Immunodiffusion Reactions Research Articles

Оценка антигенной активности вакцины против инфекционных маститов и эндометритов коров инактивированной

The article presents the results of a study of the antigenic activity of components in experimental series of vaccines against infectious mastitis and endometritis of cows inactivated using the double immunodiffusion reaction according to Ukhterloni. As a result of the research, it was found that when setting up the diffusion precipitation reaction, a clearly defined precipitation line between the hyperimmune serum and the studied drug variants is formed when using experimental vaccine series with a concentration of each antigen of 3.5 billion/cm3 and 5.0 billion/ cm3. At the same time, the reaction between wells containing the antigen at concentrations of 1.0 and 2.0 billion/cm3 is practically absent, or is weakly expressed in individual antigens. In all cases of the reaction, there was a pronounced reaction between the hyperimmune serum and the positive control, or the absence of precipitation lines between the hyperimmune serum and the negative control. It was also found that the concentration of 3.5 billion /cm3 of each strain included in the vaccine is optimal. When the concentration increases to 5.0 billion / cm3, the positive effect does not become more pronounced, but, on the contrary, affects the animal's immune system unfavorably, which causes a decrease in antibody production.

Characteristics of Stenotrophomonas rhizophila Lipopolysaccharide, a Representative of Antarctica

Characteristics of Stenotrophomonas rhizophila Lipopolysaccharide, a Representative of Antarctica

Study of diagnostic value of the reaction of indirect hemaglutination in comparison with enzyme immune assay and other serological tests at cattle brucellosis

The results of comparative brucellosis diagnostic studies of blood sera from cattle at farms with different epizootic situations, performed in 2021 in the laboratory of the Caspian Zonal Research Veterinary Institute – a branch of the Dagestan Agriculture Science Centre, are presented in the paper. The diagnostic efficiency of the indirect hemagglutination test (IHA) was studied in comparison with enzyme immunoassay (ELISA), agglutination test (RA), complement fixation test (CFR), rose-bengal test (RBP) and immunodiffusion reaction with O-PS antigen (RID with O-PS antigen). The specificity of these serological tests was confirmed by the negative results of studies of 40 samples of blood serum of non-vaccinated against brucellosis animals at a brucellosis-safe farm. The sensitivity of the tested diagnostic tools was determined using 46 blood serum samples from cows immunized with the B. abortus 82 vaccine at a brucellosis-prone farm. According to the results obtained, in particular, according to a significant percentage of animals positively reacting to brucellosis in RID with O-PS antigen and high titers of RNHA, RA and RSK, it can be stated that the infection in the herd proceeds in an acute form. Thus, positive results were obtained in RNGA in the study of 18 (39.1%), ELISA (LLC NPF “Sibbiotest”) – 29 (63 %), in RSK – 18 (39.1 %), in RA – 15 (32.6 %), RBP – 15 (32.6 %) and RID with O-PS antigen – 7 (15.2 %) blood serum samples. The conducted studies have shown that RNHA is one of the most sensitive tests, which makes it possible to detect a high percentage of animals with brucellosis at a brucellosis-prone farm (39.1% of those studied) in the early stages after infection and surpasses the results of most other serological methods. The results of comparative serological studies of blood serum samples of cattle in RNHA, IEA, RSK, RA, RBP and RID with O-PS confirmed the specificity of the tests and the high sensitivity of RNHA with brucellosis erythrocyte antigen.

