Detection of specific antibodies to Schmallenberg virus using microneutralisation test

The article presents data on microneutralization test for detection of specific antibodies to Schmallenberg virus. The method is characterized by high sensitivity and specificity. It can be used for analysis of blood sera of different species of animals

Optimization of medium composition and study of growth stages of <i>Mycoplasma bovis</i> “Kaluga 2020” isolate

Mycoplasma bovis is considered one of bovine mycoplasmosis pathogens responsible for respiratory diseases, mastitis, arthritis and keratoconjunctivitis. The paper presents results of the study on optimizing the component composition of the culture medium for Mycoplasma bovis “Kaluga 2020” isolate, as well as the study of this pathogen’s growth stages. The color-changing units assay and the culture method combined with colony-forming unit quantification were used for determination of Mycoplasma activity. It was found that when cultured in an optimized nutrient medium based on modified Hayflick broth, the microorganism enters a logarithmic growth phase after first 24 hours ofgrowth, in 72 hours the Mycoplasma culture enters astability phase, and adecline phase is recorded in 84 hours. The effect of percentage content of glucose, fresh yeast extract and horse serum in the nutrient medium on accumulation of Mycoplasma bovis “Kaluga2020” isolate was evaluated using the one-factor-at-a-time approach. It was found that the greatest effect on Mycoplasma accumulation was exerted by such growth factors as fresh yeast extract and horse serum in the nutrient medium (p &lt; 0.05), while changes in the amount of glucose did not stimulate Mycoplasma bovis growth. Based on results of the conducted studies, the appropriate composition was determined and the optimal content of growth factors in the medium for culturing Mycoplasma bovis “Kaluga 2020” isolate was selected: 12.5%of fresh yeast extract and 25% of horse serum. The use of the optimized nutrient medium based on modified Hayflick broth allowed 5-fold increase in accumulation of Mycoplasma biomass (3.98 × 109CFU/ml)compared to the standard medium (0.79 × 109CFU/ml)

Metagenomic profiling of viral and microbial communities from the pox lesions of lumpy skin disease virus and sheeppox virus-infected hosts

IntroductionIt has been recognized that capripoxvirus infections have a strong cutaneous tropism with the manifestation of skin lesions in the form of nodules and scabs in the respective hosts, followed by necrosis and sloughing off. Considering that the skin microbiota is a complex community of commensal bacteria, fungi and viruses that are influenced by infections leading to pathological states, there is no evidence on how the skin microbiome is affected during capripoxvirus pathogenesis.MethodsIn this study, shotgun metagenomic sequencing was used to investigate the microbiome in pox lesions from hosts infected with lumpy skin disease virus and sheep pox virus.ResultsThe analysis revealed a high degree of variability in bacterial community structures across affected skin samples, indicating the importance of specific commensal microorganisms colonizing individual hosts. The most common and abundant bacteria found in scab samples were Fusobacterium necrophorum, Streptococcus dysgalactiae, Helcococcus ovis and Trueperella pyogenes, irrespective of host. Bacterial reads belonging to the genera Moraxella, Mannheimia, Corynebacterium, Staphylococcus and Micrococcus were identified.DiscussionThis study is the first to investigate capripox virus-associated changes in the skin microbiome using whole-genome metagenomic profiling. The findings will provide a basis for further investigation into capripoxvirus pathogenesis. In addition, this study highlights the challenge of selecting an optimal bioinformatics approach for the analysis of metagenomic data in clinical and veterinary practice. For example, direct classification of reads using a kmer-based algorithm resulted in a significant number of systematic false positives, which may be attributed to the peculiarities of the algorithm and database selection. On the contrary, the process of de novo assembly requires a large number of target reads from the symbiotic microbial community. In this work, the obtained sequencing data were processed by three different approaches, including direct classification of reads based on k-mers, mapping of reads to a marker gene database, and de novo assembly and binning of metagenomic contigs. The advantages and disadvantages of these techniques and their practicality in veterinary settings are discussed in relation to the results obtained

Development and use of indirect liquid-phase ELISA test system for detection of PRRS virus antigen during in-process control of raw materials intended for vaccine production

