 calves obtained from cows grafted with a concentrated
form-molded vaccine, the titres of agglutinins reached 1: 400 in 1 day after calving; 1:
200 in 10 and 1: 100 in 8 calves; on the 3rd day - 1: 200-4, 1: 100 - 10 calves, and on the
10th day only in the 1:50 dilution in two calves.
The control calves did not show agglutinins or a small number of calves detected
1:50 (nonspecific reaction).
Thus, the attenuated strain of S.dublin 31 causes a moderate accumulation of
agglutinins in the serum and cow colostrum (1: 200, 1: 400, 1: 800).
87
In the blood of calves, after feeding with colostrum from grafted cows, agglutinins
reached the highest titer in a day (1: 200, 1: 400) and remained with slight fluctuations
for 3-5 days.
In calves at the age of 12-14 days vaccinated with live vaccine, agglutinins were
detected in moderate titres on days 10-25 after vaccination (1: 200, 1: 400).
Differences in the agglutinin counts in vaccinated calves obtained from immune
and non-immune cows are not observed.
The lack of parallelism between the high immunity intensity created by live
vaccine from the strain S.dublin 31 and moderate indices of agglutinins in calves is in
accordance with the literature data (B. Matvienko [131], Botes, 1965).
We conducted research and production experience in three farms, which were
disadvantaged for salmonellosis of cattle: in the peasant farming Khabit of the Almaty
region, the Anisan farm of the Aktobe region, and the Turtan-Ata farm of the Kyzylorda
region. Later in the vaccine was successfully tested in a number of Kazakhstani farms
that were not healthy for salmonellosis of cattle.
These livestock farms were infected with salmonellosis for several years. Newborn
calves on farms were systematically immunized with a concentrated formulose vaccine
according to the instructions. Despite this, there were quite frequent cases of calves'
infection with salmonella. Autopsy revealed that salmonellosis was caused by
Salmonella dublin strain, which was repeatedly confirmed by bacteriological
laboratories.
Vaccination was carried out with the coverage of all young animals (regardless of
fatness and development) subcutaneously, once, in the region of the 1/3 of the neck, in a
dose of 1 ml (109CFU).
Vaccinated animals were under clinical observations. A few hours after the
vaccination, the calves experienced a short-term depression, and the appetite remained
on the same level. Local reaction was accompanied by the formation of edema (size
3x4-4x5 cm), which resolved on 4-6 days. Along with this, the vaccine was tested on
cows in the last stage of pregnancy once, in a dose of 2 ml (2-109 CFU) subcutaneously,
in the region of 1/3 of the neck. Vaccinated cows demonstrated only local reactions.
Calves from vaccinated cows were born viable and did not develop salmonellosis.
In total, during 2015-2017, a total of more than 2,000 heads of cattle were
vaccinated, including 960 cows 20-25 days before calving and 1,040 calves. During this
period, cases of calf disease and their death from salmonellosis have not been recorded.
Observation of vaccinated calves and cows showed that the vaccine against bovine
salmonellosis from the attenuated strain S. dublin 31 does not cause complications.
The epizootological data of the farms where vaccine trials were conducted in
comparison with previous years indicate the efficacy and safety of the experimental live
vaccine and indicates the possibility of its widespread use as one of the measures to
combat bovine salmonellosis.
88
Economic efficiency as a result of immunization of cattle with live vaccine from S.
dublin strain 31 can be achieved due to decrease in the incidence and mortality of calves,
labor costs and amounted to 60 tenge per one spent tenge..The economic effect of
routine preventive vaccination of animals against salmonellosis by live vaccines is 2-3
times higher than when immunized with animals killed by vaccines.
The effectiveness of immunization of cattle in disadvantaged for salmonellosis
farms was studied by conducting an epizootic analysis before and after its use and taking
into account a decrease in the incidence of morbidity and calf death.
89
CONCLUSION
1. A study of the prevalence of salmonellosis in cattle in the farms of Almaty,
Aktobe, Kzylorda regions was conducted. The study was subjected to 350 samples taken
from calves (from 140 patients, 160-dead and 50 from faeces of healthy calves).
2. As a result of the studies of organs from diseased and dead calves, as well as
from the faeces of healthy calves, we isolated and identified 179 Salmonella cultures,
including from diseased calves - 45, from dead - 116 and from healthy calves - 18.
3. Identification of 179 Salmonella cultures isolated from diseased and dead calves,
as well as from the faeces of healthy calves revealed that 121 (68.0%) cultures are
related to Salmonella dublin, S. typhimurium - 38 (21.0%),Salmonella enteritidis - 20
(11.0%). The cultures isolated from dead calves had a rather high virulence, causing
100% death of the experimental white mice and calves. The results of the conducted
studies testify to the etiological role of the studied salmonella in the disease of calves.
4. An attenuated Salmonella dublin strain 31 was obtained using the attenuation
method developed by us. The strain meets all the requirements for vaccine strains: it
possesses stability of biological properties, moderate reactogenicity and residual
virulence, high immunogenicity for animals, is epizootically safe for use, it has three
genetic markers for distinguishing them from wild type strains. The presence of three
mutations in the strain with known mechanisms of action serves as a convincing genetic
proof of the stability and safety of the attenuated strain.