Characterization of Azospirillum brasilense Lipopolysaccharides

Azospirillum brasilense is a gram-negative, nitrogen-fixing bacterium that colonizes the rhizosphere of various types of grasses and cereals. Lipopolysaccharides (LPS) are a class of complex glycolipids present in the cell membrane of gramnegative bacteria and mediate plant-bacteria interactions. Although the effects of LPS of pathogenic plant bacteria on the induction of plant defense mechanisms have been characterized, the role of LPS of beneficial rhizobacteria on plant growth is less clear. Therefore, a very important point is the study of the chemical, biological, and functional activities of A. brasilense LPS, which was the aim of this work. Methods. A. brasilense LPSs were isolated from dry bacterial mass by the phenol-water method. The carbohydrates were analyzed by the Dubois method, nucleic acids — by Spirin, protein content — by Lowry and 2-keto-3-deoxyoctonic acid (KDO) — by Osborn. Pyrogenicity of LPS was tested observing the rules of bioethics in rabbits. Serological studies were performed by the Ouchterlony method. The identification of monosaccharides and fatty acids in LPS preparations was carried out on an Agilent 6890N/5973 inert chromatomass spectrometry system. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAAG electrophoresis) was performed according to Laemmli. Results. LPS of 3 strains of A. brasilense were isolated from dry bacterial mass and purifi ed from nucleic acids by ultracentrifugation. The purified LPSs were characterized by different relative yields from 2.44% to 4.75%, which is slightly higher than other strains of the A. brasilense (1—3%). The studied preparations were characterized by a rather high content of carbohydrates from 50.1% to 72.1%. All LPS contained up to 0.17% KDO, which is a specific component of the LPS of gram-negative bacteria. Analysis of the monosaccharide composition indicates that the LPSs of the studied A. brasilense strains turned out to be heterogeneous. At the same time, such monosaccharides as mannose, galactose, glucose, and heptose were recorded in the LPS of all tested strains. The study of the fatty acid composition of LPS showed the presence of fatty acids containing from 14 to 18 carbon atoms. Нydroxylated, saturated, and monounsaturated acids and their cis isomers were found. In the investigated LPS, the dominant fatty acids were 16:0, 18:1, 14:0(3-OH), and 16:0(3-OH), which coincides with the literature data. The research of the pyrogenic effect of LPS of A. brasilense studied strains showed that LPS solutions are apyrogenic. The double immunodiffusion reaction in Ouchterlon agar showed that all tested LPS in homologous systems exhibited ancultitigenic activity. Serological cross-reactions can be used as an approach in classifying different bacteria. Thus, we found that antisera to A. brasilense 18-2 and 61 react with all LPSs of the studied strains, which may indicate the presence of common antigenic determinants in them and that these strains belong to the same serogroup. The electrophoretic distribution data indicate that A. brasilense produces S-forms of LPS which differ in the length of O-specifi c polysaccharide chains. Conclusions. For the first time, LPS were isolated from cells of A. brasilense 10/1, 18-2 and 61. A characteristic feature of these LPS is their heterogeneity in monosaccharide and fatty acid composition, all of them were apyrogenic. The results obtained during biological-functional studies of three strains of A. brasilense LPS contribute to the biological characteristics of this species.

Значение иммуноферментного анализа в серологической диагностике бруцеллеза крупного рогатого скота

For the serological diagnosis of brucellosis in cattle in veterinary laboratories of the Russian Federation, one or several tests are used simultaneously. The sensitivity and specificity of the determination of antibodies to the pathogens of this disease using various methods varies significantly, none of the serological tests is suitable for all stages of the infectious process in brucellosis. ELISA is a modern serological test that has not found wide application in the state veterinary laboratories of the Russian Federation, although the number of studies conducted by this method increases annually. The use of enzyme-linked immunosorbent assay makes it possible to confirm the diagnosis of brucellosis at all stages of the infectious process and to identify more seropositive animals than classical serological methods. Among serological methods, enzyme-linked immunosorbent assay (hereinafter referred to as ELISA) is the most sensitive, specific and easy-to-perform test. In accordance with the Order of the Ministry of Agriculture of the Russian Federation dated 08.09.2020 No. 533 «On approval of Veterinary rules for the implementation of preventive, diagnostic, restrictive and other measures, the establishment and cancellation of quarantine and other restrictions aimed at preventing the spread and elimination of foci of brucellosis (including infectious epididymitis of sheep)» the ELISA method is one of the confirmatory in the diagnosis of brucellosis. The data obtained as a result of the studies indicates that the results of ELISA coincide with the complex of classical serological methods (BRS – Bengal roses sample; AR – agglutination reaction; CFR – complement fixation reaction; RIH – reaction of indirect hemaggtination; IDR – immunodiffusion reaction) and a higher diagnostic sensitivity of the test.

ДИАГНОСТИЧЕСКОЕ ЗНАЧЕНИЕ РЕАКЦИИ НЕПРЯМОЙ ГЕМАГГЛЮТИНАЦИИ (РНГА) В СРАВНЕНИИ С ИФА И ДРУГИМИ СЕРОЛОГИЧЕСКИМИ ТЕСТАМИ ПРИ ДИАГНОСТИКЕ БРУЦЕЛЛЕЗА КРУПНОГО РОГАТОГО СКОТА

