- Genomic Data Specialist at APHL; Lead Research Scientist at NSC IECVM; Former Postdoc at USDA-ARSedit
Aim. The analysis of single nucleotide polymorphisms (SNPs) C677T, A1298C, and A2756G of MTHFR and MTR one-carbon metabolism genes in patients with various forms of psoriasis in the Ukrainian population. Methods. Molecular genetic SNP... more
Aim. The analysis of single nucleotide polymorphisms (SNPs) C677T, A1298C, and A2756G of MTHFR and MTR one-carbon metabolism genes in patients with various forms of psoriasis in the Ukrainian population. Methods. Molecular genetic SNP analysis of 77 patients with vulgaris and arthropathic psoriasis was performed by PCR-RFLP. Results. The analysis of the genotype frequency distribution in patients with vulgaris and arthropathic psoriasis showed a statistically significant difference between them for C677T polymorphic variants. The analysis of genotype distribution of series in the two genes as a whole in patients with psoriasis showed a statistically significant difference between the theoretically expected and actual frequencies for A1298T and A2756G SNPs in MTHFR and MTR genes. Conclusions. Among patients with arthropathic psoriasis, which is the most severe form of psoriasis, the homozygotes for the wild-type allele (GG) of A2756G of the MTR gene were rare. Conversely, the homozygotes (TT) of C667T of the MTHFR gene were found to be more common among patients with arthropathic than vulgaris psoriasis, which may indicate the contribution of other genes to the development of arthropathic psoriasis.
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Aim. The aim of this study was to analyze the effects of CAPN316 and CAST282 SNPs in calpain and calpastatin genes on offspring productive traits produced by dairy and beef bulls. Methods. Data on offspring productivity included milk... more
Aim. The aim of this study was to analyze the effects of CAPN316 and CAST282 SNPs in calpain and calpastatin genes on offspring productive traits produced by dairy and beef bulls. Methods. Data on offspring productivity included milk performance traits (for dairy bulls), birth weight, and average daily gain (for beef bulls). Molecular genetic analysis was performed by PCR-RFLP. The χ²-, t-tests, and Pearson correlation coefficient were used for statistical analysis at signific-ance levels of 0.05. Results. In the progeny of dairy bulls, the number of alleles C in both SNPs demonstrated a negative correlation with milk yield (r=-0.577), fat yield (r=-0.794), and protein yield (r =-0.798). G allele of CAPN316 (“wild type”) was associated with an increased number of beef bull progenies. Body weight and average daily gain were better in GG-bull offspring. Conclusions. C alleles of CAPN316 and CAST282 SNPs being associated with meat quality traits were found to be ineffective in selection for milk production traits.
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The article highlights the results of the analysis of the L127V GH, F279Y, and A257G GHR gene's polymorphic variants, that influence milk and meat bull’s offspring characteristics. The effects of genotype SNPs L127V F279Y on fat (genotype... more
The article highlights the results of the analysis of the L127V GH, F279Y, and A257G GHR gene's polymorphic variants, that influence milk and meat bull’s offspring characteristics. The effects of genotype SNPs L127V F279Y on fat (genotype VV SNP L127V) and protein (genotype FF SNP F279Y) content in milk was demonstrated. The effects of L127V and F279Y genotypes on the fat content in milk (genotype VV SNP L127V) and milk yield (genotype FF SNP F279Y) were shown. Birth weight was determined higher in calves from bulls with genotypes FY (F279Y) and AG (A257G).
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Aim. Due to the improvement of cattle breeding methods the aim of the study was to assess the relationship between exterior parameters of Aberdeen-Angus and genes associated with meat tenderness – CAPN1 (calpain) and CAST (calpastatin).... more
Aim. Due to the improvement of cattle breeding methods the aim of the study was to assess the relationship between exterior parameters of Aberdeen-Angus and genes associated with meat tenderness – CAPN1 (calpain) and CAST (calpastatin). Methods. Livestock valuation methods were used to measure the exterior parameters of the animals. Molecular genetic analysis was performed using PCR-RFLP. Statistical testing of the hypothesis of an association between the studied genotypes and exterior parameters was conducted using ANOVA and Pearson product-moment correlation coefficient at significance levels of 0.05, 0.01, and 0.001. Results. Desirable (associated with meat tenderness) alleles C of CAPN1 and CAST genes are associated with an increased chest (CAPN1) and muscle (CAST) size. G alleles of these genes are associated with a better-balanced body. Conclusions. Selection directed to increase the C alleles of CAPN1 and CAST frequency allows to improving in the meat quality accompanied by increasing of body parts with a high content of muscle tissue and muscles.
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Aim. Analysis of single nucleotide polymorphisms C677T, A1298C, and A66G of MTHFR and MTRR one-carbon metabolism genes in patients with various forms of psoriasis in the Ukrainian population. Methods. Molecular genetic analysis of 77... more
Aim. Analysis of single nucleotide polymorphisms C677T, A1298C, and A66G of MTHFR and MTRR one-carbon metabolism genes in patients with various forms of psoriasis in the Ukrainian population. Methods. Molecular genetic analysis of 77 patients with vulgaris and arthropathic psoriasis by PCR-RFLP was carried out. Results. Analysis of the genotype distribution frequencies of A66G and C677T polymorphic variants in patients with vulgaris and arthropathic psoriasis showed a statistically significant difference between them. In patients with psoriasis, analysis of genotype distribution of series in the two genes as a whole showed a statistically significant difference between the theoretically expected and actual frequencies for single nucleotide polymorphisms A1298T and A66G of MTHFR and MTRR genes. Conclusions. Among patients with arthropathic psoriasis, which is the most severe form of psoriasis, the homozygotes for the wild-type allele AA of the MTRR gene A66G are more common, homozygotes of the TT of the MTHFR gene C667T are found more rarely than among patients with psoriasis vulgaris, which may indicate the contribution of other genes to the development of arthropathic psoriasis.
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Here, we report the complete genome sequence of a virus of a putative new serotype of avian paramyxovirus (APMV). The virus was isolated from a white-fronted goose in Ukraine in 2011 and designated white-fronted... more
Here, we report the complete genome sequence of a virus of a putative new serotype of avian paramyxovirus (APMV). The virus was isolated from a white-fronted goose in Ukraine in 2011 and designated white-fronted goose/Ukraine/Askania-Nova/48-15-02/2011. The genomic characterization of the isolate suggests that it represents the novel avian paramyxovirus group APMV 13.
