Radial Immunodiffusion Method Research Articles

MICROBIAL CARIOGENIC FACTORS AND THE MECHANISMS OF THEIR COUNTERACTION IN THE ORAL FLUID OF CHILDREN AT EARLY AGE

Objective – to determine the balance between pro- and antibacterial agents influencingupon the development of a carious process in children at an early age.Materials and methods. 117 children 2-3 years of age were examined. Two observationgroups were formed depending on the condition of the hard dental tissues. І groupincluded 52 children without caries, group II– 65 children with caries. The oral hygieneof the children from the observation groups was assessed by means of Kuzmina EM index.Lysozyme activity in the oral fluid was studied by means of bacteriological method. sIgАcontent was determined by means of radial immunodiffusion method. Streptococcusmutans concentration in the oral fluid of children was determined by means of «SalivaCheck Mutans» set. All the manipulations were carried out after an informed consent ofparents was obtained.Results. Oral hygiene in children during the period of the temporal occlusion formationis found to be unsatisfactory – (0,48±0,20) points according to Kuzmina EM index. Thelevel of colonization of the oral cavity with Streptococcus mutans is high in 34,48 %of stomatologically healthy children and in 73,33 % of children with caries. Underconditions of caries process development, the main protective components in the oralfluid change. In particular, sIgA level 17,14 % increases concerning the index of healthychildren (р<0,05), and lysozyme activity 1,25 times decreases respectively (р<0,05).Conclusions. All the children examined present soft dental plaque, which is confirmedby the values of hygienic indices and levels of Streptococcus mutans concentration,which is reliably higher in children from group II. Under conditions of caries processdevelopment, changes of the major protective components of the oral cavity appear. Theypromote effective action of cariogenic factors.

Characteristics for the third molar extraction with orthodontic indications in the aspect of age

The research analyzes the effectiveness of third molar extraction with orthodontic indications in children of different ages. The study was conducted on 95 patients divided into three groups: the I group included 30 children aged 11–13 years; to II – 35 children aged 13–16 years, and III – 30 children aged 16–18 years. Clinical examinations were carried out according to generally accepted methods immediately after tooth extraction, 3 and 6 days later. The paraclinical parameters for the study were the level of lysozyme activity in the oral fluid of observation groups’ children, which was determined according to Horin H. method modified by Levitskyi A. and Zhigina O., as well as the concentration of secretory IgA, which was determined due to simple radial immunodiffusion method. Statistical processing of data was carried out using generally accepted methods of variational statistics. The results of the study show that the age of 11–13 years old is the most reasonable period for third molars extraction with orthodontic indications, which is justified by a shorter duration of postoperative period, fewer complications and a better protective capacity of children's oral fluid in this age period. In particular, in the dynamics of observation of children of the I group in the postoperative period, significantly fewer signs of the inflammatory process were noted, which can be explained by the less traumatic surgical manipulations. On the other hand, in children of II and III groups, the above-mentioned clinical symptoms had a higher degree of manifestation, were diagnosed in a larger number of children and indicated the development of the postoperative inflammatory process. The level of lysozyme activity in the oral fluid of children in the observation groups ranged from (40.87±2.01) units/l to (26.13±1.76) units/l, and secretory immunoglobulin A from (0.35±0.02) g/l up to (0.32±0.02) g/l, which generally indicates a deterioration of local protective reactions with increasing age of children. Keywords: сhildren, hermectomy, postoperative period, lysozyme, immunoglobulins.

The CombE-IDMS Assay as an Alternate Potency Method for Adjuvanted Quadrivalent Influenza Vaccines.

The potency of all currently licensed inactivated influenza viral vaccines is assayed by the single radial immunodiffusion (SRID) method. SRID relies upon antisera and reference antigen reagents which are produced, standardized, and distributed in the mass quantities needed for vaccine manufacturers only after a significant amount of time has elapsed from the seasonal strain recommendations issued by the WHO; this time delay is exacerbated under conditions of an emerging pandemic. Previously, the limited trypsin digestion isotope dilution mass spectrometry (LTD-IDMS) method, which does not require antisera or reference antigens, demonstrated comparable quantitation of immunologically active hemagglutinin, the primary viral antigen, to SRID in stressed vaccine materials. Here, we demonstrate a streamlined improvement to the LTD-IDMS method by eliminating the need for its precipitation and washing steps, saving time and labor in the sample preparation process while paving the way for plate-based high-throughput analysis. This is accomplished using dissimilar proteases in the pretreatment (a combination of chymotrypsin and elastase) and analytical (trypsin) digestion steps so that any pretreatment digests will not cause interference while monitoring analytical tryptic digests by IDMS. The combination of enzymes (CombE)-IDMS method is tested alongside LTD-IDMS and SRID for the first time on MF59 adjuvanted seasonal cell-based quadrivalent influenza vaccines (aQIVc) under stressed conditions of heating, oxidation, lowered and elevated pH, and freeze-thaw. Overall, a correlation in the degradation trend is observed between CombE-IDMS and SRID in the four strains of the quadrivalent formulation, highlighting the method's stability indicating capability as a rapid alternate potency assay in a highly complex formulation of aQIVc.