5. The strain Salmonella dublin 31 has been deposited in the Collection of
Microorganisms of the Republican State Enterprise "Scientific Research Institute for
Biological Safety" of the Ministry of Education and Science of the Republic of
Kazakhstan (RSE SRIBS MES RK). Collection number M-42-15 / D.
6. The normative and technical documentation (the Technical condition, the
Temporary instruction for the production of live vaccine against bovine salmonellosis,
the Manual on the use of the vaccine), approved by the Institute of Problems of
Animation of the NAO of the Kazakh National Agrarian University in 2017.
7. We have applied for a patent for the invention of the Republic of Kazakhstan on
the subject "the method of preventing bovine salmonellosis with a vaccine from the
strain Salmonella dublin 31" , registration number 2018/0296-1, from 14.05.2018.
8. Production tests of live vaccine against bovine salmonellosis in disadvantaged
for this diseasefarms testify to the efficacy and safety of live vaccine and indicates the
possibility of its wide application as one of the measures to combat salmonellosis of
cattle.
90
PRACTICAL OFFERS
1.The method of obtaining an attenuated strain of Salmonella for the purpose of
designing a genetically safe live vaccine for the prevention of bovine salmonellosis is
proposed.
2.Developed normative and technical documentation (Technical condition,
Temporary instruction for the production of live stock against salmonella and large
cattle, Manual on the use of vaccine), approved by the Institute of Problems of
Animation NPJSC Kazakh National Agrarian University of 2017.
3. The recommendation "Salmonellosis of cattle and measures of struggle,
approved on. NTS Institute of Problems of Animation KazNAU from, which provides a
set of veterinary and sanitary measures against salmonellosis of farm animals.
91
REFERENCES
1. Fabrega A, Vila J. Salmonella enterica serovar Typhimurium skills to succeed in
the host: virulence and regulation. Clinical microbiology reviews. 2013;
26(2):308–41. doi: 10.1128/CMR.00066-12 PMID: 23554419; PubMed Central
PMCID: PMC3623383
2. Report of a WHO Expert Committee The fight against salmonellosis: the role of
veterinary medicine and food hygiene // Geneva, 1991. - S. 18 - 19.
3. Hurley D, McCusker MP, Fanning S, Martins M. Salmonella-host interactions—
modulation of the host innate immune system. Frontiers in immunology. 2014;
5:481. doi: 10.3389/fimmu.2014.00481 PMID: 25339955; PubMed Central
PMCID: PMC4188169.
4. The State Report "On the sanitary-epidemiological situation in the Republic of
Kazakhstan in 2003". - Almaty, 2003. - S. 115-117.
5. Матвиенко Б. А. Актуальные вопросы иммунопрофилактики
сальмонеллезов животных: сб. научных трудов АЗВИ. - Алма-Ата, 1986. - С.
54
6. Бияшев К. Б. Иммунопрофилактика сальмонеллёза сельскохозяйственных
животных в Казахстане: автореф. док. сел.-х. наук. - Алматы, 1991. - 25 с.
7. Centers for Disease Control and Prevention. 2016. Pertussis cases by year (1922–
2015). CDC, Atlanta, GA. https://www.cdc.gov/pertussis/ surv-reporting/cases-
by-year.html.
8. Plotkin SA. 2016. The importance of persistence. Clin Infect Dis 63: S117–S118.
https://doi.org/10.1093/cid/ciw525.
9. Klein NP. 2014. Licensed pertussis vaccines in the United States: history and
current state. Hum Vaccin Immunother 10:2684 –2690. https://doi
.org/10.4161/hv.29576.
10. Biyashev KB Immunoprophylaxis of salmonellosis of farm animals in
Kazakhstan: Abstract. Doc. sel.-h. Sciences./ - Almaty, 1991. - 25 p.
11. http://www.who.int/ru/news-room/fact-sheets/detail/salmonella
12. Malder R. W. Salmonella in poultry is a worldwide problem // Poultry.-1989. -Р.
5.
13. Тутельян В.А., С.А. Шевелева, Н.Р. Ефимочкина. 8-й доклад Программы
ВОЗ по контролю за пищевыми инфекциями и интоксикациями в Европе за
1999-2000 гг. Раздел: Российская Федерация. /FAO/WHO. - Берлин, 2003. –
С. 45.
14. Enwere G, Biney E, Cheung YB, Zaman SM, Okoko B, Oluwalana C, Vaughan
A, Greenwood B, Adegbola R, Cutts FT. 2006. Epidemiologic and clinical
characteristics of community-acquired invasive bacterial infections in children
aged 2–29 months in The Gambia. Pediatr Infect Dis J 25:700 –705.
http://dx.doi.org/10.1097/01.inf.0000226839.30925.a5.
92
15. Blanc-Potard AB, Solomon F, Kayser J, Groisman EA. The SPI-3 pathogenicity
island of Salmonella enterica. Journal of bacteriology. 1999; 181(3):998–1004.
PMID: 9922266; PubMed Central PMCID: PMC93469.
16. Strugnell RA, Scott TA, Wang N, Yang C, Peres N, Bedoui S, et al. Salmonella
vaccines: lessons from the mouse model or bad teaching? Current opinion in
microbiology. 2014; 17:99–105. doi:10.1016/j.mib.2013.12.004 PMID:
24440968.
