Cryopreservation of primary trypsinized fibroblast cells of chicken embryos using various cryoprotectants

Cryopreservation is the optimal way to store cells at ultra-low temperatures. Cryoprotectants are added to cell culture suspension to reduce cell death due to exposure to low temperatures. Cryoprotective media contain combinations of various cryoprotectants. Ethylene glycol, glycerin, dimethyl sulfoxide, sucrose, dextran, propylene  glycol, albumin, polyvinylpyrrolidone and blood serum can be used as cryoprotectants. For cryopreservation it is necessary  to select a cryoprotectant that ensures the highest survival of cells after storage and thawing. The paper presents  the results of experiments  on comparing the effectiveness of dimethyl sulfoxide, ethylene  glycol and glycerin in cryopreservation  of primary trypsinized chicken embryo fibroblasts. As a result of cell suspension  equillibration  (incubation  at room temperature) with serum and the specified cryoprotectants at different concentrations, the suspension variants containing different cryoprotectant and serum ratios were selected for freezing. Previously, it was found that after 12 months of observation, when using dimethyl sulfoxide as a cryoprotectant,  the largest number of surviving cells (46%) was observed in a suspension  containing  20% fetal serum and 10% dimethyl sulfoxide. The amount of surviving  cells if 10% fetal serum  and 5% ethylene  glycol were included in the cryoprotective mixture was slightly lower and amounted  to 36% after 12 months of observation. Glycerin is shown  to have weak protective properties as regards chicken embryo fibroblast cells. After 8 months of storage, the amount of surviving cells in a suspension containing 10% serum and 5% glycerin was 22%, no live cells were found in this mixture if stored longer. The proliferative properties of cells and their sensitivity to viruses remained within the 12 months  of the experiment

Testing of chickens experimentally infected with A/H9N2 avian influenza virus isolates for their immune responses

Data on tests of chickens for their immune responses to infection with low pathogenic А/Н9N2 avian influenza virus isolates belonging to Y-280 and G1 genetic lines are presented in the paper. CD4⁺/CD8⁺ ratios were determined with flow cytometry for initial immune status examination and for detection of apparent immune system disorders. Quantitative analysis of peripheral blood lymphocyte subpopulations in chickens revealed changes characteristic of the immune suppression. Analysis of dynamics of T- and B-lymphocyte levels in blood of the infected chickens revealed decrease in relative T-lymphocyte counts and increase in relative B-lymphocyte counts. T-lymphocyte subpopulation composition expressed as CD4⁺/CD8⁺ ratio (%) changed after the infection: CD4⁺ cell proportion was found to decrease whereas CD8⁺ cell proportion increased. According to literature data, immune response activated by vaccination induces the reverse dynamics towards to increase in CD4⁺/CD8⁺ ratio. Both cell-mediated immunity and humoral immunity play role in development of the immune response in chickens infected with avian influenza viruses. Apparent humoral immune response was detected by serological tests of sera taken from chickens on day 14 after infection. Mean specific anti-A/H9N2 AIV antibody titre in all groups of test chickens infected with low pathogenic avian influenza virus isolates was higher than 6 log₂ . High level of specific antibodies to avian influenza virus was indicative of postvaccinal humoral immune response development

Immunobiological properties of inactivated anti-highly pathogenic avian influenza vaccines based on antigens of А/Н5N1 avian influenza virus strains of different virulence