Characterization of lyophilized equine colostrum

Colostrum is the first milk secretion at birth and is indispensable for the equine newborn. In addition to being the only natural source of immunoglobulins (Igs) available, it has excellent nutritional importance in helping the gastrointestinal system's maturation. Thus, foals need to ingest maternal colostrum in the first hours of life to acquire immunity so that failure of passive transfer of immunity (FPTI) does not occur. The quality of the colostrum supplied to the animals must be evaluated, and innovation in its storage methods would greatly benefit management. Lyophilization is a differentiated process of dehydration that retains a large concentration of colostrum immunoglobulins. The only lyophilized colostrum for foals is based on bovine colostrum) This study aimed to compare the IgG concentration and the physicochemical characteristics of frozen equine colostrum after the lyophilization process. Thirty-one pregnant mares were used. Colostrum samples were collected manually from both mammary glands immediately after delivery. The udder was cleaned with 70% alcohol solution to avoid sample contamination. About 20 mL of colostrum were collected, and the first two jets were discarded. The IgG concentration of frozen and lyophilized colostrum was determined by the Simple Radial Immunodiffusion method (IDRS) and also, indirectly, by Brix refractometry. The samples' physical-chemical composition (pH, total protein (TP),fat, lactose, salts, total solids (TS), and density) was evaluated,and the lyophilized colostrum reconstitution test was performed. The ways of preserving colostrum were compared using a t-test at 5% probability. Pearson correlations were utilized between variables with Sisvar®, version 5.7 statistical software. No significant differences were seen in the measurements of IgG, fat, lactose, salts, TS, density, and pH before and after lyophilization. No difference was seen in the IgG concentration measured by IDRS (frozen 30.5 g/L vs lyophilized 28.5 g/L). A strong positive correlation was observed between colostrum density and IgG concentration by IDRS (r = 0.76) and between Brix and IgG concentration by IDRS (r= 0.77). In the reconstitution test, lyophilized colostrum was efficiently rehydrated in water, with total dilution, and remained stable. The results suggest that lyophilization can be an alternative for conservation of colostrum from mares, because it is a very efficient process for the retention of the physicochemical characteristics of the product, with no significant loss of IgG concentration. In addition, it is easy to reconstitute, can be preserved for a long period without the need for refrigeration and easily transported. This research should be expanded to make an innovative equine product available. This will facilitate the management of newborn foals and help to reduce the rates of failure of passive immunity transfer.

Serum immunoglobulin levels in children with acute bacterialmeningitis

To assess serum level of immunoglobulins (IgG, IgA, IgM) in children with acute bacterial meningitis before therapy and at the end of intensive antibiotic therapy and in COMparison 1 heallity age aiid scx mittchicd vonirsls. Out of 37 vases adinived ta Thi-Als Atheer pediatric hospital (Mosul city) between January 2005 and July 2007, 29 cases proved to be a case of Haemophilus influenzae meningitis. Two died during the follow up period and 27 completed the study. Also included 30 apparently healthy age and sex matched subjects taken as control group. Initially from both group (patients and control) Sml venous blood samples were taken and assay of serum immunoglobulin (IgG, IgA, IgM) were done by single radial immunodiffusion method using commercial kits. After two weeks of intensive antibiotic therapy another blood samples were taken from the patients and assay of serum immunoglobulin levels were done using the same method and the same commercial kits. Initially before therapy there was a significant reduction in all immunoglobulin levels (IgG, IgA, IgM) in children with acute bacterial meningitis in comparison to controls. After 2 weeks of intensive antibiotic therapy, there was no significant difference in the mean serum immunoglobulin levels between the patients and controls. Acute bacterial meningitis as a disease and the intensive antibiotic therapy do affect the humoral immune response as reflected by the serum immunoglobulin levels (IgG, IgA, IgM).

