Background. Diseases caused by clostridia are widespread in cattle. In addition to pathogenic clostridia, non-pathogenic species are constantly living in the organism of animals, and to make a correct diagnosis it is necessary to differentiate isolated cultures of these bacteria. A promising direction for solving this problem is the search for highly specific and sensitive methods of genetic analysis. Based on the application of real-time polymerase chain reaction (PCR), which makes it possible to detect and determine the species affiliation of microorganisms in a short period of time. Purpose. To develop a multiplex real-time polymerase chain reaction for the detection of toxigenic Clostridium species: Clostridium sporogenes, Clostridium perfringens and Clostridium sordellii in mixed and pure bacterial cultures, to determine its sensitivity and specificity. Materials and methods. In our studies we used samples of biological material from sick animals, from which clostridia cultures were isolated on artificial nutrient media with their subsequent identification based on the results of culture and biochemical properties. Primers and probes were designed using the PrimerQuest tool, and then the sensitivity and specificity of PCR were determined using reference strains and bacterial isolates. Results. In the period from 2023 to 2024, 90 samples of biomaterial from cattle collected in livestock farms of the Novosibirsk Region were investigated. As a result of bacteriological studies, 44 isolates of clostridia of nine species of the genus Clostridium were obtained. For the selection of primers and probes, we analysed three pairs of primers and probes, which were used to detect the gerKA gene in C. sporogenes, C. perfringens - plc, C. sordelli - NanS. In addition, a pair of primers was used to detect species of the genus Clostridium by the 16S RNA gene. In order to determine the working concentrations of primers and probes that would provide the necessary sensitivity of the analysis, a series of studies were carried out to optimise the conditions of the analysis. Analysing the results of the studies, we determined the sensitivity of the reaction, which was not less than 102 CFU/ml for pure bacterial cultures of C. sporogenes, C. perfringens and C. sordelli species, and for other species of Clostridia spp. 101 CFU/ml. Conclusion. The developed multiplex real-time PCR allows short-term diagnosis of bovine clostridiosis and species identification of pathogens. In addition to C. perfringens, C. sporogenes, C. sordellii, other species of Clostridium spp. may be involved in the etiology of bovine clostridiosis. It is necessary to improve diagnostic methods of clostridiosis and expand the range of detectable clostridium species. Sponsorship information. This work was financially supported by the Russian Science Foundation under the project 23-26-00009 “Species composition and toxigenic properties of clostridia in cattle in the West Siberian region and development of a test system for their rapid identification”. EDN: PSCOZR

Introduction. Bovine respiratory syncytial virus (Pneumoviridae: Orthornavirae, Orthopneumovirus; Bovine orthopneumovirus, Bovine respiratory syncytial virus, BRSV) is one of causative agents of respiratory diseases in animals. The study of the occurrence and genetic diversity of this pathogen is of particular importance. Objective. To study the frequency of virus in animals using RT-PCR and genetic heterogeneity of isolates based on determining the complete nucleotide sequence of glycoprotein G gene. Materials and methods. A 381-bp region of glycoprotein F gene was used for identification of virus genome, while complete nucleotide sequences of G gene were used for phylogenetic analysis. Phylogenetic trees were constructed using the maximum likelihood method in MEGA 7.0 software. Results. During outbreaks of respiratory diseases, BRSV RNA was detected in animals of all ages in samples of lungs, nasal secretions, pulmonary lymph nodes. Complete nucleotide sequences of glycoprotein G gene, 771 bp in length were obtained for five isolates and 789 bp in length ‒ for two isolates. Nucleotide similarity between them was 87–100%. Phylogenetic analysis assigned the isolates to subgroups II and III, each of which included two isolates. A separate clade formed by K18 isolate from animals imported from Canada and sequences from vaccines containing the attenuated «375» strain. Conclusion. The virus genome was identified in cows and heifers (20.0 and 14.3%), in calves up 1 month of age (3.05%), and in calves from 1 to 6 months of age (6.7%). Complete G gene nucleotide sequence analysis is a useful tool for studying the molecular epidemiology of BRSV on particular territories.