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The calpain proteolytic system, the micromolar calcium-activated neutral protease μ-calpain encoded by the CAPN1 gene, and its inhibitor — calpastatin, encoded by the CAST gene, are involved in biological processes regulated by partial... more
The calpain proteolytic system, the micromolar calcium-activated neutral protease μ-calpain encoded by the CAPN1 gene, and its inhibitor — calpastatin, encoded by the CAST gene, are involved in biological processes regulated by partial degradation of the following substrates — cytoskeletal proteins, signaling molecules, and enzymes. Calpain system dysfunction caused by mutations or Ca2+ elevation is primarily associated with diseases that affect central nervous and cardiovascular systems, as well as skeletal muscles. For cattle breeding purposes, calpain hyperactivity in the presence of excess calcium ions (calcium passes out due to cell death) is considered to be desirable, offering the possibility to obtain more tender meat. This study is aimed to analyze the polymorphic variants CAPN316 and CAST282 of calpain and calpastatin genes in Aberdeen-Angus cattle bred within the Kharkiv region and to compare the obtained results with commercial herds of other countries. A PCR-RFLP method was used for the SNP genotyping. Testing deviation from the Hardy-Weinberg equilibrium was performed using Pearson’s chi-squared test. Spearman’s correlation coefficient was used to measure the strength of association between two characteristics. Cluster analysis was used to classify the allele frequencies obtained within similar data from cattle in other countries. The allele and genotype frequencies of SNP CAPN316 (AF252504.2:g.5709C>G) in calpain gene are: C — 0.398 and G — 0.602; CC — 13.6%, CG — 52.3% and GG — 34.1%. The allele and genotype frequencies of SNP CAST282 (AY_008267.1:g.282C>G) in calpastatin gene are: C — 0.807 and G — 0.193; CC — 63.6%, CG — 34.1%, and GG — 2.3%. Group-to-group variability in allele C frequency for SNP CAPN316 (0.37%) is higher than CAST282 (0.06%) due to the modulating effect of the calpastatin. The Aberdeen-Angus herds of the Kharkiv region are comparable to the European commercial beef cattle herds by allele frequencies. Association between latitudinal zonation and frequency of allele C in cattle herds was demonstrated (R = 0.53, p < 0.05).
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Two complete genome sequences of Newcastle disease virus (NDV) are described here. Virulent isolates pigeon/Pakistan/Lahore/21A/2015 and pigeon/Pakistan/Lahore/25A/2015 were obtained from racing pigeons sampled in the Pakistani province... more
Two complete genome sequences of Newcastle disease virus (NDV) are described here. Virulent isolates pigeon/Pakistan/Lahore/21A/2015 and pigeon/Pakistan/Lahore/25A/2015 were obtained from racing pigeons sampled in the Pakistani province of Punjab during 2015. Phylogenetic analysis of the fusion protein genes and complete genomes classified the isolates as members of NDV class II, genotype VI.
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Our study demonstrates the repeated isolation of vaccine-derived Newcastle disease viruses from different species of wild birds across four continents from 1997 through 2014. The data indicate that at least 17 species from ten avian... more
Our study demonstrates the repeated isolation of vaccine-derived Newcastle disease viruses from different species of wild birds across four continents from 1997 through 2014. The data indicate that at least 17 species from ten avian orders occupying different habitats excrete vaccine-derived Newcastle disease viruses. The most frequently reported isolates were detected among individuals in the order Columbiformes (n = 23), followed in frequency by the order Anseriformes (n = 13). Samples were isolated from both free-ranging (n = 47) and wild birds kept in captivity (n = 7). The number of recovered vaccine-derived viruses corresponded with the most widely utilized vaccines, LaSota (n = 28) and Hitchner B1 (n = 19). Other detected vaccine-derived viruses resembled the PHY-LMV2 and V4 vaccines, with five and two cases, respectively. These results and the ubiquitous and synanthropic nature of wild pigeons highlight their potential role as indicator species for the presence of Newcastle disease virus of low virulence in the environment. The reverse spillover of live agents from domestic animals to wildlife as a result of the expansion of livestock industries employing massive amounts of live virus vaccines represent an underappreciated and poorly studied effect of human activity on wildlife.
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Here, we report the circulation of highly related virulent Newcastle disease viruses (NDV) in Bulgaria and Ukraine from 2002 until 2013. All of these NDV isolates have the same virulence-associated cleavage site ('' 113 RQKR;F 117 ''),... more
Here, we report the circulation of highly related virulent Newcastle disease viruses (NDV) in Bulgaria and Ukraine from 2002 until 2013. All of these NDV isolates have the same virulence-associated cleavage site ('' 113 RQKR;F 117 ''), and selected ones have intracerebral pathogenicity index values ranging from 1.61 to 1.96. These isolates are most closely related to viruses circulating in Eastern Europe, followed by viruses isolated in Asia during the same period of time. Interestingly, the majority of the viruses were isolated from backyard poultry, suggesting the possibility of a ''domestic'' or ''urban'' cycle of maintenance. The molecular characterization of the nucleotide sequence of the complete fusion protein gene of the studied viruses suggests continued circulation of virulent NDV of sub-genotype VIId in Eastern Europe, with occasional introductions from Asia. Furthermore, the high level of genetic similarity among those isolates suggests that the NDV isolates of sub-genotype VIId from Bulgaria and Ukraine may have been part of a broader epizootic process in Eastern Europe rather than separate introductions from Asia or Africa. The continuous monitoring of backyard poultry flocks for the presence of circulating virulent NDV strains will allow early identification of Newcastle disease outbreaks.
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A high level of inbreeding has deleterious effects on animal productive and reproductive traits. The effect of inbreeding by CAPN316, CAST282, GH L127V, GHR F279Y, GHR A257G, and CYP3A28 C994G markers on reproductive characteristics,... more
A high level of inbreeding has deleterious effects on animal productive and reproductive traits. The effect of inbreeding by CAPN316, CAST282, GH L127V, GHR F279Y, GHR A257G, and CYP3A28 C994G markers on reproductive characteristics, weight dynamics (0-5 years of age), and exterior traits were studied. The genealogical analysis showed that the population includes seven lines of cattle. For the SNP genotyping, PCR-RFLP methods were set up. Testing deviation from the Hardy-Weinberg equilibrium was performed using Pearson's chi-squared test. The estimation of inbreeding depression by Fst coefficients of SNPs for traits studied was performed via multiple regression models with forward stepwise. The tested population was analyzed in Hardy-Weinberg equilibrium for all SNPs, except GHR F279Y. The cattle lines Bryalhil Sau and Southome Extra were considered the best lines as they showed the lowest level of heterozygosity. A negative effect of inbreeding level for different markers was seen for regression coefficients: calf birth weight =-1.08±0.30 (GHR F279Y), body weight at 8 months =-0.71±0.11 (CAPN316),-0.49±0.11 (GHR F279Y), and 15 months =-0.54±0.04 (CAST282),-0.36±0.06 (GHR F279Y). Increased inbreeding had a positive influence on regression coefficients for calf average daily gain = 0.86±0.26 (GH L127V), weight at 8 and 15 months = 1.43±0.13, 1.05±0.05 (GHR A257G), and exterior traits-shoulder, back and low back, rump, and hindquarter = 1.09±0.11, 0.77±0.22, and 1.10±0.30 (CYP3A28 C994G), respectively.
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Bovine growth hormone (bGH) has effects on animal growth and metabolism. Therefore, it plays a vital role in the regulation of body weight, fertility, and lactation performance of cattle. This enables the bGH gene to be used as a... more
Bovine growth hormone (bGH) has effects on animal growth and metabolism. Therefore, it plays a vital role in the regulation of body weight, fertility, and lactation performance of cattle. This enables the bGH gene to be used as a candidate marker for improving growth, meat/milk production, and for the marker-assisted selection programs of cattle as well. The aim of this study was to analyze the polymorphic variant L127V of the bGH gene in Aberdeen-Angus bred in the Kharkiv region and to compare the obtained results with commercial herds from other countries. A PCR-RFLP method was used for the SNP genotyping. Testing deviation from the Hardy-Weinberg equilibrium was performed using Pearson’s chi-squared test. Pearson’s correlation coefficient r was used to measure the strength of association between two characteristics. The allele and genotype frequencies of SNP L127V (rs41923484; g.2141C>G) are: L — 0.319 and V — 0.681; LL — 8.6%, LV — 46.6%, and VV — 44.8% (n=58) in the population is within the Hardy-Weinberg equilibrium. The studied population was found to be close to beef herds of Russian selection. A positive correlation of L‑allele with birth weight was determined within the moderate climatic zone (r=0.93).