Adaptive immune response in patients with atherosclerosis before and after coronary bypass surgery

Objective: to assess changes in the immune status before and after coronary bypass surgery. Materials and methods: included 70 male patients with coronary atherosclerosis (group 1) and 30 volunteers (group 2) without signs of coronary artery disease comparable in age to group 1. Immunity parameters were studied before surgery, after 4–5, 9–10 and 28–30 days by the method of phenotyping of CD3+ populations; CD3+CD4+; CD3+CD8+; CD3+CD25+; CD3+CD45+; CD3+CD95+; CD4+CD25+; CD4+CD154+; CD19+, CD19+CD40+. The levels of Ig A, M, G were assessed by the method of radial immunodiffusion in the gel. Serum precipitation in polyethylene glycol was used to determine circulating immune complexes. Statistical analysis of the study results was performed using the Statistica 12.0 program (StatSoft, USA). Statistical significance was considered significant at p<0.05. Results: in patients of the IHD group, uncoupling of the processes of T-lymphocyte activation, their maturation and apoptosis was noted; suppression of immunoregulation and activation of intercellular cooperation. After CABG, in dynamics, these processes reflect the multidirectional changes. Conclusions: in patients with coronary atherosclerosis, there is a discoordination of the processes of T-lymphocyte activation, their maturation and apoptosis. Suppression of immunoregulation and activation of intercellular cooperation. In the dynamics after CABG, immunity parameters differ in different directions in different periods of observation and demonstrate different degrees of involvement of adaptive mechanisms of immune defense.

Features of the immune and metabolomic profile in overweight children associated with polymorphic variants of the IL-1B and TP53 genes

Background. The growing number of overweight children is an important problem of modern healthcare. Excess body weight causes the risk of severe metabolic disorders developing. Analysis of changes in the immune, metabolic, and genetic profile of overweight children as markers of the early development of metabolic disorders is an effective tool for the prevention, diagnosis, and treatment of these pathologies.
 Aim. To identify the features of the immune and metabolomic profile in overweight children aged 711 years associated with polymorphic variants of the IL-1B (rs1143634) and TP53 (rs1042522) genes.
 Material and methods. 132 children aged 711 years were examined: 54 children with overweight and 78 children with normal body weight. The lymphocytes subpopulation composition was analyzed by flow cytometry; the content of leptin, cortisol, superoxide dismutase, lipid hydroperoxide and interleukin-1 using enzyme immunoassay, immunoglobulin G using the method of radial immunodiffusion method by Mancini. Polymorphic variants of the IL-1B (rs1143634) and TP53 (rs1042522) genes were identified by real-time polymerase chain reaction. Statistical data processing was implemented in the Statistica 10, SNPStat and Gen-Expert programs.
 Results. The immune profile of the examined children was characterized by an increased content of CD3+CD8+-, CD19+-lymphocytes and immunoglobulin G against the background of CD3+CD4+-lymphocytes, p53 deficiency and a decrease in the number of CD4+/CD8+. The metabolomic profile of children was characterized by an excess content of leptin, cortisol, lipid hydroperoxides, and superoxide dismutase. The identified changes in immune and metabolomic regulation in overweight children were significantly associated with the G allele and GG genotype of the IL-1B gene (rs1143634), as well as with the C allele and CC genotype of the TP53 gene (rs1042522), which cause the initiation of inflammation and inhibition of apoptosis.
 Conclusion. The established features of immune and metabolomic regulation associated with the G allele and GG genotype of the IL-1B gene (rs1143634) and with the C allele and CC genotype of TP53 gene (rs1042522) represent a complex of markers of overweight formation in children aged 711 years.

Lactoferrin concentration in bovine milk: validation of radial immunodiffusion technique, sources of variation, and association to udder health status

