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AGRITECH-III-2020
IOP Conf. Series: Earth and Environmental Science 548 (2020) 022073
IOP Publishing
doi:10.1088/1755-1315/548/2/022073
1
Improvement of allergic diagnostics of animals under the
conditions of Uzbekistan
A D Ulugmuradov, A A Saidov and M A Ruzimurodov
Research Institute of Veterinary Medicine, Samarkand, Uzbekistan
E-mail: nivi@vetgov.uz
Abstract
. This article presents preliminary results of our own scientific and experimental studies
of specificity, antigenic activity, safety, reactogenicity, and other properties of various allergic
diagnostic tests made from strains of brucella with different species and virulent affiliation as
the expected standards of industrial crops.
As a result of the studies, Uzbek analogues of
the standards of production strains of brucella strains intended for the manufacture of
allergy diagnostics have been developed.
1.
Introduction
Brucellosis (Brucellosis) - mainly a chronic disease of animals caused by bacteria of the genus
“Brucella” which belongs to the 2nd group in terms of infection and is a very dangerous infectious and
allergic disease for people. The specific clinical signs of animal brucellosis are characterized by massive
abortions, infertility, decreased fetal viability and animal productivity.
At present, the epizootic and epidemiological situation of brucellosis in Uzbekistan is not entirely
favorable, since it is determined by the presence of brucellosis among farm animals, which are the main
causes of brucellosis in humans.
Sporadically, brucellosis is registered everywhere in the Republic. On average, the prevalence in the
republic is among cattle (cows) - 0.01%, small cattle - 0.04%. This trend has persisted over the past 10
years.
Nowadays, the country's existing complex of organizational, economic, veterinary, sanitary and
general preventive measures are expensive, time-consuming and not always effective because they are
based on measures intended for large livestock farms complexes, etc. In Uzbekistan, the method of
introducing livestock is different in the number of livestock, mainly it is found in private farmsteads of
dekhkans and farms, where grazing is predominant.
In the economically developed United States, Germany, the Netherlands, Denmark, China, Russia,
South Korea, Turkey, and other countries, animal brucellosis also continues to be registered with
different levels of intensity, although these countries are mainly farmers and farms, however, it is mainly
found in complexes where stall content prevails.
Therefore, brucellosis in all countries is perceived as a global problem where control and prevention
are the basis of the fight against this disease.
For this purpose, in Uzbekistan and around the world it is recommended to widely use serological
diagnostic methods: RBP - rose bengal test, KR - ring reaction with milk, RA - agglutination reaction,
RSK - complement fixation reaction, RDSK - long-term complement fixation reaction (for infectious
sheep epididymitis ), ELISA - enzyme immunoassay, PCR - polymerase chain reaction, and along with
AGRITECH-III-2020
IOP Conf. Series: Earth and Environmental Science 548 (2020) 022073
IOP Publishing
doi:10.1088/1755-1315/548/2/022073
2
them - allergic diagnostics. All of these methods are recommended by the FAO / WHO and OIE for the
diagnosis of brucellosis. (Terrestrial Animal Health Code, 2018).
Recently, the vaccination of animals has become the basis of the strategy for combating brucellosis
in many countries of the world. This method has proven itself in many European countries, where the
cases of the isolation of brucellosis animals are minimized and in some countries it was completely
possible to eliminate this disease among cattle and small cattle (USA, France, Germany, Holland, etc.).
Vaccination of animals against brucellosis requires a strict attitude to the issues of accounting,
livestock migration, compensation to animal owners in case of detection of cattle infected with
brucellosis, timely diagnostic tests, the competence of specialist laboratories and other issues.
In Uzbekistan, over the past 20 years, an allergic diagnosis of brucellosis has not been used. Although
this method is most acceptable as an additional test for farms where vaccination is not used and is very
convenient for distant cattle breeding.
The allergic method is convenient, simple and reliable in performance and affordable for practical
veterinary medicine.