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The calpain proteolytic system, the micromolar calcium-activated neutral protease μ-calpain encoded by the CAPN1 gene, and its inhibitor — calpastatin, encoded by the CAST gene are involved in biological processes regulation by partial... more
The calpain proteolytic system, the micromolar calcium-activated neutral protease μ-calpain encoded by the CAPN1 gene, and its inhibitor — calpastatin, encoded by the CAST gene are involved in biological processes regulation by partial degradation of substrates — cytoskeletal proteins, signaling molecules, and enzymes. Calpain system dysfunction caused by mutations or Ca2+ elevation is associated primarily with diseases affecting central nervous and cardiovascular systems, as well as skeletal muscles. For cattle breeding purposes the calpain hyperactivity in the presence of excess calcium ions (calcium passes out due to cell death) is considered to be desirable offering the possibility to obtain more tender meat. This study is aimed to analyze the polymorphic variants CAPN316 and CAST282 of calpain and calpastatin genes in Aberdeen-Angus bred within the Kharkiv region and to compare obtained results with commercial herds of other countries. A PCR-RFLP method was used for the SNP genotyping. Testing deviation from the Hardy-Weinberg equilibrium was performed using Pearson’s chi-squared test. Spearman’s correlation coefficient was used to measure the strength of association between two characteristics. Cluster analysis was used to classify the allele frequencies obtained within similar data obtained for cattle in other countries. The allele and genotype frequencies of SNP CAPN316 (AF252504.2:g.5709C>G) in calpain gene are: C — 0.398 and G — 0.602; CC — 13.6%, CG — 52.3% and GG — 34.1%. The allele and genotype frequencies of SNP CAST282 (AY_008267.1:g.282C>G) in calpastatin gene are: C — 0.807 and G — 0.193; CC — 63.6%, CG — 34.1%, and GG — 2.3%. Group-to-group variability in allele C frequency for SNP CAPN316 (0.37%) is higher than CAST282 (0.06%) due to the modulating effect of the calpastatin. The Aberdeen-Angus herds of the Kharkiv region are comparable to the European commercial beef cattle herds by allele frequencies. Association between latitudinal zonation and frequency of allele C in cattle herds was demonstrated (R = 0.53, p < 0.05).
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Aim. To analyze the single nucleotide polymorphism G742A in the betaine homocysteine methyltransferase S (BHMT) gene in patients with psoriasis. Methods. Genealogical information was obtained from 72 participants and 60 people from the... more
Aim. To analyze the single nucleotide polymorphism G742A in the betaine homocysteine methyltransferase S (BHMT) gene in patients with psoriasis. Methods. Genealogical information was obtained from 72 participants and 60 people from the control group. Molecular genetic analysis was performed by PCR-RFLP method. The hypothesis of an association between the studied genotypes, disease occurrence, and assessment of the distribution series equality was statistically checked using χ2 test at significance levels of 0.05, 0.01, and 0.001. Results. Genotyping showed that the allele and genotype frequencies in the control group were: pG = 0,700, qA = 0,30, GG – 45,0 %, GA – 50,0 %, AA – 5,0 %, while in patients with psoriasis – pG = 0,743, qA = 0,257, GG – 50,0 %, GA – 48,6 %, AA – 1,4 %. The actual distribution of genotypes was statistically different from the theoretically expected distribution of BHMT G742A polymorphism in a general group of patients. The frequency distribution of genotypes showed a statistically significant difference between the group of patients with psoriasis and the control group (p = 0.007). The frequency of the AA genotype in the control group was higher than in the group of patients with psoriasis without arthropathy (5.0 % vs. 2.2 %, p = 0.029). Among patients with an age of disease onset of up to 25 years, the actual distribution of G742A polymorphism was significantly different from the theoretically expected when balanced (p <0,01). Conclusion. The results of the BHMT G742A polymorphism study in patients with psoriasis revealed a lower frequency of the allele А homozygous genotype among these patients, which highlights the need for further consideration of implementing genetic screening in the planning of preventive and therapeutic measures.
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Brucellosis is an especially dangerous infectious disease of mammals, which is caused by bacteria of Brucella genus. Currently, the genus consists of 10 species of Brucella, but B. melitensis, B. abortus, and B. suis have the most... more
Brucellosis is an especially dangerous infectious disease of mammals, which is caused by bacteria of Brucella genus. Currently, the genus consists of 10 species of Brucella, but B. melitensis, B. abortus, and B. suis have the most zoonotic potential. Multiple methods are used for the diagnosis of brucellosis in veterinary practice, but they are all labor-intensive and time-consuming. The use of PCR in the early stages of research can significantly speed up the process of diagnosis. The aim of our study was to determine the minimum number of primers used that allow differentiation of Brucella from Enterobacteria in native suspensions and samples of biological material. The experiments examined the possibility of detecting DNA of Brucella abortus, Brucella suis, Brucella ovis, and Enterobacteria, with whom they share an affinity for the lipopolysaccharide antigens in the inactivated bacterial suspensions, milk samples, and blood serum. The inactivated Brucella (B. abortus 544, 19-770; B. suis 1330; B. ovis 67 / B, 76/982) and Enterobacteriaceae (Y. enterocolitica 09, E. coli 099, S. Enteritidis M ) strains suspensions, as well as samples of blood serum and milk contaminated with live Brucella and Enterobacteriaceae cultures, were used for this experiment. The sterile samples of blood serum, milk, and saline were used as a negative control. DNA isolation was performed with the commercial kit “DNA-sorb-B”. Amplification was performed with the commercial kit “AmpliSens”. The research was conducted in accordance with laboratory regulations for the identification of Brucella strains by PCR. The research established that designed Brucella specific primers are well suited for the differentiation of Brucella species, such as B. abortus, B. suis, and B. ovis, from Enterobacteriaceae species, such as Y. enterocolitica, S. enteritidis, and E. coli, in samples of biological material.
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This study was aimed at (i) the creation of a molecular-genetic control system of pestivirus contamination in biotechnology objects, (ii) identification of animals persistently infected with bovine viral diarrhea virus (BVDV), and (iii)... more
This study was aimed at (i) the creation of a molecular-genetic control system of pestivirus contamination in biotechnology objects, (ii) identification of animals persistently infected with bovine viral diarrhea virus (BVDV), and (iii) genetic typing of selected BVDV isolates. RNA extraction, cloning, polymerase chain reaction (PCR), real-time PCR, enzyme-linked immunosorbent assay, serum neutralization test, and sequencing were performed in this study. It was shown that recombinant plasmids with an insertion of the Erns gene fragment (826 base pair) of BVDV-1 or BVDV-2 were constructed. Also, we developed and optimized parameters of the duplex PCR for the simultaneous indication of Mollicutes DNA and BVDV RNA, with the possibility of nested PCR for further identification of BVDV genotypes. Specific BVDV antibodies were detected in 725 of 1,042 (69.6%) analyzed samples. In this study, 5 animals persistently infected with BVDV were detected on farms B and C from the Kharkiv region. The genetic typing of viral isolates revealed that only BVDV-1 viruses were present. The phylogenetic analysis confirmed two BVDV-1 subtypes, namely b and f. All viruses isolated from farm B samples and biotechnological objects were typed as BVDV-1b, while viruses from farm C from the Kharkiv region and farm A from the Kherson region were typed as BVDV-1f. In conclusion, the obtained recombinant plasmids can be used as a positive control in the PCR test system for the detection of pestivirus contamination in biotechnology objects. Our results indicate that the BVDV infection is widespread in cattle herds in Eastern Ukraine, which requires the application of new approaches to improve the current BVDV situation in Ukraine.