Antigen of H5N1 low pathogenic avian influenza virus Yamal strain included in the inactivated emulsion vaccine is able to induce strong immunity against highly pathogenic avian influenza in chickens. Inactivated emulsion vaccines based on antigen of H5N1 low pathogenic avian influenza virus Yamal strain and antigen of H5N1 highly pathogenic avian influenza virus А/chicken/Primorsky/85/08 strain are capable of inducing dose-dependent cross immunity against current Н5N1 and H5N8 highly pathogenic avian influenza viruses. Thus, inoculation dose of H5N1 low pathogenic avian influenza virus Yamal strain antigen required for pro­tection of 95% of chickens against H5N1 highly pathogenic avian influenza virus А/chicken/Primorsky/85/08 strain and against H5N8 highly pathogenic avian influenza virus A/duck/KChR/1590-20/20 in the vaccine inoculation volume shall be at least 609 HAU and 255 HAU, respectively. The inoculation dose of H5N1 highly pathogenic avian influenza virus А/chicken/Primorsky/85/08 strain antigen for protection from H5N8 highly pathogenic avian influenza virus A/duck/KChR/1590-20/20 strain shall be at least 294 HAU. Protective effect of the tested inactivated vaccines was associated with humoral immunity level in poultry. Predicted titre of antibodies to homologous virus antigens conferring expected 95% protection of vaccinated poultry was 1:538 or 9.1 log2. Inactivated vaccine based on H5N1 low pathogenic avian influenza virus Yamal strain antigen demonstrates its high immunogenicity in chickens infected with Н5N1 and Н5N8 highly pathogenic avian influenza influenza virus

Serological monitoring of avian influenza and Newcastle disease in the Russian Federation in 2020

Within the framework of the Rosselkhoznadzor measures aimed at control of highly dangerous  diseases and development of timely recommendations for disease prevention and control, 36,986 serum samples to be tested for the presence of avian influenza virus antibodies and 30,325 serum samples to be tested for the presence of Newcastle disease virus antibodies were submitted to the FGBI “ARRIAH” Reference Laboratory for Avian Viral Diseases  in 2020. The samples were collected from domestic, wild and synanthropic birds in 60 Subjects of the Russian Federation. As a result of the laboratory diagnosis, antibodies against type A influenza virus were found in vaccinated chickens from two poultry farms in the Primorsky Krai. Typing of sample sera using hemagglutination inhibition test showed that the detected antibodies were specific to the haemagglutinin subtype of the vaccine antigen  (A/H9). Antibodies to the H9 subtype  avian influenza  virus were detected in sera of non-vaccinated geese from two poultry farms in the Kurgan Oblast and from one poultry farm in the Republic of Bashkortostan. As for the  backyards where  scheduled  vaccination  against  avian influenza  A/H5 is carried out, a low level of immunity  was seen in the Republics of Adygea and Chechnya (0 and 15%, respectively), while a high immunity level was observed in the Rostov Oblast (74%). High seroprevalence  of Newcastle disease virus was found in adult poultry in indoor industrial farms, which was associated with mass vaccination against the disease. In broiler chickens, post-vaccination antibodies were observed, on average, in 44% of the tested sera samples. The antibodies  against  Newcastle  disease  virus and avian influenza  virus subtype  H5 detected in wild and synanthropic birds indicate the circulation of these viruses in the Russian Federation. The insufficient level of post-vaccination antibodies suggests that the risk of epidemic among poultry in industrial poultry farms and backyards remains

Infectious bursal disease virus: identification of the novel genetic group and reassortant viruses

The results of the phylogenic analysis of the nucleotide sequence of the IBDV A and B genome segments have been presented. Traditionally the IBDV isolates are classified based on the phylogenic analysis of the hypervariable region of the VP2 gene. The analysis of the VP2 gene segments of the isolates detected in the Russian Federation demonstrated that most of them belong to the genetic group comprising highly virulent IBDV isolates. However, not all isolates belonging to one genetic group have the same phenotypic characteristics. This is related to the fact that the virulence is determined not only based on the characteristics of the VP2 gene (A segment) but on the characteristics of the VP1 gene (B segment) as well. The IBDV genome segmentation allows formation of reassortant viruses which can be identified as a result of the genome segment analysis. The phylogenic analysis of the nucleotide sequences of VP2 and VP1 genes of 28 IBDV isolates detected at RF, Ukrainian and Kazakh poultry establishments in 2007 and 2019 showed that 15 of them are reassortant viruses. Different combinations of the genome segments have been identified among these reassortant viruses. Detection of different combinations of IBDV genome segments is indicative of the fact that the heterogeneous virus population circulates on the poultry farms. Pathogenicity studies of the three IBDV isolates showed that the most virulent was an isolate having two genome segments characteristic of the highly virulent virus. Two reassortant viruses having only one genome segment A or B, characteristic of the infectious bursal disease, demonstrated less pronounced virulent properties