The article presents the results of development of multiplex polymerase chain reaction in real time for detection and differentiation of three species of bovine pestiviruses (Pestivirus A, B and H). Synthetic oligonucleotide primers and probes complementary to the highly conserved 5/-UTR region of the genome, specific for all species of the genus Pestivirus and for each species separately were selected. The main parameters of the reaction were worked out. The analytical sensitivity of the developed real-time PCR was 1,6×102 GE for Pestivirus spp., 7,2×10 GE for Pestivirus A, 9,0×10 GE for Pestivirus B and 1.5×103 GE for Pestivirus H. PCR has high specificity and does not detect RNA of other viruses. Using the developed method, 18 samples of embryonic serum, 12 types of continuous cell culture lines, 10 series of live vaccines for immunization of cattle, 3050 samples of biomaterial from sick animals were studied. As a result of the studies, the Pestivirus H genome was detected in 7 samples of embryonic serum, 18 samples of internal organs of calves and 1 live vaccine. Pestivirus A and Pestivirus B RNA were detected in 173 and 5 samples of biomaterial from animals, 2 and 1 samples of continuous cell lines, respectively. Multiplex real-time PCR can be used for express diagnostics of cattle diseases caused by pestiviruses and control of biological products.

The results of comparative phylogenetic analysis of 52 isolates of bovine pestiviruses of three species circulating among highly productive dairy cattle in Siberia, as well as those present in the samples of commercial fetal serum, transfected cell culture lines and live vaccines on four genes: Npro, Erns, E1 and E2 are presented. The obtained data were compared with the results of the 5'UTR gene sequencing obtained earlier. The results confirmed the circulation in Siberia of eleven subtypes of BVDV-1 (a,b,c,d,f,g,i,j, k,p,r), three subtypes of BVDV-2 (a,b,c) and one BVDV -3(a). The genetic profiles of the isolates for the 5'UTR, Npro, Erns and E1 genes matched completely. The exceptions were three BVDV-1a isolates (R/FBS/96, N/MDBK/08 and SA/FBS/08) detected in infected cell cultures that clustered in a clade formed by BVDV-1j reference sequences. The remaining isolates previously shown to have 100% sequence identity of the 5'UTR had nucleotide sequence similarities of the Npro, Erns, E1, and E2 genes ranging from 96-99%, which also implies that they are closely related. The results of the BVDV-2 isolates sequencing showed a complete correspondence with the genome sequences identified in the previous studies. Comparative sequencing also confirmed the circulation of H Italian-Brazilian pestivirus isolates (BVBD-3a) in Siberia, but all the isolates were divided into two subclades. The first included the isolates isolated from fetal sera and cell cultures, while the second included those isolated from the vaccines and internal organs of diseased animals during outbreaks in farms. The genetic diversity of bovine pestiviruses, their origin and variability are discussed.

The article presents the results of isolation of Clostridium spp. bacteria and their associations from cattle with different clinical forms of clostridiosis. From 2016 to 2023, 910 samples of biomaterial collected from cows, heifers and calves under 6 months of age, as well as from stillborn calves and aborted fetuses in case of reproductive pathologies in cows were investigated by bacteriological methods. The following species play a major role in the etiology of bovine clostridiosis: C. perfringens, C. septicum, C. novyi (C. oedematiens), C. histolyticum and C. sordellii. C. histolyticum (65.2%), C. septicum (45.6), C. perfringens (29.7) and C. sporogenes (26.1) were isolated most frequently from cows and heifers, while C. sordellii (1.7) and C. novyi (0.9%) were isolated less frequently. Clostridium spp. bacteria of six species were isolated from animals in enterotoxemia and enteritis, and five species in vaginitis and endometritis. In other clinical forms of the disease, bacteria of four species were isolated from cows and heifers. The spectrum of bacteria involved in the development of abomasitis, enteritis and enterotoxemia in calves was similar and represented by four bacterial species: C. histolyticum, C. septicum, C. perfringens and C. sporogenes. Bacteria of five species were isolated from the calves with septicaemic form of the disease. The peculiarities of clinical manifestation and the course of clostridiosis in cattle depended on the species composition of pathogens and their associations. Diseases caused by Clostridium spp. bacteria were acute or subacute, characterized by visible lesions of organs and tissues in a few hours after the animal showed signs of disease and in most cases ended in death.