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This study was focused on (i) detection of specific BVDV-antibodies within selected cattle farms, (ii) identification of persistently infected (PI) animals, and (iii) genetic typing of selected BVDV isolates. Specific BVDV antibodies were... more
This study was focused on (i) detection of specific BVDV-antibodies within selected cattle farms, (ii) identification of persistently infected (PI) animals, and (iii) genetic typing of selected BVDV isolates. Specific BVDV antibodies were detected in 713 of 1,059 analyzed samples (67.3 percent). This number is in agreement with findings in many cattle herds around the world. However, the number of positive samples differed in the herds. While 57 samples out of 283 (20.1 percent) were identified in the first herd, 400 out of 475 (84.2 percent) and 256 out of 301 (85 percent) animals were positive in the second and third herd. BVDV RNA was detected in a high number of animals within all herds. The real-time PCR assay detected BVDV RNA in 5 of 1,068 samples analyzed (0.5 percent): 4 positive samples out of 490 (0.8 percent) and 1 out of 301 (0.33 percent) were found in the second and third herd, respectively. BVDV genetic materials were not detected in the first herd. Data on the number of PI animals were in accordance with serological findings in the cattle herds involved in our study. The genetic typing of viral isolates revealed that only BVDV-1 type viruses were present. The phylogenetic analysis confirmed two BVDV-1 subtypes, namely b and f. All 4 viruses from the second farm were typed as BVDV-1b and all of them had identical 5’-UTR sequences. However, the virus from the third farm was typed as BVDV-1f. Conclusion. Our results indicated that the BVDV infection is widespread in cattle herds in eastern Ukraine, which requires further research and the development of new approaches to improve the current situation.
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Background: Salmonellosis is one of the most dangerous diseases that is caused by Salmonella agents and has a wide spectrum of clinical manifestations - from asymptomatic to severe septic forms. In the majority of Salmonella infection... more
Background: Salmonellosis is one of the most dangerous diseases that is caused by Salmonella agents and has a wide spectrum of clinical manifestations - from asymptomatic to severe septic forms. In the majority of Salmonella infection cases, the enterica subspecies serovars are isolated from animals and humans. According to the FAO, 20% of poultry products in the world are contaminated with salmonella. Every year 21 million cases of typhoid fever are registered wherein 216 thousand are lethal. Traditional microbiological methods for Salmonella typing (cultivation) are usually time-consuming. This necessitates the development of modern methodology for food safety. Goal: Development of a multiplex PCR protocol enabling identification of Salmonella spp. and typing of Enterisa Salmonella Enteritidis, Salmonella Enterisa Typhimurium, Salmonella Typhi, Salmonella Dublin, and Salmonella Gallinarum. Methods & Materials: For amplification the following primers were used: Salmonella spp.: Salm3-Salm4 (Ferretti, 2001); Salmonella enteritidis: SentF-SentR (Agron, 2001); Salmonella typhimurium: StypF-StypR (O’Regan, 2008); Salmonella Typhi: StyphiF-StyphiR (Kumar, 2008); Salmonella Dublin: SdubF-SdubR; Salmonella Gallinarum: SgalF-SgalR (Akiba, 2011). Optimization of multiplex PCR protocol was performed according to Elnifro (Elnifro, 2000). Results: To determine the optimal PCR primer's annealing temperature the assays were performed at 58◦C, 60◦C, 63◦C, and 65◦C. The optimal amplification mode was determined to be as follows: Initial denaturation - 94◦C-2 min; Denaturation - 94◦C-45s; Annealing - 63◦C-45s; Extension - 72◦C-60s (40 cycles); Final extension - 72◦C-5 min. The optimal composition of the reaction mixture for multiplex PCR was: 10×DreamTaq Buffer-2,5ul, dNTP Mix, 2mM each-2,5ul, 25mM MgCl2-0,5ul, Primers 20pM, Template DNA-5,0ul, DremTaq DNA Polymerase-2,0ul Water, nuclease-free-3,5ul. The resulting protocol allowed for the detection of Salmonella spp. DNA in the samples, as well as the simultaneous typing of Salmonella Enterica Enteritidis, Salmonella Enterica Typhimurium, Salmonella Typhi, Salmonella Dublin, and Salmonella Gallinarum.The simultaneous amplification of all 6 expected fragments occurred in the multiplex PCR. Conclusion: The developed protocol is promising for the biological control of food safety, as well as in routine investigations.
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Goal. The topology of constructed phylogenetic trees based on the evolutionary distance allows the ability to obtain reliable information about the genotypic characterization and the spectrum of the infectious agents circulating in a... more
Goal. The topology of constructed phylogenetic trees based on the evolutionary distance allows the ability to obtain reliable information about the genotypic characterization and the spectrum of the infectious agents circulating in a particular area. The aim of this work was to study the animal pestiviruses' phylogenetic relationships. Methods. The Mega 4.0.2 and PhyML 3.0 software were used for the phylogenetic analysis. The methods of linking nearest neighbors (Neighbor-joining) and maximum savings (Maximum parsimony) were used for dendrograms construction. Results. The phylogenetic relationships of classical swine fever viruses (CSFV) circulating in different geographic regions were studied. It was clearly shown that each of the currently known CSFV genotypes formed a separated cluster. The variability of the E2 gene encoding the coat protein of the CSFV was observed. This study demonstrated that phylogenetic analysis is a necessary component for effective vaccination, establishing long-term forecasting of outbreaks, and the prevention of further CSFV spread. Conclusion. A phylogenetic analysis of animal pestiviruses based on E2 gene sequences demonstrated the possibility of successful genotyping on the basis of this gene. The use of phylogenetic analysis for the molecular labeling of the CSFV was demonstrated. Keywords: classical swine fever virus, genotype, phylogenetic analysis.
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Bovine viral diarrhea (BVD) - pestivirus disease of ruminants, accompanied by various lesions of epithelial tissue, abortion, pneumonia, vaginitis, enteritis, diarrhea, acute syndrome of exhaustion, and sometimes high mortalities. Bovine... more
Bovine viral diarrhea (BVD) - pestivirus disease of ruminants, accompanied by various lesions of epithelial tissue, abortion, pneumonia, vaginitis, enteritis, diarrhea, acute syndrome of exhaustion, and sometimes high mortalities. Bovine viral diarrhea virus (BVDV) causes infection of the fetus, leading to persistent infection in the calves born, whose rates of growth and development are unsatisfactory. There are several genetically heterogeneous subpopulations of the pathogen within cattle populations. A thorough molecular epizootiology study will help in the prevention and diagnosis of BVDV infection. Because of the exclusive economic importance of this disease, the main purposes of the presented study were the investigation of the BVD epizootic situation in China and Ukraine, as well as the genetic characterization and the phylogenetic comparison of the BVDV circulating there.