The results of comparative diagnostic studies of blood sera from cattle for brucellosis from farms with different epizootic situations, performed in the laboratory of the Caspian zonal NIVI - a branch of the FGBNU "FANC RD" and the State Budgetary Institution "Kropotkin Regional Veterinary Laboratory", are presented. The diagnostic efficiency of the indirect hemagglutination test (IHA) with the use of erythrocyte antigen was studied in comparison with other serological methods, such as enzyme immunoassay (ELISA), agglutination test (RA), complement agglutination test (RSC), rose bengal test (RBP), immunodiffusion reaction with O-PS antigen (RID with O-PS antigen). In order to determine the specificity of serological tests, 52 blood serum samples from non-vaccinated animals from a safe farm were examined for brucellosis in RNGA, ELISA, RA, RSK, RBP and RID with O-PS antigen. All blood serum samples were tested with negative results. The sensitivity of the tested diagnostic tools was determined using 82 blood serum samples from cows immunized with the B. abortus 82 vaccine from a brucellosis-prone farm. The results of the studies showed that the infection in the herd proceeds in an acute form, as evidenced by a significant percentage of positively reacting to brucellosis of animals in the RID and high titers of RNHA, RA and RSK. Thus, a positive RNHA was obtained in 20 cases (24.4%), ELISA with the test system of LLC SPF "Sibbiotest" revealed 31 sick animals (37.8%), RSK - 20 (24.4%), RA and RBP - 17 (20.7%), ELISA test system from Biok - 15 (18.3%), RID with O-PS antigen - 9 (11%). It is important to note that the RNGA additionally identified animals that respond positively to brucellosis: to ELISA "Biok" - 5 (6.1%), RA and RBP - 3 (3.7%), RID - 11 (13.4%). The results of our studies have shown that the RNGA method is one of the most sensitive tests that allows to identify a high percentage of animals with brucellosis in a dysfunctional farm (24.4% of the studied ones), which is superior to the results of other serological methods. The results of comparative serological studies of bovine blood sera in RNHA, ELISA, RA, RSK, RBP and RID with O-PS confirmed the specificity of the tests and the high sensitivity of the RNHA method with brucellosis erythrocyte antigen.

Immune Repertoire of Sheep Blood B-Cells in the Postvaccination Immune Response

The main specific functions of B-cells are carried out with the help of membrane receptors. Binding of the receptor to its ligand activates a cascade of reactions leading to the formation of memory cells and protective antibodies. The study was conducted to analyze the phenotypic profile of sheep blood B-cells in the process of postvaccination immune response to an inactivated vaccine against pathogens of acute intestinal diseases of animals. Romanov sheep at the age of 1.5 years were immunized twice with a 2-week interval. Studies were conducted before vaccination and on the seventh, 14th, and 21 days of the immune response. The phenotype of B-cells was determined by immunoperoxidase staining using monoclonal antibodies to CD receptors. A simple radial immunodiffusion reaction was used to assess IgG levels. Vaccination of sheep with an inactivated vaccine caused an increase in the level of the general population of leukocytes and a subpopulation of B2 cells (p < 0.05). The absolute number of leukocytes increased on the seventh and 14th days of the primary immune response and on the seventh day of the secondary immune response compared to the initial values. The number of B2 lymphocytes with the CD5 – IgM+ phenotype increased and amounted to 9.0 × 106 cells/mL on day 7 and 11.2 × 106 cells/mL on day 14 of the primary immune response (4.5 × 106 cells/mL before the introduction of the vaccine). An increase in the level of the CD5 – CD19 + IgM + CD20 + lymphocyte subpopulation was noted in the first 2 weeks of the immune response and was 2.5 times higher than the initial value. Data were obtained on the absence of the effect of vaccination on the level of lymphocytes with the CD5 + CD19 + IgM + phenotype (B1-cells), which did not change during either the primary or secondary immune response, which indicates the independence of priming of the two main subpopulations of B-cells. The structural components of the immune system in the process of immunogenesis are not activated simultaneously, and when evaluating the effectiveness of vaccination, functional interrelations of immunological indicators are of particular importance. A strong correlation between the indicators of B2‑cells and the level of total immunoglobulins of class G (r = 0.9) indicates a positive effect of vaccination.