The present study aimed to evaluate analytical performances of radial immunodiffusion (RID) technique for the quantification of milk lactoferrin (LF), and to investigate the main sources of variation of LF concentration in individual milk samples of Holstein Friesian (HF, n = 1516) and Simmental (SI, n = 230) cows. Repeatability (RSDr) and reproducibility (RSDR) of RID method were assessed as relative standard deviation of 15 measurements within the same day and 45 measurements across 3 d, respectively. Sources of variation of milk LF were investigated through a mixed linear model which included the fixed effects of classes of stage of lactation and parity (and their interaction), cow breed, udder health status (UHS, based on somatic cell count and differential somatic cell count), and the random effects of the herd-test day nested within breed and the residual. Method performances were satisfactory in terms of repeatability (RSDr < 9%) and reproducibility (RSDR < 8%), suggesting that the RID can be considered as a consistent cow-side test for the quantification of milk LF. Milk LF was positively associated with somatic cell score (r = 0.40) and negatively with lactose content (r = −0.33). Lower concentration of LF was observed in milk of ‘healthy’ and ‘susceptible’ cows (13.03 and 12.87 mg/dL, respectively) compared to ‘mastitic’ and ‘chronic’ cows (17.91 and 17.33 mg/dL, respectively). In this light, milk LF has the potential to be a useful biomarker to detect early mastitis. Further research is advisable to deepen the association between milk LF content and observed clinical mastitis. Highlights Radial immunodiffusion method for milk lactoferrin quantification is repeatable and reproducible. Somatic cell count is positively associated with milk lactoferrin content. Milk lactoferrin is less concentrated in milk of healthy and susceptible than potentially mastitic and chronic cows.

Immunogenic characterization of AlPO4 adsorbed Td vaccine and liposome-mediated Td vaccine.

The purpose of this study was to compare the antigenic potency and stability of tetanus and diphtheria (Td) vaccines when combined with aluminum phosphate (AlPO4) and liposome adjuvants. In vitro and in vivo analyses were conducted using the single radial immunodiffusion method and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Td vaccines were prepared with AlPO4 adsorption and liposome-mediated delivery, and protein antigens were characterized using these methods. The results revealed that the liposome-mediated Td vaccines exhibited higher immunogenicity compared to the AlPO4-adsorbed Td vaccines. Additionally, the liposome-mediated Td vaccines demonstrated higher stability as native antigens. This study highlights the importance of utilizing liposome adjuvants in vaccine development. The liposome-mediated Td vaccines showed enhanced immunogenicity and stability, making them a promising approach for improving vaccine efficacy. Understanding and optimizing adjuvant strategies can contribute to the development of effective vaccines against various diseases.

Genetic characteristics of colostrum refractive index and its use as a proxy for the concentration of immunoglobulins in Holstein cattle

BackgroundColostral concentration of immunoglobulins (Ig) is crucial for the passive transfer of antibodies from the cow to the new-born calf. Direct determination of Ig by the gold standard radial immunodiffusion method is demanding in terms of time and costs. For this reason, a refractometer is commonly used at the farm level for an indirect estimation of colostrum quality, which is given as the Ig concentration. In this study, colostrum samples were collected from 548 Italian Holstein cows within 6 h of calving. The refractive index (BRIX, %) of these samples was assessed using a portable optical refractometer, as well as the concentration of total protein, IgG, IgA, and IgM by radial immunodiffusion. A four-trait animal model was used to estimate genetic parameters for BRIX and the different immunoglobulin isotypes. A receiver operating characteristic analysis was carried out to evaluate the BRIX diagnostic accuracy.ResultsColostral BRIX was moderately heritable (0.26) and its genetic and phenotypic correlations with IgG (0.91, 0.78), IgA (0.57, 0.57), and IgM (0.71, 0.61) were all positive and of similar order, although the genetic correlations were generally higher than the phenotypic correlations. Low-quality colostrum samples, defined as those with an IgG concentration lower than 50 g/L, were accurately identified by the refractive index on the BRIX scale, with an area under the curve of 0.90.ConclusionsThe use of a refractometer is recommended on dairy farms to produce a proxy for colostral Ig concentration. BRIX is a useful phenotyping tool that can be used in cattle to improve the quality of colostrum for first feeding of calves through both traditional genetic and genomic strategies. Improving colostrum quality will reduce the incidence of failure of passive transfer of immunity in young stock.

Bayesian latent class analysis of the characteristics of diagnostic tests to assess the passive immunity transfer status in neonatal Belgian Blue beef calves