17. Vogel J. A rough guide to the non-coding RNA world of Salmonella. Molecular
microbiology. 2009; 71 (1):1–11. doi:10.1111/j.1365-2958.2008.06505.x PMID:
19007416.
18. Rathman M, Sjaastad MD, Falkow S. Acidification of phagosomes containing
Salmonella Typhimurium in murine macrophages. Infection and immunity. 1996;
64(7):2765–73. PMID: 8698506; PubMed Central PMCID: PMC174137.
19. Wood MW, Jones MA, Watson PR, Hedges S, Wallis TS, Galyov EE.
Identification of a pathogenicity island required for Salmonella
enteropathogenicity. Molecular microbiology. 1998; 29(3):883–91. PMID:
9723926.
20. https://365info.kz/2015/06/astana-stala-stolicej-salmonelleza-kishechnyx-
infekcij/
21. Шевелева С.А., Ефимочкина Н.Р. Анализ микробиологического риска как
основа для совершенствования системы оценки безопасности и контроля
пищевых продуктов //Мат. X Всероссийского съезда гигиенистов и
санитарных врачей. - М., 2007. - С.21-24.
22. Белова Т. Н., Бруснигина Н. Ф., Быкова С. А., Залесских Н. В. и др. О роли
предприятий промышленного птицеводства в распространении
сальмонеллёзов // Матер. Международной конф. «Эпизоотология,
эпидемиология, средства диагностики, терапии и специфической
профилактики инфекционных болезней, общих для человека и животных». –
Гродно, 2012. -С. 254 – 255.
23. Государственный доклад «О санитарно-эпидемиологической ситуации в
Республике Казахстан в 2003 году». - Алматы, 2003. - С. 115-117.
24. Бияшев К.Б., Тулкибаев К.А., Досанова А. Вакцинопрофилактика
сальмонеллеза овец //Вестник сельскохозяйственной науки Казахстана. -
2004. - № 10. - С.36-37.
25. Консультативное совещание экспертов ВОЗ: специфические методы
профилактики сальмонеллеза животных и борьбы с ним. - Мюнхен, 1986. -
С. 17 – 18.
26. Nabel GJ. 2013. Designing tomorrow’s vaccines. N Engl J Med 368: 551–560.
https://doi.org/10.1056/NEJMra1204186.
27. Jones BD, Ghori N, Falkow S. Salmonella typhimurium initiates murine infection
by penetrating and destroying the specialized epithelial M cells of the Peyer's
93
patches. The Journal of experimental medicine. 1994; 180(1):15–23. PMID:
8006579; PubMed Central PMCID: PMC2191576
28. Uren T., Wijburg O.L.C., Simmons C., Johansen F., Brandtzaeg P., Strugnell R.,
Vaccine-induced protection against gastrointestinal bacterial infections in the
absence of secretory antibodies, Eur. J. Immunol. (2005) 35:180–188.
29. Chakraborty S, Gogoi M, Chakravortty D. Lactoylglutathione lyase, a Critical
Enzyme in Methylglyoxal Detoxification, Contributes to Survival of Salmonella
in the Nutrient Rich Environment. Virulence. 2014:0. doi:
10.4161/21505594.2014.983791 PMID: 25517857.
30. Lober S, Jackel D, Kaiser N, Hensel M. Regulation of Salmonella pathogenicity
island 2 genes by independent environmental signals. International journal of
medical microbiology: IJMM. 2006; 296 (7):435–47. doi:
10.1016/j.ijmm.2006.05.001 PMID: 16904940.
31. Cirillo DM, Valdivia RH, Monack DM, Falkow S. Macrophage-dependent
induction of the Salmonella pathogenicity island 2 type III secretion system and
its role in intracellular survival. Molecular microbiology. 1998; 30(1):175–88.
PMID: 9786194.
32. Feng X, Oropeza R, Kenney LJ. Dual regulation by phospho-OmpR of ssrA/B
gene expression in Salmonella pathogenicity island 2. Molecular microbiology.
2003; 48(4):1131–43. PMID: 12753201.
33. Richardson EJ, Limaye B, Inamdar H, Datta A, Manjari KS, Pullinger GD, et al.
Genome sequences of Salmonella enterica serovar typhimurium, Choleraesuis,
Dublin, and Gallinarum strains of welldefined virulence in food-producing
animals. Journal of bacteriology. 2011; 193(12):3162–3. doi: 10. 1128/JB.00394-
11 PMID: 21478351; PubMed Central PMCID: PMC3133203.
34. Рахманин П.П., Куликовский А.В. Эпизоотическое состояние и меры
борьбы с сальмонеллезом // Ветеринария. -1989. - № 7. - С. 40-44. - № 1. - С.
46-48.
35. Sabbagh SC, Lepage C, McClelland M, Daigle F. Selection of Salmonella
enterica serovar Typhi genes involved during interaction with human
macrophages by screening of a transposon mutant library. PloS one. 2012;
7(5):e36643. doi: 10.1371/journal.pone.0036643 PMID: 22574205; PubMed
Central PMCID: PMC3344905.
36. Попова П.П., Ременцова М.М., Ким А.А. Экология сальмонелл и
эпидемиология сальмонеллезов. Издательство «Наука» Каз. ССР. Алма-ата.
1987. 126 с.