Containment of Cattle Brucellosis Outbreaks With a Small Dose of Vaccine From Strain 82

Improving the system of anti-brucellosis measures is the ultimate goal of all studies devoted to the problem of brucellosis. Cattle brucellosis outbreaks in the brucellosis-free regions in the Russian Federation indicate a premature cessation of immunization procedures and the need for their continuation. Meanwhile, there is an urgent problem of developing anti-brucellosis immunity in the adult cattle: the initial vaccination of cattle with a full dose of the vaccine from strain B. abortus 82 is fraught with post-vaccination abortions. In order to solve this problem, in the production conditions of one of the farms where the current epizootic situation for brucellosis required alternative approaches for containment of a brucellosis outbreak, a small dose of the vaccine was tested on the cattle without specific immunity. This resulted in immunogenicity, loss of abortogenicity and the presence of provocative properties of the vaccine in small doses. This allowed the detection of antibodies in animals with a hidden infection in the early stages after vaccination. Serological methods (AT, RBT and IDR with O-polysaccharide antigen), which are generally accepted in veterinary practice, were used as diagnostic tests. The reimmunization of the cows with a small dose of the vaccine from strain 82 allowed diagnostic tests to be carried out earlier after vaccination than after the use of a full dose of the vaccine, which made it possible to diagnose brucellosis in a short period of time after vaccination. The provocation of immunological reactions in latently sick animals after the vaccination with small doses, and the isolation of those who responded under the condition of supported immunity made it possible to achieve the recovery of farm animals in a short period of time, which was proved by negative indicators of the immunodiffusion reaction with O-polysaccharide antigen.&#x0D; Keywords: brucellosis, small doses of the vaccine, abortogenicity, immunity

Получение и оценка эффективности диагностической агглютинирующей сыворотки для идентификации возбудителя листериоза

Listeriosis is a saprozoonotic natural anthropurgic infectious disease caused by Listeria monocytogenes, with multiple pathways and factors of its transmission, characterized by pronounced clinical polymorphism from carriage and subclinical to severe generalized forms, meningitis and meningoencephalitis, and high mortality among newborn children and individuals with immunodeficiency. Express diagnostics of listeriosis is based on the use of immuno-chemical (immunodiffusion reaction, enzyme immunoassay) and molecular genetic (polymerase chain reaction) methods. In the practice of laboratory diagnostics, serological methods for the study of listeriosis remain in high demand for verifying the clinical diagnosis. One of the specific, reliable and available serological methods for laboratory diagnostics of causative agents of infectious diseases remains the agglutination test (AT), to conduct which, listeria agglutinating serum is required. Obtaining diagnostic agglutinating listeria serum with a high level of diagnostic sensitivity and specificity will allow timely identification of the pathogen in the test tube AT and on glass, based on the specific interaction of the antigen with the anti-body, with the formation of agglutinate visible to the naked eye. In this study a scheme has been developed for immunization of animal producers to obtain diagnostic listeria agglutinat-ing serum. Evaluation of the clinical efficiency of two series of the serum was carried out based on the indices of diagnostic sensitivity with different serovariants of L. monocytogenes and diagnostic specificity with closely related and heterologous strains in the test tube AT and on glass. During the study of 25 strains of L. monocytogenes serogroups I and II (serotypes 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4b, 4c, 7) isolated on the territory of the Russian Federation from various sources (clinical material, food products, wastewater), the serum titer did not differ and amounted to 1:800 in the course of in vitro agglutination reaction; a positive result was obtained on glass in 100% of cases. The assessment of the statistical reliability of the ob-tained test results was at least 89 % with a statistical significance of 95 %, which indicates a high diagnostic efficiency of the serum. In order to establish intra-stage convergence and inter-series reproducibility of studies, two probabilities of binomial distributions were compared. The convergence of staged studies for all positive samples has been proven. Based on the re-sults obtained, listeriosis serum can be used as a medical preparation for in vitro diagnostics of the causative agent of listeriosis.

Analysis of the effectiveness of the use of enzyme-linked immunosorbent assay for the diagnosis of leukemia in cattle during health-improving activities

The aim of the work is to analyze the efficiency of using enzyme-linked immunosorbent assay in the diagnosis of cattle leukemia when carrying out health-improving measures in livestock enterprises that are unfavorable for this disease. Indicators of infection of cattle with leukemia virus on 6 farms of agricultural enterprises of the Tomsk region are presented. For serological diagnostics, the immunodiffusion reaction and enzyme immunoassay were used. It has been established that while carrying out health-improving measures in enterprises unfavorable for leukemia in cattle, the use of enzyme-linked immunosorbent assay for serological diagnosis of the disease enabled to identify a greater number of seropositive animals in comparison with a low-sensitivity immunodiffusion reaction. With a decrease in the herd infection rate to single cases of detection of animals infected with the leukemia virus at the final stages of rehabilitation of the enterprise, the number of additional seropositive animals detected by enzyme immunoassay increases. In the final period of herd recovery, the number of animals with a low level of antibodies to the leukemia virus, inaccessible to detection by immunodiffusion, increases. The use of an expensive and labor-consuming delivery of an enzymelinked immunosorbent assay to detect antibodies to the cattle leukemia virus is advisable at the final stages of an enterprise’s recovery. This enables to identify animals with a low level of antibodies to the leukemia virus, to speed up the negative result of a serological test of the entire herd and to exclude repeated outbreaks of infection of animals with this virus in a rehabilitated enterprise.