The aim of the current study was to assess the diagnostic characteristics of radial immunodiffusion (RID), capillary electrophoresis (CE) and digital brix refractometry (Bx) for the diagnosis of failure of passive transfer (FPT) of immunity in neonatal Belgian Blue beef calves in the absence of a gold standard using a Bayesian latent class model. Belgian blue beef calves (n = 202) from a large farm in the south of Belgium were blood-sampled at 48–72 h of age and tested for FPT. The true prevalence of FPT in this population of calves was 34.5 % (95 % BCI: 26.1–44.3) using a FPT cut-off point of 10 g IgG/L. This true prevalence increased to 66.3 (95 % BCI: 56.9–74.8) and 88.9 % (95 % BCI: 83.1–93.2) at FPT cut-off points of respectively 18 and 25 g IgG/L serum. The Bland-Altman plot comparing the RID and CE methods, revealed that the serum IgG concentrations obtained by RID were on average 2.25 (95 % CI 1.62–2.88) g/L higher than those measured by CE. Optimal cut-off values for CE, corresponding to the FPT values as measured by RID of 10, 18, and 25 g IgG/L serum, were respectively 10, 15, and 20 g IgG/L. The overall diagnostic accuracy of the three diagnostic tests was comparable at the FPT cut-off point of 10 g IgG/L serum (i.e. 85 %). At higher cut-off points for FPT, the RID and CE assays presumably performed better that the Bx method. In conclusion, we demonstrated that: (1) the CE method is a good alternative for the RID assay, the latter having important constraints when considering its practicality, and (2) the Bx method is a cheap and user-friendly indirect method to evaluate the FPT in new-born Belgian Blue beef calves.

DYNAMICS OF SIgA CONTENT IN BREAST MILK WHEN RISK FACTORS AND LACTATIONAL CHARACTERISTICS ARE CONSIDERED

The leading role associated with an anti-infective action of breast milk belongs to secretory IgA (SIgA). Therefore, the determination of the level of SIgA in colostrum and milk of mothers with different lactation levels and mothers at risk at different levels and duration of lactation has a practical interest.&#x0D; Aim. The research aims at studying the dynamics of SIgA content in breast milk, taking into account risk factors and features of lactation.&#x0D; Material and Methods. The content of SIgA in colostrum and breast milk of 372 mothers with full lactation and 208 with hypogalactia, of which 72 with early and 146 with late hypogalactia at different times of lactation (1-8 days and 1-3 months) was determined. The SIgA content was also studied in mothers; 65 with preeclampsia, including 12 with early hypogalactia, 24 with late hypogalactia, 23 with full lactation, and 44 with anemia of pregnancy, including 12 with early hypogalactia, 19 with late hypogalactia and 13 with full lactation. The determination of SIgA content in colostrum and milk was performed using the method of simple radial immunodiffusion in a gel by G. Mancini et al.&#x0D; Results and Discussion. The SIgA concentration in colostrum and breast milk decreased during the secretion process. In early hypogalactia, the content of SIgA did not differ significantly from the level of SIgA in colostrum and breast milk with full lactation in the early neonatal period and at 1-3 months of secretion. In the late hypogalactia, SIgA content did not differ significantly from its level in the control group. Due to the lower amount of milk received by the mother's child with hypogalactia, the supply of children with SIgA is insufficient, and the deficit is higher the sooner the hypogalactia develops. Similar changes in the dynamics of SIgA content have been found in mothers with preeclampsia and at different levels of lactation. The exception was the content of SIgA in colostrum and milk of mothers with anemia of pregnancy and early hypogalactia, which in the first five days of lactation was lower than in mothers with full lactation.&#x0D; Conclusion. The SIgA concentration in colostrum and breast milk decreases during the secretion process. The level of lactation does not affect the dynamics of SIgA content, except for the SIgA content in colostrum and breast milk of mothers with anemia of pregnancy and hypogalactia in the early stages of secretion. Despite the absence of a significant difference in SIgA levels in mothers with different levels of lactation due to less colostrum and milk received by children with early and late hypogalactia, the supply of SIgA is insufficient, and the cumulative deficit is higher the faster the hypogalactia develops.

Application of Thoracic Immunogen of Musca domestica on Immunoglobulin-G Level of Goats Detected Through a Single Radial Immuno-Diffusion Test

The purpose of this study was to detect the IgG level of goats treated with immunogen thoracic of Musca domestica (ITMd). We reared twelve young goats aged two months old under extensive treatments, divided into two equal groups for a comparison experimental design. The animals in the first group (R0) served as the control without immunogen injection, and the second group (R1) was the treatment group receiving a subcutaneous injection of 10 mL of thoracic antigens per head. The parameter observed was IgG (immunoglobulin-G) antibody serum level. The quantification of goat immunoglobulins was carried out using a single radial immuno-diffusion (SRID) technique. The data obtained from the two groups were analyzed with a t-student test. The results showed that the total IgG antibody serum of goats in the treatment group was significantly higher than that of the control group (P&lt; 0.05). This difference may be due to the thoracic M. domestica immunogen response which increased antibody synthesis of experiment goats. We concluded that the treatment of thoracic immunogen of M. domestica at a level of 10 µL injected subcutaneously could significantly increase IgG antibodies in goat blood which were detected using a single radial immuno-diffusion method.