37. Chakraborty S, Chaudhuri D, Balakrishnan A, Chakravortty D. Salmonella
methylglyoxal detoxification by STM3117-encoded lactoylglutathione lyase
affects virulence in coordination with Salmonella pathogenicity island 2 and
phagosomal acidification. Microbiology. 2014; 160(Pt 9):1999–2017. doi:
10.1099/mic.0.078998–0 PMID: 24961952.
94
38. Shah DH, Lee MJ, Park JH, Lee JH, Eo SK, Kwon JT, et al. Identification of
Salmonella Gallinarum virulence genes in a chicken infection model using PCR-
based signature-tagged mutagenesis. Microbiology. 2005; 151(Pt 12):3957–68.
doi: 10.1099/mic.0.28126–0 PMID: 16339940.
39. Jha AK, Huang SC, Sergushichev A, Lampropoulou V, Ivanova Y, Loginicheva
E, et al. Ne*twork Integration of Parallel Metabolic and Transcriptional Data
Reveals Metabolic Modules that Regulate Macrophage Polarization. Immunity.
2015; 42(3):419–30. doi: 10.1016/j.immuni.2015.02.005 PMID: 25786174.
40. Blanc-Potard AB, Solomon F, Kayser J, Groisman EA. The SPI-3 pathogenicity
island of Salmonella enterica. Journal of bacteriology. 1999; 181(3):998–1004.
PMID: 9922266; PubMed Central PMCID: PMC93469.
41. Котова А.Л., Белозеров Е.С. Сальмонеллезы. - Алматы.: - «Галым» -
1992.-215 с.
42. Lee EJ, Groisman EA. Control of a Salmonella virulence locus by an ATP-
sensing leader messenger RNA. Nature. 2012; 486(7402):271–5. doi:
10.1038/nature11090 PMID: 22699622; PubMed Central PMCID: PMC3711680.
43. Lee EJ, Pontes MH, Groisman EA. A bacterial virulence protein promotes
pathogenicity by inhibiting the bacterium's own F1Fo ATP synthase. Cell. 2013;
154(1):146–56. doi: 10.1016/j.cell.2013.06.004 PMID: 23827679; PubMed
Central PMCID: PMCPMC3736803.
44. Ахмедов А.М. Сальмонеллезы молодняка - М. Колос, 1983, 239 с.
45. Wood MW, Jones MA, Watson PR, Hedges S, Wallis TS, Galyov EE.
Identification of a pathogenicity island required for Salmonella
enteropathogenicity. Molecular microbiology. 1998; 29(3):883–91. PMID:
9723926.
46. Knodler LA, Celli J, Hardt WD, Vallance BA, Yip C, Finlay BB. Salmonella
effectors within a single pathogenicity island are differentially expressed and
translocated by separate type III secretion systems. Molecular microbiology.
2002; 43(5):1089–103. PMID: 11918798.
47. Knodler LA, Vallance BA, Hensel M, Jackel D, Finlay BB, Steele-Mortimer O.
Salmonella type III effectors PipB and PipB2 are targeted to detergent-resistant
microdomains on internal host cell membranes. Molecular microbiology. 2003;
49(3):685–704. PMID: 12864852.
48. Knodler LA, Steele-Mortimer O. The Salmonella effector PipB2 affects late
endosome/lysosome distribution to mediate Sif extension. Mol Biol Cell. 2005;
16(9):4108–23. doi: 10.1091/mbc.E05-04-0367 PMID: 15987736; PubMed
Central PMCID: PMC1196323
49. Blondel CJ, Jimenez JC, Contreras I, Santiviago CA. Comparative genomic
analysis uncovers 3 novel loci encoding type six secretion systems differentially
distributed in Salmonella serotypes. BMC genomics. 2009; 10:354. doi:
95
10.1186/1471-2164-10-354 PMID: 19653904; PubMed Central PMCID:
PMC2907695.
50. Pezoa D, Yang HJ, Blondel CJ, Santiviago CA, Andrews-Polymenis HL,
Contreras I. The type VI secretion system encoded in SPI-6 plays a role in
gastrointestinal colonization and systemic spread of Salmonella enterica serovar
Typhimurium in the chicken. PloS one. 2013; 8(5):e63917. doi: 10.1371/
journal.pone.0063917 PMID: 23691117; PubMed Central PMCID: PMC3653874.
51. Mulder DT, Cooper CA, Coombes BK. Type VI secretion system-associated gene
clusters contribute to pathogenesis of Salmonella enterica serovar Typhimurium.
Infection and immunity. 2012; 80 (6):1996–2007. doi: 10.1128/IAI.06205-11
PMID: 22493086; PubMed Central PMCID: PMC3370595.
52. Matulova M, Havlickova H, Sisak F, Babak V, Rychlik I. 2013. SPI1 defective
mutants of Salmonella enterica induce cross-protective immunity in chickens
against challenge with serovars Typhimurium and Enteritidis. Vaccine 31:3156 –
3162. http://dx.doi.org/10.1016/j.vaccine.2013.05.002.
53. Levine MM, Ferreccio C, Black RE, Lagos R, San Martin O, Blackwelder WC.
2007. Ty21a live oral typhoid vaccine and prevention of paratyphoid fever caused
by Salmonella enterica serovar Paratyphi B. Clin Infect Dis 45(Suppl 1):S24 –
S28. http://dx.doi.org/10.1086/518141.