Pathogenetic aspects of rabbits’ experimental infection caused by bovine leukemia virus

Bovine leukemia virus (BLV) is an infectious disease of cattle, causing high economic losses worldwide, especially in the field of dairy farming. There is no common vision on the problem of interspecies transmission of BLV. Therefore, a detailed study of the etiologic relationship between leukemia in cattle and other animal species is relevant. Various laboratory animal models provide insight into the pathogenesis of viral infections. The article presents the research results of two series rabbits’ intravenous infection with bovine leukemia virus (BLV) using the culture antigen FLK-BLV and the blood of rabbits with clinical, hematological and immunological signs of viral tumor growth. Blood from all animals was taken from the ear vein after 14, 21, 30 days, and then monthly for six months: to study the morphological parameters of blood and to determine the titer of antibodies to BLV. Blood serum for the presence of antibodies to BLV was examined using a diagnostic kit for the indication of animals infected with the leukemia virus in an immunodiffusion reaction produced by LLC “SRE Veterinary Medicine”, Kharkiv. It was found that the stage of the BLV provirus in the blood leukogram of infected animals was characterized by pronounced lymphocytosis on the 21st day of the experiment. The highest concentration of antibodies to BLV in the blood serum was found on the 90th day after the administration of the virus-containing material, which disappeared from the blood on the 150–180th day after infection. In experimental rabbits, after five months for thirty days, in the absence of antibodies to leukemia in the blood serum, multiple tumors of a dense consistency began to develop throughout the body. Such clinical signs and changes in the of rabbits’ blood of the experimental group are characteristic of serologically positive cows on the hematological development stage of leukemic process and correlate with the results of domestic and foreign authors. The presence of a large number of lymphoblasts, as well as leukolysis cells, in the histological preparation of lymph nodes, lungs, heart and the accumulation of lymphocytes’ immature forms around the interlobular vessels of the liver, which were found in pathohistological studies of the experimental rabbits’ organs, may indicate the development of the leukemia process on early stage in them. The results obtained indicate the ability of BLV to overcome successfully the interspecies barrier upon parenteral ingestion of heterologous individuals from infected lymphocytes and in the form of a culture antigen.

Features of immune regulation and specific sensitization in children of the Northern Siberia

This paper highlights the features of immune regulation and specific hypersensitivity in children residing in the Northern Siberia being exposed to the extreme climatic and exogenous chemical risk factors. An immunological diagnostic examination of 255 children permanently residing in the region of exogenous hapten exposure was conducted. The control group consisted of 130 children residing on the territory not being exposed to exogenous chemical factors. Level of serum class G, A, M immunoglobulins was assessed by using a Mancini radial immunodiffusion reaction. Amount of total serum IgE, neuron-specific enolase, and VEGF was measured by using the ELISA. Changes in level of IgG specific to phenol and copper as well as IgE specific to nickel and benz(a)pyrene were measured by using enzyme-linked allergosorbent test. Peripheral blood lymphocyte subsets were analyzed by using membrane-bound immunofluorescence with a panel of labeled specific monoclonal antibodies. High-quality statistical data analysis was performed by using Microsoft® Office Excel 2003 software as well as Statistica 6.0 application software package (StatSoft, USA). Statistical significance was determined by using Student’s t-test. Relation between signs was estimated by using correlation and regression analysis as well as Fisher’s test. Immunological study demonstrated that immunogram parameters were significantly changed in the observation group by containing increased percentage of CD16+ effector cells absolute and relative CD19+ cell count (p ≤ 0.05), relative and absolute count of CD25+ lymphocytes (p ≤ 0.05) as well as CD95+T cells as compared to the reference values and control group (p ≤ 0.05). Moreover, level of neuron-specific enolase and VEGF (vascular endothelial growth factor) was increased compared to control group (p ≤ 0.05). It points that regulatory mediators potentially mapped to the glia and vascular intima in children from the observation group were extremely activated. The level of both total (total IgE) and specific sensitization was significantly increased by assessing immunoglobulin G specific to phenol as well as immunoglobulin E specific to nickel and benz(a)pyrene (p 0.05). Parameters of programmed cell death were significantly altered that was paralleled with simultaneous activation of general and specific sensitization highlighting features of the immunological status in children examined distinguished by excessive stress of immune regulatory arms in case of combined exposure to climate-geographic as well as exogenous chemical risk factors.