A lot of research has been done in the world on the creation and use of allergens. One of the most
deserved is the creation in 1968 by E.S. Orlov and A.N.Kasyanovs from a non-agglutinogenic strain -
brucellin VIEV, which is still used in the diagnosis of brucellosis in sheep, goats, pigs and deer.
It is known that the allergic state of a sick animal occurs after the formation of specific antibodies in the
div and persists for a long time, that is, up to 16 months (P.P. Samoilov). Although in the literature
there are other works on brucellosis allergen intended for the diagnosis of brucellosis.
Thus, the issues of studying allergic diagnostics are relevant and promising.
In Uzbekistan, allergic diagnostics is included in the general complex of a scientifically based system
for the diagnosis of brucellosis used in both prophylactic and health-improving measures for brucellosis,
goats and pigs. However, due to the fact that these allergens are not produced in the country and there
are no commercial allergens, allergy testing is not conducted.
The aim of the research was the following:
•
Comparative study of the cultural, morphological, biochemical, virulent and other properties of
the local epizootic and selection vaccine strains of Br.abortus 104M UZ, 2017/1 UZ and
Br.melitenzis Rev-1 UZ, 9 UZ from the collection of the NIIV museum in comparison with the
reference strains Br.abortus 544, Br.melitenzis 16M and Br.suis 1330 with the aim of selecting
the most suitable for the manufacture of anti-brucellosis allergens.
•
from the selected brucella strains to make experimental microseries of anti-brucellosis allergens
using different methods and conduct a comparative study of the activity, sensitivity, sensitizing
properties, safety, reactogenicity, agglutinogenicity in experiments on laboratory animals
(guinea pigs, white mice, sheep).
Materials and methods. All selected strains of Br.abortus 104M UZ, 2017/1 UZ and Br.melitenzis
Rev-1 UZ, 9 UZ, as well as three reference Br.abortus 544, Br.melitenzis Novocherkassk-102 (VGNKI)
and Br.suis 1330 adapted to the nutrient medium MPPGGA (meat-peptone hepatic glucose-glycerin
agar) by three reseeding of isolated colonies and growing in an incubator for 72 hours. After obtaining
pure cultures, each strain was seeded with 5 matry flasks containing MPPGBH on medium with
MPPHGB (meat-peptone hepatic glucose-glycerin broth). All flasks with a nutrient medium were placed
in a thermostat for further incubation at 37 ° C. The terms of incubation were different and ranged from
10 to 20 days with the aim of maximum accumulation of waste products.
After a certain time, a nutrient medium containing brucella and their decay products was taken from
the kolbot and subjected to various treatment methods (heating to 95-1000С for 1-2 hours, heating in
the same mode in the presence of 1% phenolized saline solution, freezing and thawing after inactivation
at 95-1000С and other methods.). The growth characteristic of brucella was monitored visually. The
obtained concentrates of allergens manufactured by different methods were centrifuged at 5 thousand
rpm 1 fraction and 10 thousand rpm. in an hour. The precipitated bacterial mass was not used further in
AGRITECH-III-2020
IOP Conf. Series: Earth and Environmental Science 548 (2020) 022073
IOP Publishing
doi:10.1088/1755-1315/548/2/022073
3
the production of allergens. The rest of the allergen concentrates containing brucellin extracts were
preserved with phenol until a final dilution of 0.3% was obtained. The obtained allergen samples with
1% phenol were resuspended with saline to obtain a final phenol concentration of 0.3% in the extract.
All obtained samples of allergens were placed in the refrigerator for 15-20 days to stabilize.
After stabilization, a quantitative determination of the protein was carried out by the nephelometric
method.
The purity of the prepared allergens was checked by microscopic methods using Gram stain and
Kozlovsky stain.
The purity of growth was determined by seeding on MPPGGA and MPPGB. The pH density was
determined by potentiometric methods and the pH was adjusted to 7.0-7.2 by alkalization with a 4%
sodium hydroxide solution.