PSX-B-22 Humoral factors of protection of the vaginal mucosa in healthy cows and with mycoplasmosis

Abstract Maintaining reproductive health is an urgent task in intensive livestock farming. The study of the influence of pathogens of the Mycoplasmataceae family on the microecology and protective properties of the vagina is of particular interest. The studies were carried out on non-pregnant cows 3–4 years old. Was formed 2 groups of 8 animals each. The first group is healthy cows in which the PCR test for Mycoplasma spp. was negative. The second group - cows with a positive PCR test and serological identification of M. bovigenitalium, without pronounced clinical signs of vaginitis. In both groups of animals, vaginal secretions were collected from the vaginal wall using a special spoon. In secret, by the method of radial immunodiffusion in a gel according to Mancini, the content of immunoglobulins of classes Ig G, Ig M, Ig A and secretory immunoglobulin A (sIgA) was determined, as well as the activity of lysozyme - by the nephelometric method. The study showed that the content of Ig G and the total content of immunoglobulins in the vaginal secretion in healthy cows and cows with mycoplasmosis did not have significant differences. The concentration of Ig A in cows with mycoplasmosis was 0.018±0.001 g/l, which was 25% less than in healthy cows, but it turned out to be insignificant (P &amp;gt; 0.05). The content of Ig M and sIgA in secretion in healthy cows was 0.039±0.002 and 0.067±0.005 g/l, while in cows with mycoplasmosis it significantly increased by 38.5 and 43%, respectively. The activity of lysozyme in the secretion of healthy cows was 11.71±0.41%, while in infected cows it decreased by 2 times. With genital mycoplasmosis in cows, a quantitative redistribution of immunoglobulin classes and a decrease in lysozyme activity are observed in the vaginal secretion.

Effect of phytobiotic - Germivit on the functional state of cattle

Plant-based feed additives, also known as phytobiotics, show great promise to compensate deficiencies of important biologically active substances in the diet. Therefore, this study aimed to evaluate the effect of Germivit on the feed supplement of cattle of different ages. In the first experiment, four groups of 9-month-old Simmental calves (10 animals in each group) were fed with Germivit at 0.0, 0.5, 0.7, and 0.9 g kg-1 live body weight, respectively. In the second experiment, three groups of pregnant cows (10 animals in each group) received Germivit at 0.0, 0.25, and 0.50 kg-1 live body weight, respectively. The morphological composition of the collected blood was studied using a PCE-90vet automatic hematology analyzer. The blood biochemical composition was studied using a Stat Fax 1904 biochemical analyzer, and the immunological status of the animals was evaluated using generally accepted methods in veterinary medicine: immunocompetent cells by spontaneous rosette formation; immunoglobulins by the radial immunodiffusion method; phagocytosis by using S. aureus culture; serum lysozyme activity by the photoelectrocolorimetric method; and bactericidal activity of serum by determining the degree of growth inhibition of the mixture of daily culture of E. coli in a nutritive culture broth. Germivit contributed to the improvement of the biochemical and immunological parameters in the calves. In the cows, an increase in the morphological parameters of blood was observed, their immune status improved, and their calves were born with high rates of natural resistance and health. Germivit had a positive effect on the functional state of cattle.

Measurement of ITIH4 and Hp levels in bitches with pyometra using newly developed ELISA methods

Measurement of acute phase proteins (APPs) as biomarkers in canine medicine is in increasing demand. In the present study, the development and validation of two ELISA methods for the quantification of canine inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) and haptoglobin (Hp) are shown. The adequate imprecision and accuracy and wide analytical range make the developed methods appropriate to quantify ITIH4 and Hp in serum samples. The inter- and intra-assay CVs were lower than 10 %, and the assays maintained linearity under dilution and showed analytical equivalence with the method of radial immunodiffusion. The measurement of CRP, Hp and ITIH4 in sera from bitches affected by pyometra allowed us to determine that ITIH4 behaves as a moderate APP in dogs. The group of bitches affected by pyometra showed very high levels of CRP (147 ± 91 mg/L), corresponding to a strong inflammatory process, which resulted in a moderate increase in the concentrations of Hp (7 times) and ITIH4 (3 times) compared to the control group.