Spread Dynamics of Leucosis in Cattle in Livestock Farms of the Russian Federation for 2000–2018

Leucosis occupies a leading position in the modern nosological structure of cattle infectious diseases. This is due to the high infection of livestock and large economic damage in affected farms [1–3]. Leucosis is a chronic tumor disease caused by an RNA-containing virus of the Retroviridae family. The disease is characterized by uncontrolled reproduction of immature hematopoietic cells [4, 5]. The objective is to assess in dynamics the indicators of cattle infection with the leucosis virus and the incidence of leucosis in farms of all categories of the Russian Federation in the period of 2000–2018. The data of the analysis of the epizootic situation for leucosis in cattle in farms of all categories of the Russian Federation for 2000-2018 are presented. Over this period of time, the number of diagnostic serological studies in the immunodiffusion reaction increased 1.88 times in all subjects. According to the results of epizootological monitoring in farms of all categories of the Russian Federation, the rates of infection of the bovine leucosis virus decreased 1.89 times, the incidence rates decreased 2.02 times. At present, the epizootic situation has improved markedly, but so far there are problems with incomplete release of farms from leucosis in cattle. It is necessary to continue work on the elimination of this dangerous chronic disease in farms of all categories in the territory of the Russian Federation.

Detection of specific antibodies of classes G and M to bovine leukemia virus in the blood serum.

Bovine leukemia is a widespread infection worldwide, the causative agent of which is the bovine leukemia virus (BLV) in structural structure and functional features similar to human T-cell leukemia virus (HTLV-1 and HTLV-2) and It is considered as an actual medical and social problem. The study of the immune response in experimentally infected calves at an early stage of the disease development, synthesis of specific antibodies of classes G and M (IgG and IgM), diagnostic informativeness of detection of IgM in cattle leukemia is relevant and determines the purpose of this study. Samples of blood and serum of cattle: animals experimentally infected with VLCRS, patients with cattle leukemia; control negative; specific to heterologous pathogens of cattle diseases. Indirect and sandwich variant enzyme-linked immunosorbent assay (ELISA); commercial ELISA kits (IDEXX, USA; Hema LLC, FKP Kursk Biofactory Firm BIOK, Russia) for the detection of specific IgG and IgM for BLV in the agar gel immunodiffusion reaction (RID). The humoral immune response develops shortly after infection - by 1-8 weeks. IgM are detected starting from the 3rd day, and IgG from the 7th day after infection. Up to 97% of coincidence of positive results in RID and indirect variant of TF ELISA based on monoclonal antibodies to cattle IgM (IgMbovine) were found. The dynamics of the synthesis of antibodies of classes M and G to the glycoprotein gp 51 BLV has a dosedependent wave-like character, is consistent with the levels of increase / decrease in the absolute and relative number of leukocytes / blood lymphocytes of infected calves. Serum specific IgM was detected starting 3 days after infection with BLV. Early detection of IgM in serum of cattle can be used as an additional test for the detection of sick animals.

INFLUENCE OF INTERFERON INDUCER (CYCLOPHERON) ON NONSPECIFIC AND HUMORAL IMMUNITY FACTORS IN BRONCHIAL ASTHMA IN CHILDREN

Bronchial asthma (BA) remains one of the most pressing problems in modern pediatrics. The incidence of this pathology is increasing annually and, according to WHO, about 300 million people in the world suffer from asthma. Despite the recent advances in the diagnosis and treatment of asthma, complete control over the course of asthma has failed. The objective of the workwas to evaluate the effect of immunomodulatory therapy on nonspecific and humoral immunity in children with BA by including interferon inducer (cycloferon) in a standard base therapy. There were 120 children with bronchial asthma aged 5 to 14 years, with an average age of (11.6 ± 1.5) years. To determine the diagnosis and effectiveness of therapy used the international classification of diseases of the 10th examination, protocol for the diagnosis and treatment of bronchial asthma in children (order of the Ministry of Health of Ukraine № 767 from 27.12.2005), GINA 2011. The main group (n = 60) included children, who, in addition to basic therapy, received in addition interferon inducer (cycloferon) according to the scheme: 150 mg on 1-, 2-, 4-, 6- and 8-th day of therapy (No. 5) and then 150 mg after 72 hours. (No. 5) (1500 mg total). The comparison group included children (n = 60) who received only basic therapy, depending on the severity of the disease. The control group consisted of 25 healthy children. The level of serum interferon, virus-induced production of interferon-a (IFN-a), mitogen-stimulated production of interferon-g (IFN-g), the level of total serum IgE were determined by the enzyme-linked immunosorbent assay. The phagocytic activity of neutrophils (NG) was evaluated. The circulating immune complexes (CECs), immunoglobulins (Ig) A, M and G in serum were determined by the immunodiffusion reaction method. It was found that the additional use of cycloferon had a corrective effect on phagocytes, which led to an increase in the synthesis of IFN-a and IFN-g, a decrease in the severity of the allergic component due to the decrease in IgE and circulating immune complexes and a normalization of the synthesis of IgA, IgM, IgG with further development of long-term clinical remission of the disease.