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Type I and II bovine viral diarrhea viruses were used in this work. Erns gene fragment obtained using polymerase chain reaction was inserted in the cloning vector (pTZ57R/T). In addition, E. coli DH10B bacteria were transformed for... more
Type I and II bovine viral diarrhea viruses were used in this work. Erns gene fragment obtained using polymerase chain reaction was inserted in the cloning vector (pTZ57R/T). In addition, E. coli DH10B bacteria were transformed for recombinant plasmid uptake. The presence of gene Erns insertion was confirmed using restriction enzyme digestion analysis. During the studies, recombinant plasmids carrying 826 bp Erns gene insertion of genotypes I and II bovine viral diarrhea virus were constructed.
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Aim. The method of amplifying and cloning the Erns gene of Bovine Viral Diarrhea Virus was developed to obtain the positive controls for a polymerase chain reaction. Methods. The strains used in this study were BVDV-1b (Ossloss) and... more
Aim. The method of amplifying and cloning the Erns gene of Bovine Viral Diarrhea Virus was developed to obtain the positive controls for a polymerase chain reaction. Methods. The strains used in this study were BVDV-1b (Ossloss) and BVDV-2 (Kosice). Viral RNA was extracted using the silica-based extraction method. A part of Erns gene was amplificated using the specific primers. The PCR product was inserted into the cloning vector pTZ57R/T. Furthermore, E. coli DH10B bacteria were transformed to amplify the recombinant plasmid. Recombinant clones were identified by antibiotic selection on an agar plate and confirmed by PCR. Moreover, the insert of the Erns gene was verified by restriction enzyme digestion assay using EcoRI and HindIII. Results. It was shown that we had constructed the recombinant plasmids with the insertion of the BVDV-1 and BVDV-2 Erns gene fragment (826 base pair). Conclusion. The obtained recombinant plasmids can be used as a positive control for PCR.
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Bovine viral diarrhea is a widespread infection of cattle that has a wide range of clinical symptoms in domestic and wild ruminants. It is a major problem in cattle and causes significant economic losses in the cattle industry. The virus... more
Bovine viral diarrhea is a widespread infection of cattle that has a wide range of clinical symptoms in domestic and wild ruminants. It is a major problem in cattle and causes significant economic losses in the cattle industry. The virus infects bovines of all ages and causes both immunosuppression and reproductive, respiratory, and digestive disorders. Persistently infected cattle are the main factor in the transmission of the disease between and among herds. Comparative results of antibody presence were obtained utilizing enzyme-linked immunosorbent assay (ELISA) and virus neutralization tests (VNT). During the work, 1010 blood serum samples of cattle from three farms in the Kharkiv region were selected and analyzed. Antibodies against bovine viral diarrhea virus were detected in 704 samples (69.7%) using a commercial ELISA kit and in 690 samples (68.3%) using an in-house ELISA method. Results were clarified using VNT, which detected antibodies against the bovine viral diarrhea virus in 712 samples (70.5%). Enzyme-linked immunosorbent assay is recommended for mass screening of cattle for viral diarrhea occurrence. The results confirmed that the sensitivity of ELISA satisfies the requirements of the diagnostic methods. The virus neutralization test, the «gold standard» of serological methods, is an appropriate method to verify the results of ELISA. Since the results contain a significant number of false-positive results, it is necessary to carry out comprehensive studies using both serological and molecular genetics methods.
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Bovine viral diarrhea is a widespread infection of cattle caused by bovine viral diarrhea virus (BVDV), a member of the Pestivirus genus of the Flaviviridae family. The virus persists in the cattle population via a unique combination of... more
Bovine viral diarrhea is a widespread infection of cattle caused by bovine viral diarrhea virus (BVDV), a member of the Pestivirus genus of the Flaviviridae family. The virus persists in the cattle population via a unique combination of transient and persistent infections. Persistently infected animals may succumb to mucosal disease, which is characterized by lesions in the gastrointestinal tract and its abrupt lethal outcome. This study was focused on the identification of persistently infected animals in cattle farms of Kharkiv region. For this reason, 1080 blood samples from three different farms were tested for the presence of BVDV specific antibodies by ELISA and viral genetic materials by real-time RT-PCR. In this study, 5 persistently infected animals were detected in two farms. The phylogenetic analysis of 5'-UTR (245 bp fragment) was used for the genetic typing of revealed BVDV isolates into subgenotypes. The genetic typing indicated that all 4 viruses from the second farm were identical and belonged to the BVDV-1b subgenotype. The viral isolate from the third farm was typed as BVDV-1f genotype.
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Bovine viral diarrhea is a widespread infection of cattle caused by bovine viral diarrhea virus (BVDV), a member of the Pestivirus genus of the Flaviviridae family. The virus persists in the cattle population via a unique combination of... more
Bovine viral diarrhea is a widespread infection of cattle caused by bovine viral diarrhea virus (BVDV), a member of the Pestivirus genus of the Flaviviridae family. The virus persists in the cattle population via a unique combination of transient and persistent infections. Persistently infected animals may succumb to mucosal disease, which is characterized by lesions in the gastrointestinal tract and its abrupt lethal outcome. This study was focused on the identification of persistently infected animals in cattle farms of Kharkiv region. For this reason, 1080 blood samples from three different farms were tested for the presence of BVDV specific antibodies by ELISA and viral genetic materials by real-time RT-PCR. In this study, 5 persistently infected animals were detected in two farms. The phylogenetic analysis of 5'-UTR (245 bp fragment) was used for the genetic typing of revealed BVDV isolates into subgenotypes. The genetic typing indicated that all 4 viruses from the second farm were identical and belonged to the BVDV-1b subgenotype. The viral isolate from the third farm was typed as BVDV-1f genotype.
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In this study, we tested the PCR parameters for typing field isolates and museum strains of Brucella species (B. abortus and B. suis). Specific and generic differences between their genomes were established.
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The phylogenetic relationships between all known paramyxoviruses serotypes of poultry and wild birds circulating in different geographical regions were studied. The phylogenetic relationships between different types of paramyxoviruses... more
The phylogenetic relationships between all known paramyxoviruses serotypes of poultry and wild birds circulating in different geographical regions were studied. The phylogenetic relationships between different types of paramyxoviruses from wild birds were established. The importance of phylogenetic analysis for the molecular genotyping of microorganisms was demonstrated.