Research results. The cultural, morphological biochemical, and other properties of the local epizootic
and selection vaccine strains of Br.abortus 104M UZ, 2017/1 UZ and Br.melitenzis Rev-1 UZ, 9 UZ
were tested in comparison with the reference cultures of Br.abortus 544, Br.melitenzis Novocherkassk-
102 (VGNKI) and Br.suis 1330 in accordance with the methods recommended by the FAO / WHO for
working with brucella cultures.
The affiliation of brucella cultures of one kind or another was studied by the ability to form hydrogen
sulfide (H2S) with lead acetic acid, the tinctorial properties of sensitivity to basic fuchsin and thionine
when it was contained in various concentrations in a nutrient medium (1: 25000, 1: 50000, 1: 100000 )
The virulent properties of the studied cultures will be evaluated by growth and sensitivity to various
concentrations of penicillin in the nutrient medium (0.5, 5, 10, 50 IU / ml) and erythritol.
It was found that all the studied strains have characteristic species properties according to these
indicators. The strain Br. abortus 2017/1 actively produced hydrogen sulfide, and the average total
indicators of the size of the blackening of the indicator paper was more than 12 mm (counting was
carried out every two days).
Strains Br. abortus104M UZ and Br. melitenzis Rev-1 UZ, 9 UZ did not produce hydrogen sulfide.
For the strain Br. abortus 104M UZ, this trait is characteristic and serves as a marker that allows you to
identify it as a biotype -6. The results obtained using the reference strains Br. abortus 544, Br. melitenzis
Novocherkassk-102 (VGNKI) and Br. suis 1330 also indicate their compliance with the passport data.
When studying the virulent properties of cultures in preliminary tests for growth and sensitivity to
various concentrations of penicillin in a nutrient medium (0.5, 5, 10, 50 IU / ml) and erythritol, it was
found that cultures of field strains of Br.abortus 2017/1 Br.melitenzis 9 UZ are not sensitive to this
antibiotic at its concentration in MPPGA 50 U / ml, while vaccine strains of the strain Br.abortus 104M
UZ and Br.melitenzis Rev-1 UZ, to concentrations of 5 and 10 U / ml. were sensitive and did not grow
on these media. All cultures of reference strains (Br. Abortus 544, Br. Melitensis Novocherkask-102,
and Br. Suis 1330) were also not sensitive to penicillin and grew well when it was kept in MPPGA at a
concentration of 50 U / ml. On a medium with erythritol (1: 1000), all cultures of the studied field strains
grew well in a sowing dose of 100 microns. cells / ml Reference strains of Br. abortus 544, br. Melitensis
Novocherkask-102, and Br. suis 1330 were also not sensitive to erythritol at a dose of 100 μl / ml i.e.
observed their rapid growth.
The vaccine strains of Br.abortus 104M UZ and Br.melitenzis Rev-1 UZ did not grow on the medium
containing erythritol when sowing 100 μl, but single colonies were observed when the strain of Br
Melitensis Rev-1 was inoculated with a dose of 1 million μl. class This indicates its higher virulence
compared with the culture of the species Br.abortus 104M UZ. Thus, despite the long-term storage of
museum and epizootic strains of brucella at MPPHA and the periodicity of their reseeding for a long
time, they all retained their main species and type cultural morphological, biochemical, virulent and
other properties and remain stable and epizootically significant.
The agglutinable properties of the prepared antigens from each strain separately were studied in the
classical agglutination reaction to the limit titer. It was found that the titers of detectable antibodies with
antigens from field strains were lower than the titers in the same sera with antigens from vaccine strains
of brucella.
AGRITECH-III-2020
IOP Conf. Series: Earth and Environmental Science 548 (2020) 022073
IOP Publishing
doi:10.1088/1755-1315/548/2/022073
4
Results of agglutinable, virulent and biochemical properties of epizootic strains of Br. abortus 1 /
2017UZ and Br. melitensis No. 9 allows using them as domestic reference preparations along with
commercial reference strains of Br. Abortus 544 (Weybridge), Br. Melitensis Novocherkassk-102
(VGNKI).