54. Simanjuntak CH, Paleologo FP, Punjabi NH, Darmowigoto R, Soeprawoto
Totosudirjo H, Haryanto P, Suprijanto E, Witham ND, Hoffman SL. 1991. Oral
immunisation against typhoid fever in Indonesia with Ty21a vaccine. Lancet
338:1055–1059. http://dx.doi.org/10.1016/0140 -6736(91)91910-M.
55. Roudier C, Krause M, Fierer J, Guiney DG. 1990. Correlation between the
presence of sequences homologous to the vir region of Salmonella dublin plasmid
pSDL2 and the virulence of twenty-two Salmonella serotypes in mice. Infect
Immun 58:1180 –1185. 38.
56. Suez J, Porwollik S, Dagan A, Marzel A, Schorr YI, Desai PT, Agmon V,
McClelland M, Rahav G, Gal-Mor O. 2013. Virulence gene profiling and
pathogenicity characterization of non-typhoidal Salmonella accounted for
invasive disease in humans. PLoS One 8:e58449.
http://dx.doi.org/10.1371/journal.pone.0058449.
57. Yang J, Barrila J, Roland KL, Kilbourne J, Ott CM, Forsyth RJ, Nickerson CA.
2015. Characterization of the invasive, multidrug resistant non-typhoidal
Salmonella strain D23580 in a urine odel of nfection. PLoS Negl Trop Dis
9:e0003839. http://dx.doi.org/10.1371/journal .pntd.0003839.
58. Okoro CK, Barquist L, Connor TR, Harris SR, Clare S, Stevens MP, Arends MJ,
Hale C, Kane L, Pickard DJ, Hill J, Harcourt K, Parkhill J, Dougan G, Kingsley
RA. 2015. Signatures of adaptation in human invasive Salmonella Typhimurium
ST313 populations from sub-Saharan Africa. PLoS Negl Trop Dis 9:e0003611.
http://dx.doi.org/10.1371/journal.pntd.0003611.
96
59. Carden S, Okoro C, Dougan G, Monack D. 2015. Non-typhoidal Salmonella
Typhimurium ST313 isolates that cause bacteremia in humans stimulate less
inflammasome activation than ST19 isolates associated with gastroenteritis.
Pathog Dis 73:ftu023. http://dx.doi.org/10.1093/femspd /ftu023.
60. Okoro CK, Kingsley RA, Connor TR, Harris SR, Parry CM, AlMashhadani MN,
Kariuki S, Msefula CL, Gordon MA, de Pinna E, Wain J, Heyderman RS, Obaro
S, Alonso PL, Mandomando I, MacLennan CA, Tapia MD, Levine MM, Tennant
SM, Parkhill J, Dougan G. 2012. Intracontinental spread of human invasive
Salmonella Typhimurium pathovariants in sub-Saharan Africa. Nat Genet
44:1215–1221. http://dx.doi.org/10.1038/ng.2423.
61. Kingsley RA, Msefula CL, Thomson NR, Kariuki S, Holt KE, Gordon MA,
Harris D, Clarke L, Whitehead S, Sangal V, Marsh K, Achtman M, Molyneux
ME, Cormican M, Parkhill J, MacLennan CA, Heyderman RS, Dougan G. 2009.
Epidemic multiple drug resistant Salmonella Typhimurium causing invasive
disease in sub-Saharan Africa have a distinct genotype. Genome Res 19:2279 –
2287. http://dx.doi.org/10.1101/gr.091017.109.
62. Beyene G, Nair S, Asrat D, Mengistu Y, Engers H, Wain J. 2011. Multidrug
resistant Salmonella Concord is a major cause of salmonellosis in children in
Ethiopia. J Infect Dev Ctries 5:23–33.
63. Wadula J, von Gottberg A, Kilner D, de Jong G, Cohen C, Khoosal M, Keddy K,
Crewe-Brown H. 2006. Nosocomial outbreak of extended-spectrum beta-
lactamase-producing Salmonella isangi in pediatric wards. Pediatr Infect Dis J
25:843–844. http://dx.doi.org/10.1097/01.inf.0000233543.78070.a2.
64. Tennant SM, Diallo S, Levy H, Livio S, Sow SO, Tapia M, Fields PI, Mikoleit M,
Tamboura B, Kotloff KL, Nataro JP, Galen JE, Levine MM. 2010. Identification
by PCR of non-typhoidal Salmonella enterica serovars associated with invasive
infections among febrile patients in Mali. PLoS Negl Trop Dis 4:e621.
http://dx.doi.org/10.1371/journal.pntd.0000621.
65. Enwere G, Biney E, Cheung YB, Zaman SM, Okoko B, Oluwalana C, Vaughan
A, Greenwood B, Adegbola R, Cutts FT. 2006. Epidemiologic and clinical
characteristics of community-acquired invasive bacterial infections in children
aged 2–29 months in The Gambia. Pediatr Infect Dis J 25:700 –705.
http://dx.doi.org/10.1097/01.inf.0000226839.30925.a5.
