REVIEWS | GENERAL ISSUES
A short report is devoted to the radical changes in the taxonomy of viruses. The metagenomic sequencing has revealed the presence of a vast variety of viruses in diverse environmental samples without any connections with banal parasitism, infectivity, or pathogenicity. The understanding of viruses has expanded beyond the original parasitic–pathogen model, and now virologists recognize the role of viruses in host regulation and the maintenance of natural ecosystems. Co-evolution of the viral and cellular genomes includes mutual horizontal gene transfer and joint development of new biological functions, such as the mechanism of phylogenesis and phylodynamics of coactants. The concepts of the origin of viruses and their relation to the Universal Tree of Life are formulated. In this regard, the International Committee on the Taxonomy of Viruses (ICTV) changed the previous Code of their classification hierarchy from five ranks to a fifteen-rank one, that emulates a Linnaean framework and accommodates the entire spectrum of genetic divergence in the virosphere. Changes in the rank hierarchy are based on the evolution of the recognition of virus taxa over time, from a traditional phenotype-based characterization process to a multistage process that includes comparative sequence analyses of conserved genes and proteins, including gene phylogeny, gene synteny and shared gene content. The ICTV, that oversees the official classification of viruses and nomenclature of taxa, accepts possible non-hierarchical classifications of viruses beyond taxonomic attribution. The above provisions are illustrated with schemes of chimeric polyphyletic origin of viruses and a new rank structure; the table gives examples of the modern classification of viruses that cause some socially significant infections.
BOVINE DISEASES
Despite the large number of papers dealing with the description of proposed methods for bovine tuberculosis diagnosis and mechanisms of non-specific reaction development in diseased and healthy animals, various aspects require further study. Many specialists are still of the view, formulated when studying causes of pseudoallergic reactions, that the agents of actinomycosis, trematode infections etc. can cause sensitization of the animal body to tuberculin PPD for mammals. The possibility of sensitization of cattle body to Аctinomyces bovis tuberculin was studied in 240 animals identified as actinomycosis diseased among 3,473 tested animals. Only 11 (4.6%) of the total number of diseased animals were reactors to tuberculin PPD for mammals. During bacteriological tests of material from animals euthanized for diagnostic purposes (tuberculin reactors and nonreactors with a confirmed postmortem diagnosis of actinomycosis), acid-tolerant nontuberculous mycobacterium (NTM) cultures were isolated. The results of the experiment conducted in 628 cows of a dairy holding confirmed that Actinomyces bovis lacks tuberculin-associated allergenicity: actinomycosis was detected only in one of 96 (15.2%) tuberculin reactors. The conducted clinical tests with high significance level (Р < 0.005) showed that there is no association between allergic reaction to tuberculin PPD for mammals and actinomycosis infection. The obtained results are indicative of imperfections in differential diagnosis, and further studies in this field should significantly contribute to gaining a better insight into non-specific sensitization of cattle body to tuberculin.
SMALL RUMINANTS DISEASES
The Russian Federation was officially recognized free from peste des petitts ruminants (PPR). As far as the disease infects both domestic and wild small ruminants, it is important to identify the level of the threat associated with the wild fauna diversity in the neighboring countries, where PPR outbreaks were reported. For that reason, habitats of various disease susceptible animal species were examined. Habitats of the wild susceptible animals were mapped for further examination of the interactions between different animal species using zoological research data; PPR outbreaks in wild animals were also designated in the map thus allowing for the detection of the potential routes of the infection spread in the population and introduction to the country. Analysis of the PPR epidemic situation in the country demonstrated that the disease cases were reported in wild mountain animals (ibices and moufflons) and migratory steppe animals (gazelles and saigas). Risk of this highly contagious viral disease spread in wild small ruminants in Mongolia was reported (probability 0.77). Expert survey was carried out for the determination of possible trends and factors of the infection introduction with the wild susceptible animals, through which small ruminant epizootologists assessed the risk probability. During the survey it was determined that PPR was expected to be introduced from Mongolia (probability 0.81), and of major significance were seasonal migrations of wild animal populations. The resulted semi-quantitative parameters of the potential risk can be recommended for the arrangement and implementation of measures aimed at prevention of PPR introduction and spread in the intact domestic and wild small ruminant populations inhabiting the territory of the Russian Federation.