The influenza D virus was first detected and identified in 2011. The overall amino acid sequence of influenza D virus shares approximately 50% identity with that of influenza C virus, suggesting that both viruses had a common ancestor. Cattle is considered to be the primary natural reservoir for influenza D virus. The involvement of this virus into the bovine respiratory disease complex has been confirmed. The virus causes mild to moderate disease in calves and replicates in both the upper and lower respiratory tracts, promoting bronchopneumonia. The influenza D virus can be transmitted by contact or aerosol over short distances, has a high transmission rate and can potentiate the effects of other respiratory pathogens. There are currently no vaccines or specific treatment for influenza D virus. This virus can replicate and be transmitted by direct contact in ferrets and guinea pigs, which are surrogate models of human influenza infection, as well as in well-differentiated human airway epithelial cells (hAECs). Currently five distinctive lineages of influenza D virus have been identified, co-circulating in worldwide bovine and pig populations that may facilitate genetic re-assortment between different viral strains. The virus has a zoonotic potential, and if its pathogenicity for humans changes, its importance for public health will be great. Very high seropositivity rates among persons working with cattle in the USA and Italy have been reported. There is no data in the available literature on the circulation of the influenza D virus in the Russian Federation. Research is needed to study this new virus, as well as monitoring of the virus spread and circulation in our country to understand its role in bovine respiratory disease complex and its zoonotic potential.

INTRODUCTION:Bovine coronaviruses (BCoVs) are causative agents of diarrhea, respiratory diseases in calves and winter cow dysentery. The study of genetic diversity of these viruses is topical issue. The purpose of the research is studying the genetic diversity of BCoV isolates circulating among dairy cattle in Siberia.MATERIALS AND METHODS:Specimens used in this study were collected from animals that died or was forcedly slaughtered before the start of the study. The target for amplification were nucleotide sequences of S and N gene regions.RESULTS:Based on the results of RT-PCR testing, virus genome was present in 16.3% of samples from calves with diarrheal syndrome and in 9.9% with respiratory syndrome. The nucleotide sequences of S gene region were determined for 18 isolates, and N gene sequences - for 12 isolates. Based on S gene, isolates were divided into two clades each containing two subclades. First subclade of first clade (European line) included 11 isolates. Second one included classic strains Quebec and Mebus, strains from Europe, USA and Korea, but none of sequences from this study belonged to this subclade. 6 isolates belonged to first subclade of second clade (American-Asian line). Second subclade (mixed line) included one isolate. N gene sequences formed two clades, one of them included two subclades. First subclade included 3 isolates (American-Asian line), and second subclade (mixed) included one isolate. Second clade (mixed) included 8 sequences. No differences in phylogenetic grouping between intestinal and respiratory isolates, as well as according to their geographic origin were identified.CONCLUSION:The studied population of BCoV isolates is heterogeneous. Nucleotide sequence analysis is a useful tool for studying molecular epidemiology of BCoV. It can be beneficial for choice of vaccines to be used in a particular geographic region.

The results of the study of specific features of distribution and clinical manifestation of bovine cryptosporidiosis in association with pathogens of viral and bacterial nature are presented. From 2014 to 2023, 666 samples of biomaterial (tracheal mucosa, lungs, mediastinal and mesenteric lymph nodes, contents of rennet and intestine) collected from fallen and forcefully killed calves under 6 months of age with signs of enteritis and respiratory diseases were examined. 94 farms of six regions, two territories of the Siberian region and the Republic of Kazakhstan were surveyed. The findings confirm the widespread prevalence of Cryptosporidium in calves. Cryptosporidium parvum oocysts were most frequently detected and were present in 34 (5.1%) samples of biological material from calves in 22 (23.4%) of the surveyed farms. It was found that oocysts were rarely (22.7%) present in monovariant in animals, more often (77.3%) they were found in associations with viruses and bacteria during outbreaks of viral-bacterial infections. Young animals under 30 days of age are most susceptible to infection. Newborn calves are most susceptible to infection with oocysts, and those animals that have had the disease become lifelong sources of the pathogen for susceptible animals. Cryptosporidiosis is an independent disease and runs independently of the presence of the viruses and bacteria in the body and has no synergistic interaction with them, but the presence and multiplication in the body of animals of this pathogen contributes to the severity of the course of enteritis in calves and leads to difficulties in the treatment and preventive measures. The presence of Cryptosporidium in animals should be taken into account when carrying out complex anti-epizootic measures.