Investigating passive immunodiffusion reactions in the channels of a microfluidic device

The passive immunodiffusion reaction in a microfluidic system is investigated via the interaction between fluorescein human albumin conjugate and monoclonal antibodies. The topology of a microfluidic device suitable for this reaction was designed, and the device was fabricated using LIGA technology. A system for transitioning from the macro- to the microlevel is developed and produced. The dissociation constants and specificity of interaction in complexes formed as a result of the reaction between fluorescein human albumin conjugates and monoclonal antibodies are determined. The results from our study reveal that systems based on combinations of passive immunodiffusion reactions and microfluidic technologies are applicable for detecting antigens and antibodies in the analyzed fluids.

Cell-free antigens from precocious Paracoccidioides brasiliensis culture induce a typical delayed-type hypersensitivity reaction

Cell-free antigens (CFAg) derived from Paracoccidioides brasiliensis have typically been used in immunodiffusion reactions for serodiagnosis or therapeutic follow-up of paracoccidioidomycosis patients. Thus, we investigated the usefulness of CFAg obtained from cultures at different ages, to evaluate cellular immunity by the footpad test, in experimental murine paracoccidioidomycosis. Male mice infected with P. brasiliensis 265 strain were challenged in the footpad with CFAg obtained from four- (4d CFAg) or 11-day-old cultures (11d CFAg). The increase in footpad swelling provoked by 4d CFAg and 11d CFAg was similar and showed significant difference in relation to control groups. However, the infiltrate pattern was strikingly different: 4d CFAg induced a predominant mononuclear infiltrate whereas 11d CFAg provoked a predominant polymophonuclear infiltrate. These different inflammatory patterns were associated with distinct electrophoretic characteristics. By comparison with 11d CFAg, 4d CFAg showed more numerous and intense bands, including a strong one of 43 kDa (gp43). These results suggest that CFAg derived from Pb 265 isolate can be used as a reagent to evaluate cellular immunity; however, the culture's age is critical because only young cultures are able to induce a typical mononuclear infiltrate. The efficacy of this new paracoccidioidin to assay the cellular immunity in infections caused by other P. brasiliensis isolates is under investigation.

Influence of Ionizing Radiation on Echis pyramidium Snake Venom: Biochemical and Immunological Aspects