PORCINE DISEASES
It is thought that due to the high virulence of the African swine fever virus its circulation in the Russian Federation is accompanied by a low seroprevalence. However taking into account a long-term ASF unfavourable situation, the introduction of the virus into the wild boar population, and the occurrence of attenuated viral variants, the significance of serological testing aimed at the detection of viral antibodies is increasing. To collect field samples of biological material from animals for molecular genetic, virological, and serological tests, filter paper, as well as swabs, can be used. The specificity and sensitivity of enzyme-linked immunosorbent assay when testing blood absorbed by filter paper are worse than those shown when testing sera, but they allow effective detection of African swine fever virus antibodies. It was demonstrated that blood absorbed on filter paper can be used for the immunoblot analysis, but the optimum performance could be achieved when the immunoperoxidase technique in combination with samples, taken by swabs was used. When comparing results of enzyme-linked immunosorbent assay performed on sera collected from domestic pigs (infected with ASFV isolates Antonovo 07/14 and Sobinka 07/15), and blood from ear veins absorbed on filter paper the sensitivity was 88.9%, specificity – 90.6%. However, the use of the immunoperoxidase technique for testing blood from swabs showed 100% coincidence with ELISA, while testing of sera with immunoperoxidase technique was superior to ELISA in sensitivity. This means blood sampling using swabs may be recommended for tests after proper validation. This technique can be especially useful for collecting data about infected wild boars because effective eradication strategies are impossible without such data.
AVIAN DISEASES
There is currently almost no poultry holding where avian eimerioses, both monoinvasions and those associated with cryptosporidioses, salmonelloses and colibacte- rioses, are not reported. In view of this, the disease control is an urgent challenge that shall be approached in its entirety, using various eimeriostats, antibiotics and probiotics. Searching for new effective products with broad-spectrum antiparasitic action is one of priorities in avian eimeriosis control. Comparative tests of different combinations of eimeriocidal products, namely solicox + chicktonic, maduvet + tylosin and eimeterm + enrofloxacin, for their treatment and protective effectiveness were carried out under production conditions in broiler chickens of a poultry factory located in the Republic of Dagestan. To perform the tests, four groups of broiler chickens (one control group and three test groups, each comprising 50 chickens) were formed based on the principle of analogues. The treatment and prevention scheme adopted in the said poultry holding was used for the control group chickens. Test group 1 chickens were given solicox at a dose of 2 ml per 1 liter of drinking water in combination with chicktonic (a feed supplement) at a dose of 1 ml per 1 liter of water during 4–5 days. Group 2 chickens were given maduvet at a dose of 3−5 mg/kg of body weight with feed twice and tylosin at a dose of 5 g of powder per 10 liters of water once a day during 5 days; where necessary, the procedure was repeated in 14–16 days. Group 3 broiler chickens were given eimeterm 2.5% at a dose of 7 mg/kg of body weight with water during 2 days in combination with enrofloxacin at a dose of 3 ml per 1 liter of drinking water during 5–6 days. It is shown that a decrease in the number of clinically diseased and dead chickens was observed in the test groups after the use of eimeriocidal products that helped to improve zootechnical parameters of reared broiler chicks. Product extenseffectiveness and intens- effectiveness in different groups of chickens were as follows: Group 1 – 89.2 and 96%, Group 2 – 87.6 and 94%, Group 3 –81.4 and 96%, respectively. The clinical signs of eimeriosis were observed in the control group chickens throughout the entire period of studies, invasion extensity and intensity were 87.6 and 42.6%, respectively.