HEMAGGLUTINATION AND ANTIGENIC PROPERTIES OF AVIBACTERIUM PARAGALLINARUM ISOLATES RECOVERED IN THE RUSSIAN FEDERATION AND REPUBLIC OF BELARUS

The paper demonstrates examination results of antigenic properties of Avibacterium paragallinarum reference strain and ten isolates thereof recovered in the Russian Federation and Republic of Belarus. Nine isolates and the reference strain demonstrated type L hemagglutinin (thermoliable, trypsin-sensitive, active against fresh and glutaraldehyde-treated RBC). One isolate was marked by type Hl hemagglutinin (thermoliable, trypsin-resistant, active only against glutaraldehydetreated RBC). The reference strain antigen proved to be inagglutinable by homologous antiserum, and unable to agglutinate RBCs due to hyaluronic acid in the capsular substance. Serological and hemagglutination non-reactivity was removed through the cell treatment with hyaluronidase. The agglutination test demonstrated that seven out of ten tested isolates belonged to the same serological group; herewith, the proportion of the bilateral antigenic relatedness amounted to ≥78.4%. HI results demonstrated ≥92.6% antigenic relatedness of the tested isolates being indicative of the fact that they belonged not only to the same serological group but also to the same serotype. No serological relatedness was identifed between the tested isolates and reference strain of serogroup A both using agglutination test (≤23.6%) and HI (≤12.2%). Polymerase chain reaction demonstrated that all the isolates recovered in the Russian Federation and Republic of Belarus in 2014-2016 belonged to serogroup B

RESULTS OF EXPERIMENTAL INFECTION OF TURKEYS WITH A/DUCK/ALTAI/469/14 H5N1 STRAIN OF AVIAN INFLUENZA VIRUS

The data on experimental infection of 6-week-old Big-6 cross turkeys with an epidemic A/duck/Altai/469/14 H5N1 clade 2.3.2.1c strain of avian influenza virus are presented. The characteristics of the infection process in birds inoculated intranasally at a dose of 5.0 lg EID50/0.5 cm3 are described with an indication of the incubation period and the mean time of death. The pathomorphological changes at the tissue and cellular level are shown based on histological and immunohistochemical studies of fragments of respiratory, digestive, cardiovascular, nervous, excretory, lymphoid and muscular systems of experimental birds. The testing was carried out using paired preparations of paraffin-embedded tissue sections from experimentally infected and healthy turkeys. One sample was subjected to histological staining using hematoxylin and eosin dyes, and its duplicate was subjected to immunohistochemical assay using a preparation of polyclonal antibodies as primary antibodies against the ribonucleoprotein of avian influenza virus. The results of histological and immunohistochemical studies are photodocumented and presented in the paper. Inflammatory and necrotic lesions of varying severity are detected in the preparations of the trachea, lung, muscular stomach, glandular stomach, small intestine, large intestine, pancreas, brain, cerebellum, heart, kidneys, liver and spleen of turkeys. Immunohistochemical analysis showed the greatest distribution of the influenza virus antigen in the cerebral endothelium, cerebellar Purkinje neurons, acinar cells of the pancreas and in myocardiocytes of the heart. In the course of the experiment it was established that A/duck/Altai/469/14 H5N1 caused a generalized form of infection in turkeys with clinical and pathologic lesions characteristic of highly pathogenic avian influenza

Serological monitoring of avian influenza and Newcastle disease in the Russian Federation in 2019

More than 30,000 samples of blood serum from domestic, wild and synanthropic birds from 50 regions of the Russian Federation were submitted to the FGBI “ARRIAH” (Vladimir) Reference Laboratory for Avian Viral Diseases to be tested for avian influenza and Newcastle disease within the framework of monitoring activities conducted by the Rosselkhoznadzor in 2019. As a result of the laboratory diagnosis, antibodies to type A influenza virus were detected in vaccinated chickens from two poultry farms in the Perm and Primorsky Krais (A/N9). The detected antibodies were specific to the haemagglutinin subtype of the vaccine antigen. As for the backyards in the RF Subjects, where scheduled vaccination against avian influenza A/H5 is carried out, a low level of immunity was seen in the Rostov and Astrakhan Oblasts (35 and 44%, respectively) while a high level of immunity was observed in the Republic of Altai, Krasnodar Krai, the Chechen Republic and the Primorsky Krai (69, 78, 80 and 88%, respectively). High seroprevalence of Newcastle disease virus in adult poultry in indoor holdings was associated with mass vaccination against the disease. In broiler chickens, post-vaccination antibodies were observed, on average, in 42% of the studied blood serum samples. Antibodies to the Newcastle disease virus were detected in 39% of samples from backyard chickens. Seroprevalence in wild and synanthropic birds was high. The obtained results suggest that the risk of introduction and spread of avian influenza and Newcastle disease in industrial poultry farms and in backyards remains