The effect of a single LD50 dose of native Echis pyramidum venom (27.69μg/mouse) on the activities of certain serum enzymes levels: aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), urea, creatinine, lactate dehydrogenase (LDH), creatine phosphokinase (CPK), creatine kinase isoenzyme (CK-MB) were studied. Samples from the serum were collected 4hr following LD50 venom dose intraperitonealy injected in male Swiss albino mice. The activities of these enzymes showed significant elevation compared to the non-envenomated group. In contrast, an equivalent dose of 1.5 kGy γ irradiated Echis pyramidum venom (27.69g/mouse) did not cause any significant increase compared to non-envenomated group. The effect of a dose that is equivalent to 1⁄2 LD50 (13.8 μg/50 μl) of native Echis pyramidum venom on plasma creatine phosphokinase (CPK) induced a significant increase of creatine phosphokinase (CPK) level compared to normal control (P<0.01). In contrast, an equivalent dose of 1.5 kGy γ irradiated Echis pyramidum venom showed non significant difference in creatine phosphokinase activity when compared to the normal control. Light microscopic examinations of gastrocenemius muscles of mice injected with native Echis pyramidum venom (1⁄2 LD50; 13.8μg/50μl) showed fragmentation, disorganization, loss of myofibrils in some of the muscle fibers, hemorrhage in-between the muscle fibers and mononuclear cellular infiltration. While light microscopic examinations of gastrocenemius muscles of mice injected with 1.5 kGy γ irradiated Echis pyramidum venom (13.8μg/50μl; a dose identical to that used from native venom) showed that most muscle fibers were of normal appearance except for small area of fragmentation and disorganized myofibrils and oedema of the intercellular connective tissue. Double immunodiffusion test revealed a similar reactivity for native, 1 kGy, 1.5 kGy and 3 kGy γ irradiated Echis pyramidum venoms against a commercial polyvalent Egyptian antivenin. The visible lines obtained in the immunodiffusion reactions were identical and joined smoothly at the corners, indicating that there was no change in their antigenic reactivity. These results demonstrate that the ability of the venom antigens to react with its corresponding antibodies was maintained in spite of being exposed to radiation doses of 1 kGy, 1.5 kGy and 3 kGy. Both antivenins raised against native or 1.5 kGy γ irradiated venoms recognized Echis pyramidum venom when submitted to protein blotting, but the anti 1.5 kGy γ irradiated venom show a higher intensity bands than the antivenin raised against native Echis pyramidum venom, in spite of having less neutralizing activity (native neutralize 50 LD50, 1.5 kGy γ irradiated neutralize 40 LD50), this indicates that antibodies were formed not only for toxic fraction but also for non toxic fraction. Irradiation of the whole Echis Pyramidium Venom with 1.5KGy reduced its lethality 12.5 times though keeping its immunogenicity. The 1.5KGy dose was shown to be the best radiation dose to promote detoxification without significantly affecting its immunogenicity. Thus results of this study confirm the conclusion that γ radiation is a suitable way to detoxify Echis Pyramidium Venom without affecting its immunogenicity provided that proper dose is used.

Amperometric immunosensor for diagnosis of BLV infection

A new amperometric immunosensor for detection of antibodies against bovine leukemia protein (gp51) was designed. The detection of antibody–antigen complex formation was based on application of secondary antibodies labeled with horseradish peroxidase (HRP). Ferrocenecarboxylic acid (FCA) and N,N,N′,N′-tetramethylbenzidine (TMB) were selected as suitable mediators for this immunosensor. Optimal conditions for amperometric detection were found. Sensitivity of created system was compared with the results of enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID) reaction, and was sufficient for detection of usual anti-gp51 antibody concentration present in the blood serum of BLV-infected cattle.

Protein-leakage during heparin – induced &amp;lt;i&amp;gt;in-vitro&amp;lt;/i&amp;gt; capacitation of bull sperm

Abstract. Freshly ejaculated washed spermatozoa of six cross-bred bulls were capacitated by incubating in TALP medium supplemented with BSA, heparin and hepes buffer at a sperm concentration of 6 × 109 s/ml at 37ο C for 6 h. A control was also run in 0.85% saline. Percentage motility was observed at two h interval. Sperm smears were stained with giemsa and observed under oil emulsion to study different stages of acrosome reaction. After six h of incubation, sperm membrane proteins were extracted from capacitated- and uncapacitated- spermatozoa using deoxycholate in Tris-HCl (0.02M). Spermatozoa, incubation media (IM) and sperm membrane extract (SME) were analyzed for total proteins which were further fractionated by SDS-PAGE. Antisera raised against SME of bull # 1, 2 and 3 were used for identification of antigens in SME of all the bulls using immunodiffusion technique. After incubation of spermatozoa for 6 h, there was 15–21% and 55–62% decline in motility of capacitated- and uncapacitated- spermatozoa respectively. A total of 62–87% spermatozoa showed acrosome reaction (swelling, vesiculation and shedding of acrosome) in all the bulls. A statistically significant (P&lt;0.05) decrease in protein content of spermatozoa was observed during capacitation. The percentage extraction of protein and percentage leakage of proteins into medium showed a significant correlation with percentage acrosome reaction in respective bulls. A higher and positive correlation of rate of acrosome reaction with protein content of capacitated- and uncapacitated- spermatozoa and IM indicates that percentage capacitation in correlation to fertility can be predicted from the protein content of spermatozoa and proteins released into the medium. Protein profile revealed that from 12.5 to 70 kDa proteins get exposed during capacitation. There was leakage of 4–7 proteins, mainly of medium (70–130 kDa) and low mol wt (&lt;12.5–35 kDa) from capacitated spermatozoa, whereas only 2–5 proteins of high (160–&gt;240 kDa) and low (&lt;12.5–25 kDa) mol wt proteins get leaked out from uncapacitated spermatozoa. Immunodiffusion reaction of SME of bull # 1, 2 and 3 also suggested the removal/exposure of antigens from sperm surface during capacitation and acrosome reaction.