Avian influenza is a highly dangerous viral disease that causes huge economic damage to poultry farming. Currently, highly virulent influenza virus with N8 neur- aminidase subtype is quite often detected in populations of domestic and wild birds in various countries of the world. The article provides data on complete nucleotide sequences of the neuraminidase gene of highly pathogenic avian influenza virus isolates recovered in the second half of 2020 from pathological material received from four regions of the Russian Federation. The conducted research showed that the subtype of the isolated virus was N8. According to the phylogenetic analysis, isolates of N8 virus belong to group 8C.4. During the phylogenetic analysis of the neuraminidase, we also took into account data on hemagglutinin classification, according to which H5N8 virus isolates belong to a widespread clade 2.3.4.4. Viruses of the clade were first registered in 2010 in China and they have been circulating up to now. The paper also provides data of a comparative analysis of nucleotide sequences of the studied isolates and the isolates from the international GenBank and GISAID databases, recovered in other countries from 2007 to 2020. During the analysis of the amino acid sequence of the studied isolates, no substitutions were found in the positions that affect resistance to neuraminidase inhibitors. The complete nucleotide sequences of the neuraminidase gene of the avian influenza virus subtype N8 (isolates A/domestic goose/OMSK/1521-1/2020, A/duck/Chelyabinsk/1207-1/2020, A/duck/Saratov/1578-2/2020, A/goose/Tatarstan/1730-2/2020) are published in the international GenBank and GISAID databases. Based on the analysis of the nucleotide sequences of the studied isolates, the article shows gradual evolution of the N8 subtype virus.
The paper presents results of avian influenza epidemiological monitoring in the Republic of Crimea in 2019–2020. The attention was focused on the study of water basins of the Azov and Black Seas, the Sivash Lagoon and freshwater lakes in the Feodosia Urban Okrug, Leninsky, Sovetsky, Nizhnegorsky, Chernomorsky and Saksky Raions to detect the avian influenza virus circulation. Examination of the above mentioned areas showed that some freshwater reservoirs became shallow and dry, and aquatic vegetation degraded. The natural biotope analysis conducted in 2019 and 2020 showed a decreased number of semiaquatic wild birds. The pathological material was sampled from semiaquatic and migratory wild birds, as well as from poultry kept in poultry farms and backyards. The collected samples were tested using real-time RT-PCR. In 2019, the AIV type A (H9) genome was detected in one fecal sample taken from wild birds near Kuchuk-Adzhigol Lake in Feodosia Urban Okrug. The AIV type A (H5) genome was detected in 2020 during laboratory testing of pathological material taken from the remains of a mute swan within the shoreline of a freshwater lake near the Ermakovo settlement of the Dzhankoysky Raion. The genetic analysis was performed in the FGBI “ARRIAH” (Vladimir), and the N8 subtype neuraminidase of the influenza virus isolate was determined. The comparative genetic analysis of 258 bp nucleic acid sequences of the AIV H gene fragment showed that the identified isolate belongs to the Asian genetic lineage of highly pathogenic AIV subtype H5 (clade 2.3.4.4) associated with the epidemic spread in Asia, Europe, the Middle East and Africa in 2016–2020.
PET DISEASES
Currently, vaccination is the main measure to combat rabies in domestic and wild animals. Inactivated and live vaccines are used for this purpose. Oral vaccines for wild carnivores contain live attenuated rabies viruses in liquid or freeze-dried form, which are packaged inside edible baits. Since there are no consistent data showing advantages of liquid and freeze-dried oral products for vaccine-induced immunity against rabies in animals, we compared effectiveness of these rabies vaccines produced from rabies virus strain VRC-RZ2. Immunogenicity was tested in mongrel dogs aged 3 months and older that are seronegative for rabies virus antigens. The animals were randomly divided into three groups: two experimental and one control group. Group One was fed a block-type bait containing a blister with liquid virus-containing suspension, Group Two was given a block-type bait containing a gelatin capsule with freeze-dried virus suspension. On Day 21 post vaccination, blood samples were taken from all the animals and the obtained sera were examined in virus neutralization test to measure virus neutralizing antibodies titers. The level of the immune response against rabies in the vaccinated dogs was assessed by intracerebral infection of animals with virulent rabies virus strain CVS. The carried out research demonstrated that both groups of the vaccinated dogs had approximately the same titers of virus neutralizing antibodies that ranged from 3.25 to 4.33 log2. The virus neutralizing antibodies observed in the immunized dogs ensured good protection from virulent CVS strain. All animals of the control group died after infection demonstrating clinical signs of paralytic rabies. The results obtained show that both forms of the oral rabies vaccines are effective.