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Aim. The aim of this study was to develop a duplex PCR and optimize its amplification parameters for the simultaneous detection of mycoplasma and bovine viral diarrhea virus (BVDV) genetic materials, with the possibility of further BVDV... more
Aim. The aim of this study was to develop a duplex PCR and optimize its amplification parameters for the simultaneous detection of mycoplasma and bovine viral diarrhea virus (BVDV) genetic materials, with the possibility of further BVDV genotype differentiation. Methods. Duplex and nested PCR protocols were tested using recombinant plasmid pTZ57R/T-VD, containing an insertion of an Erns gene fragment of both BVDV genotype 1 (strain Ossloss) and genotype 2 (strain Kosice), and DNA samples of M. orale N-I, M. hyorhinis BTS-7, and M. bovis PG45T reference strains. Results. The optimized duplex PCR with simultaneous use of P1/P2 and GPO-1/MGSO amplification primers allowed to differentiate BVDV and mycoplasma by forming two amplicon bands of 826 and 715 bp, correspondently. During the nested PCR amplification, specific products of amplification of 223 bp in length for BVDV of genotype 1 and 488 bp - for genotype 2 were formed. Conclusions. During the ongoing work, a duplex PCR protocol was developed and optimized for the simultaneous identification of the mycoplasma and BVDV genetic material with the possibility of utilizing nested PCR for the further identification of BVDV’s genotypes.
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This research compares the results of the Bovine Viral Diarrhoea Virus (BVDV) detection by Polymerase Chain Reaction (PCR) and antibodies against BVDV by Enzyme Linked Immuno Sorbent Assay (ELISA). During this study, 1023 samples of blood... more
This research compares the results of the Bovine Viral Diarrhoea Virus (BVDV) detection by Polymerase Chain Reaction (PCR) and antibodies against BVDV by Enzyme Linked Immuno Sorbent Assay (ELISA). During this study, 1023 samples of blood serum of cattle from three farms in the Kharkiv region were selected and analyzed. BVDV was detected in 143 samples (14%) by PCR and antibodies against this virus were detected in 694 samples (67.8%) by ELISA. Also, statistical analysis of the distribution of Bovine Viral Diarrhoea Virus in different age groups of farms was quoted.
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This paper is dedicated to the development of recombinant positive control for the detection of DNA of African swine fever virus (ASFV) based on the TA-cloning method. The VP73-gene (278 bp long) was amplified using PCR and cloned into... more
This paper is dedicated to the development of recombinant positive control for the detection of DNA of African swine fever virus (ASFV) based on the TA-cloning method. The VP73-gene (278 bp long) was amplified using PCR and cloned into the recombinant plasmid vector pTZ57R/T, which was successfully transformed into competent E. coli cells of HB10B strain. The ability to produce clones of recombinant DNA, that can be used as a positive control for PCR detection of ASFV, was proven. The research results can be applied for improving disease monitoring protocols based on PCR-screening of clinical material.
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The recombinant plasmid pTZR/T-VD carrying the 5' UTR untranslated region of Bovine viral diarrhea virus (288 bp long) was constructed. The orientation of the insert was confirmed by PCR-screening. The use of the obtained recombinant... more
The recombinant plasmid pTZR/T-VD carrying the 5' UTR untranslated region of Bovine viral diarrhea virus (288 bp long) was constructed. The orientation of the insert was confirmed by PCR-screening. The use of the obtained recombinant plasmid pTZR/T-VD as a positive control for PCR was proven.
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In the presented work, we developed and optimized the method for molecular-genetic studies of the Bovine Viral Diarrhea Virus and Mycoplasma presence in various types of biological material. 62 samples of semen, 90 veterinary... more
In the presented work, we developed and optimized the method for molecular-genetic studies of the Bovine Viral Diarrhea Virus and Mycoplasma presence in various types of biological material. 62 samples of semen, 90 veterinary immunobiological products, 1084 samples of raw materials for production of native bovine serum, and 37 FLK cell cultures were studied by this method. It was established that 8 samples of blood serum were contaminated by Mycoplasma and 6 contained genetic material of the Bovine Viral Diarrhea Virus.
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The potential of polymerase chain reaction to be used as a part of the general diagnostics system for detection of the Porcine Reproductive Respiratory Syndrome Virus (PRRSV) in pigs was studied. Additionally, different methods of nucleic... more
The potential of polymerase chain reaction to be used as a part of the general diagnostics system for detection of the Porcine Reproductive Respiratory Syndrome Virus (PRRSV) in pigs was studied. Additionally, different methods of nucleic acid extraction from biological materials were compared and optimal amplification parameters were determined. The main objectives of this study were: (a) to compare two primer sets for the genetic material of the PRRSV detection in biological materials; (b) to optimize the PRRSV amplification parameters. During this study, new experimental data were obtained; the amplification conditions for highly specific and sensitive detection of PRRSV allocation were determined; and statistical analysis of the acquired results was carried out.
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Here, we report the isolation and characterization of virulent Newcastle disease viruses (vNDV) isolated from backyard chickens in Bulgaria and Ukraine between 2002 and 2013. Nineteen isolates were submitted to the Southeast Poultry... more
Here, we report the isolation and characterization of virulent Newcastle disease viruses (vNDV) isolated from backyard chickens in Bulgaria and Ukraine between 2002 and 2013. Nineteen isolates were submitted to the Southeast Poultry Research Laboratory of the USDA in Athens, GA, USA. Viruses were propagated in 9-to-11-day-old specific-pathogen-free embryonated chicken eggs. Viral RNA was isolated from the allantoic fluids using TRIzol LS. Amplification reactions were performed with one-step reverse transcription PCR. All PCR products were subjected to DNA sequencing and Phylogenetic analysis was performed using MEGA6 software. An intracerebral pathogenicity index (ICPI) assay was conducted on 12 viruses following established procedures. All of the studied NDV isolates had the same virulence-associated cleavage site (‘‘113RQKR;F117’’), and the ones tested in ICPI assay had values ranging from 1.61 to 1.96. This is the first report, confirmed by multiple isolations, of complete fusion sequences of NDV of sub-genotype VIId from Bulgaria and Ukraine. The molecular characterization of the nucleotide sequences of the complete fusion protein gene of these viruses suggests continued circulation of vNDV of sub-genotype VIId in Eastern Europe, with occasional introductions from Asia. Furthermore, the high level of genetic similarity among those isolates suggests that the NDV isolates of sub-genotype VIId from Bulgaria and Ukraine may have been part of a broader epizootic process in Eastern Europe rather than separate introductions from Asia or Africa. Our results provide indication for both east to west, and north to south transmission of virulent NDV. This work emphasizes the importance of investigating the epidemiology of NDV and studying the mechanisms for intercontinental spread of the virus.
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INTRODUCTION. Newcastle disease (ND) is one of the most dangerous diseases of poultry, which causes the biggest economical losses and high amounts of morbidity and mortality in poultry farming. Disease control should be based on effective... more
INTRODUCTION. Newcastle disease (ND) is one of the most dangerous diseases of poultry, which causes the biggest economical losses and high amounts of morbidity and mortality in poultry farming. Disease control should be based on effective live and inactivated vaccine usage. Vaccination effectively depends on ND strains circulating in the country. Therefore, the genotype determination of circulating strains is a critical point in a vaccine's development. GOAL. The aim of our work was to develop an alternative sequencing technique for NDV genotyping and its comparison with the existing technique by Aldous E. (2003). METHODS. 13 NDVs of different origins and genotypes were used. RNA was extracted by affine absorption technique, cDNA was amplified using two primer sets: AV-primer set for 374 bp of F gene and a primer set, which was designed at the NSC IECVM, for 345 bp region of the same F gene. PCR products were purified and sequenced using the ABI sequencer. Genetic analysis of sequences was done using BioEdit7.0.1 and MEGA4.0 software. RESULTS. Both primer sets provided the synthesis of amplicons with the expected length. The isolate sequences were further used for dendrogram construction. It was determined, that 13 analyzed viruses belonged to ND genotypes I, II, IVd, Va, and Vd. All of them possess the typical cleavage sites, that were recognized as velo-, meso-, and lentogenic NDV group representatives. Isolates of IVd, Va, and Vd genotypes had amino acid sequences, typical for the velogenic group. Genotype II viruses were both avirulent and virulent, and isolates of genotype I were all non-virulent strains. The divergence between the analyzed genome region of the genotype II isolates was up to 20%; IVd genotype – 5-12%; I genotype I – 10%, Va – 5%, and Vd – 12%. Analysis of the 345 bp region of the F gene of these viruses demonstrated their belonging to the same groups in NDV topology. Genotype I group contained one isolate, genotype II – six, IVd and Va – one, and Vd – four isolates. Analysis of both dendrograms showed a non-sufficient level of divergence between both trees in genotype-representative groups and stability of topology. The amino acid sequences of cleavage sites were similar for the same strains in both sequenced fragments. CONCLUSION. Sequences obtained by using both NDV primer sets demonstrated the similarity of results concerning pathotype and genotype determination, which confirms that both sets can be used for ND molecular epidemiology study.
