Modern approaches to production of safe and effective genetically modified rabies vaccines for animals

Rabies is a dangerous zoonoticdisease that affects the central nervous system, causes encephalomyelitis and paralyses and Is almost invariably fatal. The disease causes significant economic losses associated with the death of animals, outbreak consequences, strict restrictions on domestic and international trade in livestock products, preventive and quarantine measures, laboratory tests. The World Organization for Animal Health recommends vaccination to control rabies. Taking into account that there is a lack of affordable high-quality vaccines to globally prevent and control the disease, stable, attenuated production strains of rabies virus with broad cross-activity against various variants of the pathogen shall be considered as ideal candidates to produce high-quality, safe and effective vaccines. Currently, someapproachesareappliedtoreducethevirusvirulenceandimprovesafetyof rabies vaccines. Reverse genetics is very popular now. It provides new approaches to study functions of a specific gene by analyzing phenotypic effects after direct manipulations with nucleotide sequences. The methods of reverse genetics have revolutionized molecular biology and have become apowerful tool to study genetics of RNA viruses. These methods are widely used to study rabies virus. The use of reverse genetics has made it possible to modify rabies virus production strains for manufacture of modern genetically modified rabies vaccines that induce a persistent and long-term immunity. The review briefly covers general approaches to development of viral vectors with the purpose to create genetically modified rabies vaccines

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

ENTOMOLOGICAL ASPECTS OF LUMPY SKIN DISEASE EPIZOOTOLOGY (REVIEW)

Lumpy skin disease (LSD) is a serious threat to the global cattle farming, including that in the Russian Federation where the frst outbreak was reported in 2015. Since the disease occurred for the frst time, it has continued to spread in this country; however, virus transmission mechanisms have not yet been studied. Transmission through insect bites is considered to be the most likely mechanism of virus shortrange transmission. At present, such arthropod species as stable fly (Stomoxys сalcitrans), Aedes aegypti mosquitoes, as well as Amblyomma hebraeum and Rhipicephalus appendiculatus ticks are regarded as potential vectors. Viral DNA has also been detected on the exoskeletons of house flies (Musca domestica). The available literature describes the results of many studies on the role of arthropods in LSD virus spread, but the data presented are inconsistent and do not provide an unambiguous answer concerning the level of signifcance of potential LSD virus vectors in the progression of the feld epizootic. These papers investigate the ability of gadflies, flies and ticks to act as mechanical vectors. Currently, there is no unequivocal viewpoint with respect to the proved LSD vector. This paper reviews the entomological papers aimed at studying possible LSD virus transmission by arthropods

Identification of Haemophilus parasuis using MALDI-TOF mass spectrometry

The paper covers results of MALDI-TOF mass spectrometry used to rapidly identify Haemophilus parasuis field isolates. ClinProTools software showed 15 peaks in the protein spectrum with 100% identification capability differentiating 6 Pasteurellaceae species. Peak corresponding to mass of 8407.92 Da (

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

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

DETECTION OF LUMPY SKIN DISEASE VIRUS GENOME IN FIELD SAMPLES COLLECTED FROM CATTLE IN THE RUSSIAN FEDERATION

Results of laboratory tests of pathological and biological material samples for lumpy skin disease virus genome with the FGBI “ARRIAH” lumpy skin disease real-time PCR test system are presented. Samples were collected from cattle in 16 regions of the Russian Federation in 2016–2017. A total of 848 stabilized blood, serum, skin (nodule) scrape, nasal washing samples were tested. In the process of antemortem diagnosis lumpy skin disease virus genome was detected in nasal washings (29.2%), serum (19.5%) and stabilized blood (24.4%). Lumpy skin disease virus genome was detected in skin lesion samples (77.7% of cases) during postmortem diagnosis. No lumpy skin disease virus genome was detected in trachea, spleen and aborted fetus samples. Thus, in case of lumpy skin disease suspicion serum and nasal washing samples shall be tested first for antemortem diagnosis, while nodules (lumps) shall be primarily tested for postmortem diagnosis