Toxin production and antibiotic resistance of Staphylococcus aureus strains isolated from market gardening products and their irrigation water in cotonou, Benin

Consumption of market gardening product has displayed several cases of food poisoning because they are being consumed raw often. This study aims to establish antibiotics resistance and toxins production profiles of Staphylococcus aureus strain isolated from market gardening products and irrigation water in Cotonou. In this regard, 4 kind of market gardening products (Lettuce, Carrot, Nightshade, and Cabbage) and 22 samples of irrigation water (pool, well and drilling water) were collected from four market gardeners and were analyzed. The S. aureus strains were identified using microbial method and classical biochemical tests. The antimicrobial susceptibility of 17 antibiotics was performed by the agar disk diffusion method. The toxins production was detected by the radial gel immunodiffusion method. Out of the 112 samples analyzed, 69.81% were contaminated by S. aureus . Market gardening products were contaminated by S. aureus at the rate of 82.44% and the irrigation water was at 17.56%. Among these products, nightshade was the most contaminated (32.79%), followed by lettuce (27.86%), cabbage (21.31%) and carrot (18.04%). Based on the type of water samples used, it appears that 53.85% of the well water contained S. aureus . Globally, the highest resistance proportion has been observed with lincomycin (62.06%) and none of the strains were resistant to ciprofloxacin, whereas about 20.69% of the strains were resistant to the methicillin (MRSA). ETA and LPV toxins were the most produced (32.69%) followed by ETB (28.85%), and LukE-D (5.77%). From the toxin-producing strains of S. aureus , 46.43% were MRSA. High contamination rate of toxins producer and resistant S. aureus strains isolated were recorded on market gardening products. It is important to promote monitoring and sensitizing farmers in aim to reduce the contamination rate of market gardening products. • Market gardening products and irrigation water are highly contaminated by S. aureus . • S. aureus isolated are multi-resistant to several conventional antibiotics. • Nightshade are the most contaminated (32.79%) Market gardening products. • ETA and PVL toxins were the most produced (32.69%) by isolated S. aureus.

Technical note: Capillary electrophoresis as a rapid test for the quantification of immunoglobulin G in serum of newborn lambs

Finding a rapid and simple method of serum IgG determination in lambs is essential for monitoring failure of passive transfer of immunity. The aim of this study was to assess the ability of capillary electrophoresis (CE), an instrument mainly used in blood serum protein analysis, to estimate IgG content in serum of newborn lambs through determination of only total Ig percentage by comparing the results with those obtained with radial immunodiffusion (RID), the reference method for serum IgG quantification. Serum samples were collected at 24 h after birth from 40 Sarda lambs. The IgG concentration measured by RID and serum total Ig concentration measured by CE were (mean ± standard deviation) 29.8 ± 16.1 g/L and 37.8 ± 15.0%, respectively. Data provided by RID and CE analysis showed a polynomial trend (RID = 0.02CE2 - 0.04CE + 4.13; coefficient of determination, R2 = 0.96), displaying a strong relationship between these 2 methods. Applying the polynomial equation, the IgG values were predicted. Predicted IgG values were highly correlated (r = 0.98) and related (R2 = 0.96) to IgG values obtained by RID assay. These data were subjected to Bland-Altman analysis, revealing a high level of agreement between CE and RID methods with a bias that was not different from 0 (-0.04 g/L) and agreement limits of -6.38 g/L (low) and +6.30 g/L (high). In addition, the linear regression analysis between differences (dependent variable) and average of IgG concentration by CE and RID (independent variable) did not show proportional bias (R2 = 0.01). In conclusion, CE is a reliable instrument for a lamb health monitoring program, where Bland-Altman analysis also confirmed that the CE method can be a suitable alternative to RID.