Examination of the virus-cell interactions is of both scientific and practical importance. Our study was aimed at comparative characterization of rabbit myxoma virus and Shope fibroma virus biological properties that manifested during the virus reproduction in RK-13/2-03 clonal continuous rabbit kidney cell culture. It was demonstrated that the viruses varied in infection development and cytopathic effect duration in RK-13/2-03 cell culture. Apparent lesions in cell monolayers infected by myxoma virus and fibroma virus at similar multiplicity of infection and cultivation temperature were observed on day 2 and day 3 of cultivation, respec- tively, as well as maximum cell lesions with evident degeneration were observed on day 3 and day 6 of cultivation, respectively. Myxoma virus was accumulated at titre of 6.25–6.50 lg TCID50/0,2 cm3, and Shope fibroma virusа was accumulated at titre of 5.50−5.75 lg TCID50/0.2 cm3. Shope fibroma virus demonstrated such
infectivity during three passages and myxoma virus demonstrated such infectivity during twenty passages. Prepared cultures were identified as myxoma virus and Shope fibroma virus with molecular genetic analysis. Tests of the viruses for their antigenic relatedness showed that antibodies against myxoma virus were able to neutralize Shope fibroma virus also. NT titres of antibodies against both viruses were similar (1:8). RK-13/2-03 cell culture was found to be highly permissive to Shope fibroma virus that had been isolated from the diseased rabbit and not been an attenuated variant.
Based on the official statistics the situation of parasitic diseases in the Russian Federation is still quite unfavorable. The issues of soil contamination with Echinococcus granulosus eggs are understudied that’s why the objective of the research was to study canine echinococcosis which poses a threat to animal and human disease freedom in the North Caucasian countries. Helminthological tests of the guardian dog feces were performed in seven North Caucasian Subjects: the Stavropol Krai, the Karachay-Cherkessia Republic, the Kabardino-Balkaria Republic, Republic of North Ossetia −Alania, Republic of Ingushetia, the Chechen Republic, and the Republic of Dagestan. As a result, it was determined that the average frequency index of echinococcosis occurrence was 85.07%. The moderate fecal egg count in 1,400 samples was 22.73 ± 1.49 eggs per 10 grams of dog feces. The data obtained are indicative of the disease unfavourable situation in the Subjects and the zoonosis spread at the regional level. Helminthological tests of 14,000 soil samples from near-village pastures for contamination with tapeworm eggs, including Echinococcus granulosus, showed that the invasion rate in the Stavropol Krai was 65.80%, in Karachay-Cherkessia republic − 79.00%, in Kabardino-Balkaria − 82.60%, in North Ossetia − Alania − 74.65%, in Ingushetia − 88.20%, in Chechnya − 83.75%, in Dagestan − 79.85%. The results obtained testify to the high level of soil contamination with the infective eggs. It was demonstrated that there is a relationship between the distribution of viable Echinococcus granulosus eggs in pasture soils and ecological characteristics of the Subject: the largest number of viable Echinococcus granulosus eggs was observed in submountain areas, fewer eggs were observed in flatlands, and the least number of eggs – in the mountain areas. The number of eggs detected in the soil samples from pastures is indicative of the disease persistence in humans and animals. Results of the helminthological tests of 7,500 soil samples from 119 cattle-driving routes of the North Caucasus demonstrate 100% contamination with parasitic agents which poses a threat of epidemiological and epizootological situation of echinococcosis in the Subject.
VETERINARY MICROBIOLOGY
Staphylococcus aureus is a pathogenic microorganism causing a great number of diseases in humans and animals. Many researches on genotyping Staphylococcus aureus isolated from humans and mastitis affected cows are performed, but no foreign reports on typing of Staphylococcus aureus detected in monkeys have been found. Staphylococcus-induced infections are however widely spread in primates. The paper demonstrates results of molecular and genetic examination of Staphy-lococcus aureus isolated from non-human primates. The examination was based on typing of coagulase gene and polymorphic locus of arg gene that regulates expression of pathogenicity-associated genes. Structures of coagulase gene (coa) and polymorphic types of regulatory gene (agr) were studied in 145 Staphylococcus aureus isolates recovered from various monkey species. The studies resulted in singular coagulase gene fragments of four dimensions: 600, 750, 800 and 900 bps. Following AluI endonuclease restriction results Staphylococcus aureus was classified in seven different coa-types. Coagulase gene of genotype VII predominated (31.7%), genotype II was detected less frequently (9.7%). Each Staphylococcus aureus isolate is specified by a definite coagulase gene restriction profile; therefore, at least seven Staphylococcus aureus strains are currently circulating in the monkeys in the monkey facilities. Herewith, those staphylococci that bear genotype VII coagulase gene are invasive as they are isolated from various organs and pus as well as from feces and nasal cavities of the animals. Analysis of the study results demonstrated that bacteria of this species could be transmitted between different monkey species. Apart from human Staphylococcus aureus, in whose genome agrI prevails, agrIV prevailed in the isolates outlined in this paper (59.3%); agrII and agrIII were detected in 5.5 and 2.1% of the isolates, respectively.