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Psoriasis is one of the most urgent and complex problems of modern dermatology. It is characterized by patches of abnormal skin. The search for safe treatment of psoriasis, an objective assessment of toxicity, and the long-term... more
Psoriasis is one of the most urgent and complex problems of modern dermatology. It is characterized by patches of abnormal skin. The search for safe treatment of psoriasis, an objective assessment of toxicity, and the long-term consequences of drugs that affect the lives of patients are necessary tasks of modern medicine. Genetic studies of single nucleotide polymorphisms (SNP), corresponding to genes that were associated with the development of psoriasis, reveals the possibility of extrapolating bio-testing data to predict the maximum clinical effect and to minimize adverse reactions. This can be the basis for targeted individualized pathogenetic therapy for patients with psoriasis.
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The study of genes of the calpain-calpastatin system on model objects will help to clarify the mechanisms of age-related changes in muscle tissue, as well as to assess the impact of polymorphic variants of these genes on the processes of... more
The study of genes of the calpain-calpastatin system on model objects will help to clarify the mechanisms of age-related changes in muscle tissue, as well as to assess the impact of polymorphic variants of these genes on the processes of an organism's growth and development.
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Aim. Analysis of single nucleotide polymorphisms C677T and A1298C of MTHFR genes in patients with psoriasis in the Ukrainian population. Methods. Molecular genetic analysis of 77 patients with psoriasis by PCR-RFLP was carried out.... more
Aim. Analysis of single nucleotide polymorphisms C677T and A1298C of MTHFR genes in patients with psoriasis in the Ukrainian population. Methods. Molecular genetic analysis of 77 patients with psoriasis by PCR-RFLP was carried out. Results. Population structure corresponds to the correlation of the Hardy-Weinberg balance. The actual distribution of genotypes was not significantly different from the theoretically expected at balance C677T polymorphisms (df = 2, χ2 = 3,76, χst = 5.99, p>0,05) and A1298C (df = 2, χ2 = 3,86, χst = 5.99, p> 0,05). Analysis of the genotype distribution series for the MTHFR gene in patients with psoriasis did not show a significant difference between the theoretically expected frequencies and the actual for the C677T and A1298C polymorphic variants of the MTHFR gene (df = 8, χ2 = 0.55, χst = 15.51, p <0.05), which requires further analysis of haplotypes. Patients with psoriasis had a combination of СТ/АС and СТ/АА more frequently (3,2 and 1,7 times, respectively). Conclusions. The results of the study of the C677T and A1298C polymorphic variants of the MTHFR gene among patients with psoriasis showed a low frequency of homozygous T and C alleles, respectively. This shows the possibility of polymorphic variants of the MTHFR gene being accounted for in the planning of preventive and curative measures for patients with psoriasis.
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Our data highlights the importance of continuous monitoring of wild birds in the Azov-Black Sea region and to further identify possible introductions of avian viruses from other geographic regions.
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Creation of polymerase chain reaction protocol for West Nile fever virus detection
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The aim of this study was to determine the allele and genotype frequencies of SNPs for gene calpain (CAPN316), calpastatin (CAST282), and growth hormone (GH L127V) in Aberdeen-Angus (n=52) and to evaluate their impact on body weight... more
The aim of this study was to determine the allele and genotype frequencies of SNPs for gene calpain (CAPN316), calpastatin (CAST282), and growth hormone (GH L127V) in Aberdeen-Angus (n=52) and to evaluate their impact on body weight dynamics until the age of 5 years. The allele and genotype frequencies were CAPN316: C = 0.45, G = 0.55; CC = 19.2%, CG = 51.9%, GG = 28.9%, CAST282: C = 0.75, G = 0.25; CC = 53.8%, CG = 42.3%, GG = 3.9%, GH L127V: C = 0.34, G = 0.66; CC = 9.6%, CG = 48.1%, GG = 42.3%. There is a correlation between the number of C-alleles for GH L127V and an increase in body weight from birth until 2 years. The CAPN316 and CAST282 alleles, which are associated with meat tenderness, positively affect live body weight after 2 years of age. A significant effect of genotype CC CAPN316 is observed at the ages of 3 and 4 years. It was concluded, that selection aimed to improve meat quality does not lead to a significant reduction in live body weight.
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The study of genes of the calpain-calpastatin system on model objects will help to clarify the mechanisms of age-related changes in muscle tissue, as well as to assess the impact of polymorphic variants of these genes on the processes of... more
The study of genes of the calpain-calpastatin system on model objects will help to clarify the mechanisms of age-related changes in muscle tissue, as well as to assess the impact of polymorphic variants of these genes on the processes of growth and development of the organism.
Research Interests:
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Introduction: Brucellosis is a dangerous and contagious disease affecting animals (domestic and wild) and humans. Circulating Enterobacteria in livestock complicates the diagnosis of brucellosis because the causative agent Brucella... more
Introduction: Brucellosis is a dangerous and contagious disease affecting animals (domestic and wild) and humans. Circulating Enterobacteria in livestock complicates the diagnosis of brucellosis because the causative agent Brucella demonstrates antigenic homology. The use of PCR in the early stages of diagnosis can simplify and accelerate the identification of the causative agent. Goal: Differentiation of Brucella and Enterobacteria in samples of serum and milk by standard microbiological methods and PCR. Methods: Reference Brucella strains - B.abortus 544, B.abortus 19, B.abortus В-1, B.suis 1330 and typical strains of Enterobacteria – Escherichia coli 099, Yersinia enterocolitica serovar 09, and Salmonella enterica serovar Enteritidis M. were used. Sterile bovine milk and serum samples were inoculated with Brucella and Enterobacteria at concentrations of 10^6-10^9 CFU. The samples of biological fluids were examined using standard microbiological methods and PCR. The length of testing and accuracy of the results for microbiological testing and PCR were compared. Results: Brucella strains were isolated from the inoculated blood and milk samples, and further typed by determination of the main cultural and biochemical characteristics (growth in MPLGGA, growth/no growth in SMPLGGA with fuchsin and thionine, formation H2S, TAT and TFT, WWC testing, agglutination with S- or R- Brucella serum). Additionally, Enterobacteria strains were also isolated and typed by determination of the main cultural and biochemical characteristics (growth in MPA and MPB; glucose, sorbitol, lactose, carbamide, citrate and acetate utilization; formation H2S and indole, catalase and oxidase test, FP and methyl red test). Following PCR, B.abortus strains were typed by the presence of bands of 1682, 794, 587, 450, and 152 bp and B.suis - by the presence of bands1071, 794, 587, 450, 272, 218, and 152 bp. Samples contaminated with E.coli 099, Y.enterocolitica 09, S.Enteritidis M., and control intact samples showed negative results. Conclusion: Efficiency of the molecular genetic studies usage in the differential diagnosis of Brucella and Enterobacteria in biological fluids was shown.