IMMUNOTROPIC EFFECTS OF IVERMECTIN IN PRODUCTIVE AND LABORATORY ANIMALS

The aim of this work was to identify the immunotropic effects of ivermectin in productive and laboratory animals in real and simulated conditions. The studies were carried out on hybrid pigs at the age of 144 days reared in an industrial pig complex and on Wistar rats aged 5 months with a body weight of 230 - 250 g. For the experiments there was used acaricide insecticide Ivermin (Biovet Drwalew S.A., Poland), which was administered to pigs once subcutaneously in a dose of 0,2 mg / kg and to rats in a tenfold therapeutic dose. Blood samples were taken from pigs either before the administration of Ivermin, and 1, 3, 7, 14 and 30 days after the administration. The level of immunoglobulins (IgG, IgM, IgA) was determined in serum by the method of radial immunodiffusion according to Mancini. To prepare histopreparations, samples of thymus, spleen and lymph nodes were taken from rats 14 days after the administration of the preparation. For statistical processing of experimental data, Student’s T-test for dependent samples has been used.When studying pigs’ blood serum, a decrease in the content of IgG was established in all periods of the study. A decrease in level of IgM and IgA was marked in 7 days after the experiment started. At the end of the experiment, the IgG level remained below the background value by 16,4%; IgM - by 15,2%; IgA – by 33%. In the rat thymus, a narrowing of the cortical substance was observed when a toxic dose of Ivermin was injected, with the splenic follicles reduced, and reproduction centers faintly pronounced. Hemosiderin accumulation was detected around the follicles. In the mesenteric lymph nodes, there was found a thickening of the capsule and the expansion of trabeculae. Cortical substance was narrow and paracortical zone was wide. The results of the study indicate a high risk of immunotoxic effect of ivermectin.

Оценка профилактической эффективности препаратов рекомбинантного интерферона α-2b у сотрудников скорой помощи, контактирующих с пациентами с COVID-19

Against the background of the unfavorable situation with COVID-19, the problem of sufficient medical personnel protection when delivering professional medical care in epidemiological foci remains relevant these days. Medical personnel who are in constant contact with COVID-19 patients are subjected to a strong viral load and are at risk of infection development. In this regard, adequate disease preventive measures, in addition to personal protective equipment (PPE), should include the prescription of medications with pronounced antiviral and immunomodulatory activity. Objective. Evaluation of the preventive efficacy of the combined use of VIFERON®, rectal suppositories 1 000 000 IU together with VIFERON®, ointment 40 000 IU/g or gel 36 000 IU/g in ambulance personnel contacting with COVID-19 patients. Patients and methods. Patient groups were as follows: the first main group – 100 people used personal protective equipment (PPE) and VIFERON®, rectal suppositories (IFN a-2b) 1 000 000 IU before each work shift (10 shifts) + intranasal ointment or gel IFN a-2b during one month; the second control group – 50 people (for the prevention of SARS, PPE is used). The assessment of the content of sIgA in the nasal secretion was carried out by the method of radial immunodiffusion according to Mancini G. (1965), the study of lysozyme activity – according to the method of Dorofeychuk V.G. (1968). Determination of reactive and personal anxiety was carried out according to Spielberger–Hanin (Spielberger Ch.D., 1973; Hanin Yu.L., 1976), depression was determined according to the Zung Scale (Zung W., 1965). Statistical analysis of the results was conducted using the STATISTICA v. 12.0 software package (StatSoft Inc, USA). Results. The research studied the preventive efficacy of the combined use of recombinant interferon α-2b with antioxidants (VIFERON®), rectal suppositories, ointment and gel in medical forms in different dosages for medical workers contacting with COVID-19 patients. The studies included observation of humoral immunity indicator factors and nonspecific resistance – secretory immunoglobulin A (sIgA) and lysozyme. In addition, a complex of studies to determine the degree of psycho-emotional disorders in medical personnel contacting with patients suffering from COVID-19 was conducted. The conducted research proved the high efficiency and safety of the use of combined dosages forms of VIFERON® as a preventive measure for medical workers who are at high risk of infection with COVID-19: a significant increase in local immunity indicators, absence of severe complications in confirmed cases of coronavirus infection and improvement of the psycho-emotional status of medical staff were shown. Conclusions. IFN a-2b in the main group has contributed to a significant decrease in the number of acute respiratory viral infections, including those caused by the SARS-CoV-2 virus. Only one employee in the main group has undergone SARS of COVID etiology. IFN a-2b provides a protection against the development of a complicated course of the disease. Pneumonia of COVID etiology has developed in 2 medical workers in the control group. There has been a significant increase in the indicators of local immunity of the mucous membranes of the upper respiratory tract – lysozyme and sIgA against the background of IFN a-2b. In the main group, the psychological state of medical workers has improved; situational and personal anxiety has decreased. The combined administration of VIFERON® (ointment or gel + rectal suppositories) may be recommended for widespread use among medical personnel contacting with COVID-19 patients. Key words: depression, interferon-α2b with antioxidants, interferon therapy, lysozyme, medical personnel, local immunity, psycho-emotional status, anxiety, prevention, epidemiology, COVID-19, sIgA