GENERAL ISSUES
Laboratory and field experiment on use of the feed additive based on Bacillus subtilis endo- and exometabolites for the cows in different physiological periods are described in the paper. The feed additive impact on main body systems of the tested cows (n = 30), milk production parameters, growth rate of the calves (n = 18) born to the said cows were examined. The feed additive was added to the diets for the cows of test groups, 15 g per cow. The feed additive was found to have a positive effect on immunohematological and metabolic processes in postpartum cows. Neutrophils’ phagocytic activity increased by 12.5% and 14.6% in the animals of test group 1 and test group 2, respectively, as compared to that one in control animals (42.8 ± 1.9%). Neutrophil absorbency increased by 2.5 times, 3.2 times and 2.1 times in the animals of test group 1, test group 2 and control group, respectively. The proportion of T-lymphocytes in blood of animals in test group 1 and test group 2 was 44.5 and 48.9%, respectively, proportion of T-lymphocytes in blood of control animals equaled to 37.5%. Trend for increase in total protein concentration in cow sera owing to increase in albumin fraction was observed in postpartum period: it was 72.91 ± 3.45 g/l in test group 1; 75.54 ± 4.12 g/l in test group 2; 70.95 ± 4.25 g/l in control group. Average daily milk yield in cows of test group 1, test group 2 and control group for the 150 days of lactation was 24.50 ± 1.86 kg; 25.33 ± 1.45 kg and 22.75 ± 4.41 kg, respectively. Higher growth rate was reported for the calves born to the cows received the diet supplemented with the said feed additive. Heifers of test group 1 and test group 2 have reached body weight of 193.51 ± 5.76 and 195.33 ± 3.76 kg and in control group – of 187.33 ± 4.98 kg within 6 months. Feed additive based on endo- and exometabolites of Bacillus subtilis is recommended for cow diets for highly productive dairy herd creation and food-producing animal welfare maintenance.
Chlorella shows a wide spectrum of biological activity, in particular, it exhibits a pronounced antioxidant activity and demonstrates anti-inflammatory, antitumor and antiviral properties. A number of research works have been devoted to studying feed advantages of this unicellular green algae when used in the diets of livestock animals, but the possibility of including different Chlorella species in the diet of primates has not been practically studied. The aim of this work was to assess the possibility of replacing high-protein animal and vegetable feeds with Chlorella, to calculate the digestibility coefficients for the diet nutrients and the effect of algal dry and suspension forms on hematological and serum biochemical parameters in male rhesus monkeys. The data obtained during the experiment indicate that the inclusion of Chlorella in the diet both in the dry form and cell suspension improves nutrient digestibility. Thus, the digestibility of crude protein in the animals receiving algae suspension increased by 4.18% (p < 0.05), that of crude fat – by 4.70% (p < 0.01), crude fiber – by 4.14% (p < 0.05) and crude ash – by 12.32% (p < 0.001). The digestibility coefficients of crude protein in the primates receiving compound feed supplemented with Chlorella powder were higher by 6.83% (p < 0.001), those of crude fiber – by 4.78% (p < 0.05) and crude ash – by 18.93% (p < 0.001) The hematological study results indicate the absence of side effects from long-term Chlorella consumption by primates. The introduction of dry Chlorella into the diet increased blood glucose levels to the upper limit of the control values, while Chlorella suspension did not produce this effect. Thus, Chlorella can be successfully used as a component of a balanced laboratory diet for primates or as a feed additive.
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