Porcine reproductive and respiratory syndrome (PRRS) being endemic and reported in the most countries in the world remains one of the most challenging diseases in pig industry. The main disease control measures include preventive vaccination and animal movement control within and outside the country as well as diagnostic testing of pigs in the population. Live and inactivated vaccines are used for specific prevention of porcine reproductive and respiratory syndrome. Complete and irreversible infectious agent inactivation with maximum epitope preservation and protective immunity in immunized animals are the main requirements for inactivated vaccines. Therefore, continuous improvement of methods for vaccine quality control at various vaccine production stages is of current importance. Results of development of the test system based on indirect liquid-phase enzyme-linked immunosorbent assay (ELISA) for PRRS virus antigen detection and activity testing in infectious and inactivated virus-containing cell cultures at intermediate stages of the vaccine production process are described in the paper. The test-system development process included purified and concentrated virus antigen as well as hyperimmune rabbit sera preparation. Specificity of purified and concentrated virus antigen was confirmed with real-time polymerase chain reaction. The developed test-system was shown to detect the virus antigen at initial infectivity titre of 4.87–7.21 lg TCID50/cm3 corresponding to ELISA titre (dilution) of 1:4 up to 1:64. Methodical Guidelines for detection of porcine reproductive and respiratory syndrome virus antigen with indirect liquid-phase enzyme-linked immunosorbent assay (ELISA) (2019) were developed based on the work results, commissioned and approved by the FGBI “ARRIAH” Scientific Board

IMMUNOBIOLOGICAL PROPERTIES OF LUMPY SKIN DISEASE VIRUS STRAIN LSD CATTLE/DAGESTAN/2015

Data on the study of immunobiological properties of a novel lumpy skin disease virus strain LSD Cattle/Dagestan/2015 are presented in the paper. In the course of the research it was found that the obtained strain is biologically active, free from contamination with foreign biological agents. It is type specific and with its immunobiological characteristics meets the standards and requirements set before virus strains used for vaccine and diagnostic production

Evaluation of veterinary laboratory proficiency based on results of interlaboratory comparisons organized by FGBI “ARRIAH” in 2018–2019

Laboratory diagnosis is a crucial component in implementation of the set of anti-epidemic measures aimed at contagious animal disease control. The need for unswerving trust in the quality of laboratory performance is a matter of importance not only for service providers and customers, but also for inspecting organiza­tions, accreditation bodies, etc. that establish performance requirements for diagnostic laboratories. Incorrect laboratory test results can lead to a misdiagnosis and, therefore, to grave consequences. One of the forms of experimental verification of a laboratory’s performance with a view to determine the laboratory’s competence and to verify its compliance with accreditation criteria as part of inspection control of the laboratory’s activities is interlaboratory comparison. The laboratory can prove its competence at a particular time, as well as clearly demonstrate how stable the quality of its test results is by summarizing and analyzing the results of its participation in interlaboratory comparisons. The analysis of the results of the interlaboratory comparisons (programmes for detection of causative agents or antibodies to the causative agents of avian influenza, Newcastle disease, rabies, classical swine fever, African swine fever, bluetongue, lumpy skin disease) organized by the FGBI “ARRIAH” for the veterinary laboratories of the Russian Federation in 2018–2019 is presented. The results showed that most of the laboratories had passed the tests successfully. The results submitted by participants were unsatisfactory in some interlaboratory comparison programmes (rabies virus detection using fluorescent antibody technique; detection of avian influenza, classical swine fever and lumpy skin disease viruses using polymerase chain reaction). That highlights the need for those participants who failed the tests to improve their laboratory testing quality

CULTURAL PROPERTIES OF BOVINE RESPIRATORY SYNCYTIAL VIRUS STRAIN AB1908

Cattle respiratory diseases are some of the most spread pathologies that can cause economic damage, resulting from fi nancial losses and costs of treatment and diagnostics. One of the major factors contributing to respiratory pathology development is bovine respiratory syncytial infection. The analysis of serological testing, performed by the FGBI “ARRIAH” Reference Laboratory for Cattle Diseases in 2017–2018, showed that respiratory syncytial virus seroprevalence in animals of dairy farms is 60%. Herewith, it was noted that the most susceptible animals to this infection are calves under one year of age. The eff ectiveness of bovine respiratory syncytial infection control measures depends on timely diagnosis; that is why reliable and accurate diagnostic tools are needed, including optimal techniques of virus isolation from pathological material. For successful virus isolation from clinical samples, it is necessary to adhere strictly to optimal parameters of this agent cultivation. This paper presents data on study of bovine respiratory syncytial virus strain AB 1908 cultural properties. The tests performed showed that a continuous bovine turbinate (BT) cell line, continuous bovine fetal trachea (FBT) cell line and continuous bovine calf kidney (RBT) cell line are sensitive for cultivation of this agent and can be used to prepare viral suspension, needed for further research. Virus titre in BT cell culture was 4.33 ± 0.16 – 4.66 ± 0.12 lg TCID50/ cm3, in RBT cell culture – 4.33 ± 0.33 – 4.7 ± 0.36 lg TCID50/cm3 and in FBT cell culture – 4.13 ± 0.20 – 4.78 ± 0.17 lg TCID50/cm3. The following virus cultivation optimal parameters were also determined during this study: the age of the culture for virus inoculation should be 1–2 days and multiplicity of inoculation should be 0.1 TCID50/cell