The stability, variety and degree of dissociation of colonies of epizootic cultures of brucella was
studied using the thermoagglutination reaction, the use of acryllavine, S and R positive sera and the
White-Wilson method using a crystal violet in a working dilution of 1: 2000. At the same time, the
stability of breeding colonies with clearly expressed S and R brucella forms were studied by passage
multiplicities and storage periods on nutrient media.
All selected strains of the species Br.abortus 104M UZ, 2017/1 UZ and Br.melitenzis Rev-1 UZ, 9
UZ, according to the results of preliminary studies (2018-2020) were deposited in a unique collection
of animal microorganisms of the NIIV with passports in the established form, included to the depository
list and intended for the manufacture and study of them as domestic anti-brucellosis vaccines and
diagnosticums.
The protein content in the extracts of the experimental series ranged from 18-27 mg%.
The safety test was carried out on healthy white mice weighing 18-22 g. Three heads of white mice
were taken for each microseries. Allergens were injected into the back area in a volume of 0.5 ml and
were observed for ten days. During the observation period, all experimental mice remained alive and
deviations from physiological norms were not observed.
The specificity of the tested allergens was studied on guinea pigs weighing 300-350 g. By which the
drug was injected into the depilated area of the lateral torso intradermally in a volume of 0.1 ml.
Compared to commercial brucellin containing 200 activity units (EA). As a result of the inactivation of
the experimental microseries of allergens made by different methods, after 24-48 hours, the
characteristic diffuse inflammatory processes, redness, and significant swelling were not established,
which testified to the specificity of all tested allergen samples.
The specificity test and the absence of agglutinogenic properties of the experimental allergen
microseries on healthy 15 heads of Karakul sheep when introduced under the skin in a dose of 0.5 ml
(palpebrally), taking into account the results of allergic reactions after 24 hours (primary) and 48 hours
(finally), also indicates the high specificity of the tested brucellodiagnostics. The study of agglutinogenic
properties 10-15-20 days after the introduction of allergens to these 15 sheep heads indicates the absence
of anti-brucellosis antibodies in the blood during the study of blood clots in the Rose-Bengal test and
the agglutination reaction with brucellosis antigens.
The reactogenic properties of allergens were determined during a 5-day control, at which the
following results were obtained: the sheep were kept together with free watering and feeding, while the
average daily div temperature was ∑X-39.0 ° C using an allergen made from strain 104MUZ. When
using allergens from strains Rev-1UZ, 1 / 2017UZ, 9UZ, no changes in the physiological state of the
sheep were also observed, and the average div temperature was -X-39.80С, ∑Х-39.40 ° С and ∑Х-
39, respectively. 60C.
Currently, studies on the properties of allergens made from strains of brucella with various species
and virulent accessories and made by various methods are ongoing.
2. Conclusion
As a result of the studies, Uzbek analogues of the standards of production strains of brucella strains
intended for the manufacture of allergy diagnostics have been developed.
It has been established that all microseries of prototypes of antigens produced from local strains
Br. abortus 104MUZ, 1/2017UZ and Br. melitenzis Rev-1UZ and 9UZ have sufficient specificity,
safety, arereactogenicity against anti-brucellosis allergic diagnostics.
It was established that all tested allergens from the strains Br.abortus 104MUZ, 1 / 2017UZ and
Br.melitenzis Rev-1UZ and 9UZ did not cause the production of specific anti-brucellosis antibodies
detected in the Rose-Bengal test and agglutination reaction with brucellosis antigens.
AGRITECH-III-2020
IOP Conf. Series: Earth and Environmental Science 548 (2020) 022073
IOP Publishing
doi:10.1088/1755-1315/548/2/022073
5
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