Development of the test kit for detection of SARS-CoV-2 antibodies in sera of susceptible animals

The novel coronavirus infection COVID-19, caused by the SARS-CoV-2, has triggered a pandemic, and has also been reported in animal populations – in farm minks, dogs and felines: domestic cats, lions and tigers. The susceptibility of some animal species to the SARS-CoV-2 has been proven by experimental infection. Serological methods are effectively used to detect the infection in animals. Currently, methods such as neutralization test, immunofluorescence assay and enzyme-linked immunoassay are used to detect antibodies to coronaviruses. Thanks to these studies, a test kit was developed based on an indirect enzyme-linked immunoassay to detect the SARS-CoV-2 antibodies in sera of susceptible animals. The use of a purified concentrated inactivated virus as an antigen allows the detection of antibodies to various SARS-CoV-2 immunodominant proteins (S and N). The reaction conditions were optimized, and a positive-negative threshold was established by testing of 154 negative sera from animals of six species (ferrets, minks, foxes, arctic foxes, cats and dogs). The method reproducibility analysis showed that the average value of the variation coefficient did not exceed 7%, which is an acceptable value. The specificity and sensitivity of the neutralization test, when testing 30 sera from ferrets was 100 and 92.6%, respectively. The high diagnostic sensitivity and specificity shown by testing of 50 serum samples from minks, foxes, cats and dogs with different immune status, allow us to recommend the developed test kit for screening and monitoring tests and post-vaccination immunity control

ANALYSIS OF GENETIC CHARACTERISTICS OF INFLUENZA VIRUS A/CHICKEN/CHELYABINSK/30/2019 H9N2 ISOLATED IN CHELYABINSK OBLAST

The paper presents data on the study of genetic characteristics of the infl uenza virus A/chicken/ Chelyabinsk/30/2019 H9N2 isolated from pathological material (chicken internal organs) in February 2019 and received from the poultry farm in the Chelyabinsk Oblast. The H9N2 subtype of the isolated virus was identifi ed based on virological analysis. Sequencing of the hemagglutinin gene segment revealed that the amino acid sequence at the cleavage site was RSSR/GLF, which is characteristic of a low virulent avian infl uenza virus. Phylogenetic analysis of the obtained nucleotide sequences of the hemagglutinin gene fragment (1–1539 bp open reading frame) showed that the A/chicken/Chelyabinsk/30/2019 H9N2 isolate belongs to the G1 genetic group of the low virulent infl uenza virus A/H9, the representatives of which are widely spread in the Middle Eastern and Central Asian countries. The complete nucleotide genome sequence of the studied pathogen was determined. The comparative analysis of all genomic segments using the GenBank database revealed a close relationship (over 99%) between the A/chicken/Chelyabinsk/30/2019 H9N2 virus and the A/H9 infl uenza virus isolates circulating in Israel in 2006–2012. According to the analysis of the predicted amino acid sequence of the studied isolate, the positions of some molecular markers that determine the biological properties of the virus have been identifi ed. Most amino acid positions of hemagglutinin (according to H3 subtype sequence numbering) suggest affi nity for the ACA2-3Gal-receptors of avian epithelial cells. Amino acid substitutions were detected at the site within the receptor-binding domain as compared to the A/H9N2 infl uenza virus isolates obtained in Russia in 2018. The primary structure of the A/chicken/Chelyabinsk/30/2019 H9N2 isolate demonstrates a very high level of genetic similarity to the infl uenza virus isolate A/chicken/ Israel/215/2007 H9N2 used as a vaccine strain