66. Nicol JW, Helt GA, Blanchard SG Jr., Raja A, Loraine AE. The Integrated
Genome Browser: free software for distribution and exploration of genome-scale
datasets. Bioinformatics. 2009; 25(20):2730–1. doi:
10.1093/bioinformatics/btp472 PMID: 19654113; PubMed Central PMCID:
PMC2759552.
67. Skinner ME, Uzilov AV, Stein LD, Mungall CJ, Holmes IH. JBrowse: a next-
generation genome browser. Genome research. 2009; 19(9):1630–8. doi:
97
10.1101/gr.094607.109 PMID: 19570905; PubMed Central PMCID:
PMC2752129.
68. Wagner GP, Kin K, Lynch VJ. A model based criterion for gene expression calls
using RNA-seq data. Theory in biosciences = Theorie in den Biowissenschaften.
2013; 132(3):159–64. doi:10.1007/ s12064-013-0178-3 PMID: 23615947.
69. Li B, Ruotti V, Stewart RM, Thomson JA, Dewey CN. RNA-Seq gene expression
estimation with read mapping uncertainty. Bioinformatics. 2010; 26(4):493–500.
doi: 10.1093/bioinformatics/btp692 PMID: 20022975; PubMed Central PMCID:
PMC2820677.
70. Argaman L, Hershberg R, Vogel J, Bejerano G, Wagner EG, Margalit H, et al.
Novel small RNAencoding genes in the intergenic regions of Escherichia coli.
Current biology: CB. 2001; 11(12):941– 50. PMID: 11448770.
71. Pearson WR, Lipman DJ. Improved tools for biological sequence comparison.
Proceedings of the National Academy of Sciences of the United States of
America. 1988; 85(8):2444–8. PMID: 3162770; PubMed Central PMCID:
PMC280013.
72. Walthers D, Carroll RK, Navarre WW, Libby SJ, Fang FC, Kenney LJ. The
response regulator SsrB activates expression of diverse Salmonella pathogenicity
island 2 promoters and counters silencing by the nucleoid-associated protein H-
NS. Molecular microbiology. 2007; 65(2):477–93. doi: 10.1111/j. 1365-
2958.2007.05800.x PMID: 17630976.
73. Kidwai AS, Mushamiri I, Niemann GS, Brown RN, Adkins JN, Heffron F.
Diverse secreted effectors are required for Salmonella persistence in a mouse
infection model. PloS one. 2013; 8(8):e70753. doi: 10.1371/journal.pone.0070753
PMID: 23950998; PubMed Central PMCID: PMC3741292.
74. Chao Y, Papenfort K, Reinhardt R, Sharma CM, Vogel J. An atlas of Hfq-bound
transcripts reveals 3' UTRs as a genomic reservoir of regulatory small RNAs.
EMBO J. 2012; 31(20):4005–19. doi:10.1038/emboj.2012.229 PMID: 22922465;
PubMed Central PMCID: PMC3474919.
75. McKelvie ND, Stratford R, Wu T, Bellaby T, Aldred E, Hughes NJ, et al.
Expression of heterologous antigens in Salmonella Typhimurium vaccine vectors
using the in vivo-inducible, SPI-2 promoter, ssaG. Vaccine. 2004; 22(25–
26):3243–55. doi: 10.1016/j.vaccine.2004.05.014 PMID: 15308346.
76. Brown NF, Vallance BA, Coombes BK, Valdez Y, Coburn BA, Finlay BB.
Salmonella pathogenicity island 2 is expressed prior to penetrating the intestine.
PLoS pathogens. 2005; 1(3):e32. doi:10.1371/journal.ppat.0010032
PMID:16304611; PubMed Central PMCID: PMC1287911.
77. Osborne SE, Coombes BK. Transcriptional priming of Salmonella Pathogenicity
Island-2 precedes cellular invasion. PloS one. 2011; 6(6):e21648. doi:
10.1371/journal.pone.0021648 PMID: 21738750; PubMed Central PMCID:
PMC3125303.
98
78. Rollenhagen C, Sorensen M, Rizos K, Hurvitz R, Bumann D. Antigen selection
based on expression levels during infection facilitates vaccine development for an
intracellular pathogen. Proceedings of the National Academy of Sciences of the
United States of America. 2004; 101(23):8739–44. doi:10.1073/pnas.0401283101
PMID:15173591; PubMed Central PMCID: PMC423265.
79. Vogel J. A rough guide to the non-coding RNA world of Salmonella. Molecular
microbiology. 2009; 71 (1):1–11. doi:10.1111/j.1365-2958.2008.06505.x PMID:
19007416.
80. Papenfort K, Vogel J. Small RNA functions in carbon metabolism and virulence
of enteric pathogens. Frontiers in cellular and infection microbiology. 2014; 4:91.
doi: 10.3389/fcimb.2014.00091 PMID: 25077072; PubMed Central PMCID:
PMC4098024.
81. Papenfort K, Vogel J. Regulatory RNA in bacterial pathogens. Cell host &
microbe. 2010; 8(1):116– 27. doi:10.1016/j.chom.2010.06.008 PMID: 20638647.
82. Storz G, Vogel J, Wassarman KM. Regulation by small RNAs in bacteria:
expanding frontiers. Molecular cell. 2011; 43(6):880–91. doi:
10.1016/j.molcel.2011.08.022 PMID: 21925377; PubMed Central PMCID:
PMC3176440.