An outbreak of infection caused by Pestivirus H (virus of bovine viral diarrhea – mucosal disease of the third kind, BVDV-3) in a dairy farm with high morbidity and mortality in animals of different ages is described. In some sick animals a full complex of pronounced symptoms characteristic of "classical" bovine mucosal disease was registered: erosions and ulcers on the nasal mirror and tongue, foaming from the mouth, serous discharge from the nose, hemorrhagic inflammation and pronounced longitudinal erosions on the mucosa of the esophagus, rennet stomach and intestine. Cows miscarried at different stages of pregnancy. The coefficient of effective insemination decreased to 20%. The course of the disease was complicated by the involvement of the bovine herpes virus type 4, bacteria of the family Pasteurellaceae and Clostridium spp. in the infectious process. The BVDV-3 genome was found in a wide range of internal organs of aborted fetuses, calves, and adult animals. According to sequencing data, the pathogen was classified as subtype 3a. Phylogenetic analysis of the 5'-untranslated region of the virus genome (5'-UTR) showed its close relationship to the strains isolated in Italy and Brazil, most of which were previously identified as contaminants of fetal bovine serum and live vaccines against viral infections of cattle. No specific prophylaxis against BVDV-3 infection has been developed at this time, therefore, it is necessary to update and improve diagnostic methods, optimize control measures to prevent the spread of virulent strains of the pathogen, and control the safety of the vaccines used.

The genus Pestivirus of the family Flaviviridae includes 11 species. Bovine pestiviruses are the causative agents of viral diarrhea/mucosal disease and include three genetically distinct species: pestivirus A (BVDV-1), B (BVDV-2), and H (BVDV-3). The number of BVDV-1 subtypes is 21, BVDV-2 – 4, and BVDV-3 – 4, which complicates the diagnosis of associated diseases, reduces the effectiveness of vaccination and control programs.We performed the search in the PubMed, Web of Science, Scopus, eLIBRARY.RU databases for articles published in 2000–2021.Pestivirus A is distributed everywhere, although the largest number of subtypes was found in cattle in Italy and China. The virus is widespread in the Central region of the Russia (subtypes 1a and 1m). In Siberia, eleven subtypes circulate among native and imported animals: 1a (5%), 1b (35%), 1c (5%), 1d (10%), 1f (20%), 1g, 1i (both 2.5%), 1j, 1k, 1p, and 1r (all for 5%). Pestivirus B subtype is more virulent, found less frequently and mainly in the North and South America, in some European countries, and in Asia. Three subtypes have been identified in Siberia: 2a (25%), 2b (10%), and 2c (5%). Pestivirus H circulates in Europe, Asia and South America. The main route of entry is contaminated biological products. In Russia, BVDV-3 of the Italian-Brazilian group (3a) was detected in 7 lots of fetal bovine serum.The role of the virus in the occurrence of respiratory diseases in calves, abortion, systemic infection and enteritis in calves and adult animals has been established. The source of the virus in such cases was a contaminated modified live vaccine.

ВЫЯВЛЕНИЕ И КОЛИЧЕСТВЕННАЯ ОЦЕНКА ВИРУСНЫХ И БАКТЕРИАЛЬНЫХ ВОЗБУДИТЕЛЕЙ РЕСПИРАТОРНЫХ БОЛЕЗНЕЙ КРУПНОГО РОГАТОГО СКОТА ПРИ ПОМОЩИ ПЦРВ РЕАЛЬНОМ ВРЕМЕНИ А.В.НЕФЕДЧЕНКО, А