TEST-SYSTEM BASED ON INDIRECT “SANDWICH” ELISA TO DETECT THE LUMPY SKIN DISEASE VIRUS ANTIGEN

Lumpy skin disease is an economically significant disease as it results in decrease in weight gain and milk yield, abortions, mastitis, reproduction disorders, animal emaciation, lesions of respiratory organs and in some cases – death. Today the disease is included in the OIE list and is subject to obligatory notification. The emergence and spread of the disease in the Russian Federation necessitated performance of tests in the framework of laboratory diagnosis method improvement. The test-system based on the indirect “Sandwich” ELISA for diagnosis of lumpy skin disease allowing performance of biomaterial tests within 24 hours was for the first time developed in Russia. The test-system development included antibody preparation in laboratory animals (rabbits and guinea pigs), selection of optimal dilution of capture and detection antibodies, composition of a buffer solution and conditions of the reaction procedure. 50 samples of initial culture antigens of the lumpy skin disease virus as well virus-containing suspensions collected on different stages of purification and concentration were tested using this technique. To confirm the ELISA results all analyzed samples were tested using RT-PCR. Besides, the virus infectivity titer was determined by titration in YaDK-04 (Goat gonad cells). The test specificity was 100%, and analytical sensitivity – 3.5 lg TCD50. The developed “Sandwich” ELISA allows performing tests of 24 antigen samples at 1:2–1:16 dilution simultaneously using 96-well plate and it can be used for lumpy skin disease diagnosis

Bovine mycoplasmosis occurrence on livestock farms in the Russian Federation for 2015–2018

Mycoplasmosis control remains urgent in view of wide spread of bovine mycoplasmoses in the countries with intensive animal farming and trade relations between the Russian Federation and foreign partners including import of pedigree livestock and stud bull semen. Results of testing 1,186 biomaterial samples (blood, sera, nasal swabs, milk, preputial swabs, vaginal swabs, aborted and stillborn fetuses) collected from animals that demonstrated clinical signs of respiratory and reproductive disorders in 34 different regions of the Russian Federation for 2015–2018 are presented in the paper. The samples were tested with real-time polymerase chain reaction (rtPCR) for genomes of the following mycoplasmosis agents: Mycoplasma bovis, Mycoplasma bovigenitalium, Mycoplasma dispar. As a result, M. bovis genome was detected in 10.1% of the samples, M. bovigenitalium genome was detected in 8.6% of the samples and М. dispar genome was detected in 37.15% of the samples. Also, 927 semen samples submitted from Russian and foreign breeding farms were tested with PCR. Test results showed presence of M. bovis and M. bovigenitalium genomes in semen samples collected from native bull population. Presented data support Russian scientists’ conclusions on wide mycoplasmoses occurrence in cattle in the Russian Federation territory and risk of the disease agent introduction through semen import. All of these highlight the need for control of semen products as a source for mycoplasmosis spread as well as insufficiency of single testing of semen for granting the disease-free status to the breeding farm for genetic material marketing

Optimization of cultivation parameters for bovine respiratory syncytial virus strain Vologda/2019

There are currently many controversial issues in the study of bovine respiratory syncytial infection. In this regard, it is relevant to study the biological properties of the virus, optimize the methods of its cultivation and select the most technologically advanced methods of designing diagnostic and prevention tools for this disease. The aim of this work was to select sensitive cell systems and to optimize the cultivation parameters in selected cell cultures. The Vologda/2019 strain of the bovine respiratory syncytial infection virus isolated from biological material obtained from a calf with respiratory symptoms was used in the experiment. The strain was adapted to the continuous cell culture derived from bovine turbinate tissue (BT) and deposited in the State collection of microorganism strains at FGBI “ARRIAH”. It was established that the continuous cell lines of fetal bovine trachea (FBT) and calf kidney (RBT) are the most sensitive cell systems for the reproduction of the bovine respiratory syncytial virus strain Vologda/2019, the maximum accumulation of the virus was observed in these cell cultures. The cytopathic activity of the virus in the FBT cell culture ranged from 4.78 ± 0.18 to 5.50 ± 0.16 lg TCID50/cm3, and in the RBT cell culture – from 4.00 ± 0.23 to 4.75 ± 0.20 lg TCID50/cm3 at days 4–5 of cultivation. It was determined that in case of multiplicity of inoculation of FBT and RBT cell cultures with the virus at 0.1 lg TCD50/cell and the use of 2% glutamine in the maintenance nutrient medium, as well as 2% horse or cattle blood serum, it is possible to obtain virus material with high cytopathic activity