83. Caldelari I, Chao Y, Romby P, Vogel J. RNA-mediated regulation in pathogenic
bacteria. Cold Spring Harbor perspectives in medicine. 2013; 3(9):a010298. doi:
10.1101/cshperspect.a010298 PMID: 24003243. 89. Vogel J, Luisi BF. Hfq and
its constellation of RNA. Nature reviews Microbiology. 2011; 9(8):578–89. doi:
10.1038/nrmicro2615 PMID: 21760622.
84. Papenfort K, Pfeiffer V, Mika F, Lucchini S, Hinton JCD, Vogel J. σE-dependent
small RNAs of Salmonella respond to membrane stress by accelerating global
omp mRNA decay. Molecular microbiology. 2006; 62(6):1674–88. doi:
10.1111/j.1365-2958.2006.05524.x PMID: 17427289
85. Humphreys S, Stevenson A, Bacon A, Weinhardt AB, Roberts M. The alternative
sigma factor, sigmaE, is critically important for the virulence of Salmonella
Typhimurium. Infection and immunity. 1999; 67(4):1560–8. PMID: 10084987;
PubMed Central PMCID: PMC96497.
86. Guo MS, Updegrove TB, Gogol EB, Shabalina SA, Gross CA, Storz G. MicL, a
new sigmaE-dependent sRNA, combats envelope stress by repressing synthesis of
Lpp, the major outer membrane lipoprotein. Genes & development. 2014;
28(14):1620–34. doi: 10.1101/gad.243485.114 PMID: 25030700; PubMed
Central PMCID: PMC4102768.
87. Padalon-Brauch G, Hershberg R, Elgrably-Weiss M, Baruch K, Rosenshine I,
Margalit H, et al. Small RNAs encoded within genetic islands of Salmonella
Typhimurium show host-induced expression and role in virulence. Nucleic Acids
Res. 2008; 36(6):1913–27. doi: 10.1093/nar/gkn050 PMID: 18267966; PubMed
Central PMCID: PMC2330248.
99
88. Calderon IL, Morales EH, Collao B, Calderon PF, Chahuan CA, Acuna LG, et al.
Role of Salmonella Typhimurium small RNAs RyhB-1 and RyhB-2 in the
oxidative stress response. Res Microbiol. 2014; 165(1):30–40. doi:
10.1016/j.resmic.2013.10.008 PMID: 24239962.
89. Leclerc JM, Dozois CM, Daigle F. Role of the Salmonella enterica serovar Typhi
Fur regulator and small RNAs RfrA and RfrB in iron homeostasis and interaction
with host cells. Microbiology. 2013; 159(Pt 3):591–602. doi:
10.1099/mic.0.064329–0 PMID: 23306672.
90. Ortega AD, Gonzalo-Asensio J, Garcia-del Portillo F. Dynamics of Salmonella
small RNA expression in non-growing bacteria located inside eukaryotic cells.
RNA biology. 2012; 9(4):469–88. doi: 10. 4161/rna.19317 PMID: 22336761. 97.
Hoiseth SK, Stocker BA. Aromatic-dependent Salmonella Typhimurium are non-
virulent and effective as live vaccines. Nature. 1981; 291(5812):238–9. PMID:
7015147.
91. Tedin K, Blasi U. The RNA chain elongation rate of the lambda late mRNA is
unaffected by high levels of ppGpp in the absence of amino acid starvation. The
Journal of biological chemistry. 1996; 271 (30):17675–86. PMID: 8663373. The
Transcriptome of Intra-Macrophage S. Typhimurium PLOS Pathogens |
DOI:10.1371/journal.ppat.1005262 November 12, 2015 25 / 26
92. Hinton JC, Hautefort I, Eriksson S, Thompson A, Rhen M. Benefits and pitfalls of
using microarrays to monitor bacterial gene expression during infection. Current
opinion in microbiology. 2004; 7(3):277– 82. doi: 10.1016/j.mib.2004.04.009
PMID: 15196496.
93. Hoffmann S, Otto C, Kurtz S, Sharma CM, Khaitovich P, Vogel J, et al. Fast
mapping of short sequences with mismatches, insertions and deletions using index
structures. PLoS computational biology. 2009; 5(9):e1000502. doi:
10.1371/journal.pcbi.1000502 PMID: 19750212; PubMed Central PMCID:
PMC2730575.
94. Osman D, Cavet JS. Metal sensing in Salmonella: implications for pathogenesis.
Advances in microbial physiology. 2011; 58:175–232. doi: 10.1016/B978-0-12-
381043-4.00005–2 PMID: 21722794.
95. Faucher SP, Porwollik S, Dozois CM, McClelland M, Daigle F. Transcriptome of
Salmonella enterica serovar Typhi within macrophages revealed through the
selective capture of transcribed sequences. Proceedings of the National Academy
of Sciences of the United States of America. 2006; 103 (6):1906–11. doi:
10.1073/pnas.0509183103 PMID: 16443683; PubMed Central PMCID:
PMC1413645.
96. Chilcott GS, Hughes KT. Coupling of flagellar gene expression to flagellar
assembly in Salmonella enterica serovar typhimurium and Escherichia coli.
Microbiology and molecular biology reviews: MMBR. 2000; 64(4):694–708.
PMID: 11104815; PubMed Central PMCID: PMC99010.
100
97. Saini S, Slauch JM, Aldridge PD, Rao CV. Role of cross talk in regulating the
dynamic expression of the flagellar Salmonella pathogenicity island 1 and type 1
fimbrial genes. Journal of bacteriology. 2010; 192(21):5767–77. doi:
10.1128/JB.00624-10 PMID:20833811; PubMed Central PMCID: PMC2953706.
98. GOST 7269-91 «Meat. Methods of sampling and organoleptic methods of
freshness test».
99. GOST 29112-91 «Solid culture media (for veterinary purposes). Characteristics» .
100. MU 4.2.2723-10 Laboratory diagnostics of salmonellosis, detection of
salmonella in food and environmental objects (Methodical instructions of the
Foundation of the Central Research Institute of Epidemiology of Russian
consumer supervision (S.Sh.Rozhnova, A.T.Podkolzin).
101. Ашмарин И. П., Воробьев А. А. Статистические методы в
микробиологических исследованиях // Медгиз. – 1962. - 162 с.
102. Методические указания по применению статистических методов в
эпизоотологии / Р.Ф. Сосов, А.А. Глушков. — М., 1974. — 68 с.
103. A.Y. Zholdasbekova, B.K. Biyashev, K.B. Biyashev, Sarybaeva D.A.
Koshkimbaev S.S., Zhumanov K.T. «Prevalence of enteric infection pathogens in
young cattle in Kazakhstan», Modern science, International scientific journal №4,
2018, Vol.I, pages 20-21, Moscow 2018.
104. Жолдасбекова А.Е., Бияшев К.Б., Бияшев Б.К., Сарыбаева Д.А.,
Нургожаева Г.М. «Ауырған, өлген және дені сау жаңа туылған бұзаулардан
бөлінген Salmonella өсінділерінің зардаптылық қасиетін зерттеу». Материал
ХХХ Международной научно-практической интернет –конференции
«Проблемы и перспективы развития науки в начале третьего тысячелетия в
странах Европы и Азии». Украина, 2016, стр 10-12.
105. A.Y. Zholdasbekova, K.B.Biyashev,B. K. Biyashev,D.A.
Sarybaeva,K.T.Zhumanov. «Method for producing attenuated Salmonella strain.»
Journal of Pharmaceutical Sciences and Research. India, Vol. 10(1), 2017, рages
162-163. Scopus.
106. Жолдасбекова А.Е., Бияшев К.Б., Бияшев Б.К.,Сарыбаева Д.А.
«Аттенуирленген Salmonella dublin 31 штамының қасиеттерін зерттеу ».
Ғылым жаршысы №4, Казахского агротехнического университета, Астана,
2017, стр. 57-63.
107. A.Y. Zholdasbekova , K.B. Biyashev,B.K. Biyashev,Zh.S.
Kirkimbaeva,Valdovska A. «Control of the stability of the residual virulence of
the attenuated strain Salmonella dublin 31» Известия НАН РК. Серия аграрных
наук № 3, 2018 г.
108. Жолдасбекова А.Е.,Бияшев К.Б., Бияшев Б.К.,Ермагамбетова С.Е.
«Изучение иммуногенных свойств аттенуированного штамма Salmonella
dublin 31». Материалы XXXVI международной научно-практической
101
конференции «Современные проблемы гуманитарных и естественных наук»,
Москва, 2017, стр. 129-134.
109. Бияшев К.Б., Бияшев Б.К., Макбуз А.Ж. Патент на изобретение №
32022 «Штамм бактерий Salmonella dublin 31, используемый для
приоготовления живой вакцины против сальмонеллеза крупного рогатого
скота» . Дата 31.03.2017 г.
110. Жолдасбекова А.Е., Бияшев Б.К., Макбуз А.Ж., Кошкимбаев
С.С.,Мырзекеева А.А. «Способ изготовления живой вакцины против
сальмонеллеза крупно рогатого скота».Материалы XXXVII международной
научно-практической конференции «Современные проблемы гуманитарных
и естественных наук», Москва, 2017, стр. 78-81.
111. Жолдасбекова А.Е., Бияшев К.Б., Бияшев Б.К.,Сарыбаева Д.А.
«Производственные испытания вакцины из аттенуированного штамма
Salmonella dublin 31». Исследования, результаты №1 (77) 2018, Алматы, стр
395-399.
112. Жолдасбекова А.Е., Бияшев К.Б., Бияшев Б.К., Киркимбаева Ж.С.,
Ермагамбетова С.Е., Сарыбаева Д.А. Заявка на выдаче патента на
изобретение «Способ профилактики сальмонеллеза крупного рогатого скота
вакциной из штамма Salmonella dublin 31 ».
102
Annexes 1
103
104
105
106
Annexes 2
107
108
109
Annexes 3
110
111
Annexes 4
112
113
114
115
Annexes 5
116
117
Annexes 6
118
Annexes 7
119
Annexes 8
120
Annexes 9
121
Annexes 10
122
Annexes 11
123
Annexes 12
124
125
Annexes 13
126
127
128
129
130
Annexes 14
Figure 19 - Internship at the Latvian University of Agriculture in 2016. Seeding on
Endo's medium.
Figure 20 - Reading the results of the PCR study.