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It was shown that PCR is a more specific, sensitive, and accurate diagnostic method compared to RID, as 40.6% of RID positive samples did not contain genetic material of leukemia virus when analyzed with PCR. It was also proven that PCR... more
It was shown that PCR is a more specific, sensitive, and accurate diagnostic method compared to RID, as 40.6% of RID positive samples did not contain genetic material of leukemia virus when analyzed with PCR. It was also proven that PCR analysis detected positive samples at the earlier stages of disease than RID was able to. PCR screening for leukemia virus can help to prevent the spread of infection between animals or begin the recovery of farms as soon as possible. In our work, it was proven that selective molecular genetic testing of cattle at the initial and final stages of a farm’s recovery from leukemia is necessary.
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Background: West Nile fever is a disease of horses, humans, and several avian species caused by a Flavivirus. Wild birds act as a reservoir for West Nile virus (WNV). Wildlife populations should be monitored for WNV using modern... more
Background: West Nile fever is a disease of horses, humans, and several avian species caused by a Flavivirus. Wild birds act as a reservoir for West Nile virus (WNV). Wildlife populations should be monitored for WNV using modern surveillance techniques. Ukraine is located in the migration pathways of wild birds connecting Asia, Europe, and Africa. These birds pose a risk for the introduction of WNV into Ukraine. The goal of this work was to develop a protocol for the detection of viral RNA using conventional PCR. Materials: Specific primers were developed for a 280 bp region of the gE gene of WNV based on GenBank published sequences of viral cDNA. The amplification reaction was optimized based on a gE recombinant template derived from the WNV vaccine seed. PCR master mix reagents from a Russian Federation manufacturer were used. The protocol was validated according to the OIE requirements for diagnostics techniques. Results: The gE gene sequences from the GenBank were analyzed for the conserved regions, and four of them were selected for the primer design. The forward and reverse primer pair, flanking 280 bp region of the gE gene, was designed. The protocol for the conventional PCR was a 40-cycle reaction with an annealing temperature of 57 ̊C. The reaction mix volume was 30 ml with 5 U of Taq polymerase, 1.5 mM Mg, and 20 pM of each primer. The PCR was able to detect viral cDNA in samples of vaccine virus and plasmid DNA (5-7 pg/ml). The PCR was validated with cDNA test panels and recognized as 95 % sensitive, 100 % specific, repeatable, and accurate. Screening of the blood and blood sera panels from wild birds, collected in Ukraine (n = 63), demonstrated the absence of the WNV in the specimens. Conclusion: A conventional PCR for WNV detection was developed and validated. Screening of field samples from wildlife demonstrated the absence of the virus. Further work is needed for the development of surveillance systems for WNV in wildlife and synanthropic birds in Ukraine.
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Purpose: The aim of this work was to genotype the bovine viral diarrhea virus (BVDV), which was detected in 2 cell lines, 6 biopreparations, and 4 cattle semen samples. Methods: Amplification of samples of cDNA from RNA of BVDV was done... more
Purpose: The aim of this work was to genotype the bovine viral diarrhea virus (BVDV), which was detected in 2 cell lines, 6 biopreparations, and 4 cattle semen samples. Methods: Amplification of samples of cDNA from RNA of BVDV was done using the Amplisans commercial kit and 5’-UTR specific primers, published by Prof. Vilcek (SK). Results: Sequencing data of BVDV 288 bp 5’-UTR region revealed four viral variants in the analyzed group. Obtained results demonstrated that viruses detected in blood sera and cell lines FLK and PK15 belong to the first viral variant group (pairwise distances: 0,030-0,046 inside the group, 0,130-0,373 - among groups). The second variant group contained viruses allocated in vaccines (CSF, PRRS, and PPV), blood sera for cell cultures, complex globulins, and cell cultures (distance in clad - 0,095, intraclad distance - 0,130-0,373). The third and fourth variant groups contained bovine semen isolates (distances - 0,200 and 0,373, respectively). Sequenced cDNA samples of BVDV, detected in cell lines of the first group, were classified as 1b genotype and were related to the strains Manas and 390. Contaminants of Pestivirus origin, detected in veterinary drugs (vaccines, blood sera, and globulin) were genotyped as 1a BVDV. They were most closely related to the BVDV strains NADL and Letuyi. Viruses of Bovine diarrhea detected in semen (Poltava and Cherv Veleten) belonged to subtypes 2а and 2b, respectively. Their topographic situation on the constructed dendrogram demonstrated high level of homology with the strains TR-2006 (Canada) and Kosice (Central Europe), respectively. The sequencing data were published in GenBank (No FJ223608-FJ223614) after the phylogenetic study. Conclusions: 1a, 1b, 2a, and 2b BVDV genotypes were detected in various biological matrixes by PCR amplification and sequencing.
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Purpose: Despite a large number of DNA vaccine regulations, most of them serve as guidelines. We developed the Ukrainian state standard (analog state SOP) “Molecular diagnostics. DNA-vaccine. Methods of quality control” by carefully... more
Purpose: Despite a large number of DNA vaccine regulations, most of them serve as guidelines. We developed the Ukrainian state standard (analog state SOP) “Molecular diagnostics. DNA-vaccine. Methods of quality control” by carefully considering all current national requirements, the OIE and the WHO recommendations for DNA preparations, coupled with our vast experience in recombinant DNA technologies. Methods: PCR, RFLP, electrophoresis, sequencing, spectrophotometry, microbiology, LOL-test, and МТТ-test were utilized. Results: The proposed control system of quality DNA vaccines for veterinary use involves the following steps: a) Authenticity control of the specific gene region in plasmid DNA (PCR). b) Availability control of the specific gene insertion in the plasmid material (RFLP). c) Uniformity control of the plasmid DNA (electrophoresis). d) Nucleotide sequence control of the specific gene insertion in the plasmid DNA (sequencing). e) Concentration and purity control of the plasmid DNA (spectrophotometry). f) Microbial contamination control of the DNA vaccine. g) Safety control of the DNA vaccines (testing in laboratory animals). h) Availability control of the bacterial endotoxins in DNA vaccines (LOL-test). i) Cytotoxicity control of the DNA vaccines (MTT-test). j) Immunogenicity control of the DNA vaccines (ELISA). k) Protective effectiveness control of the DNA vaccine (testing in laboratory animals). Conclusions: This developed standard is the first Ukrainian experience in the implementation of international biosafety recommendations for recombinant DNA technology in the practice of biotechnological production.
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Purpose: This study was focused on (i) detection of BVDV specific antibodies in selected cattle farms, (ii) identification of persistently infected (PI) animals, and (iii) genetic typing of selected BVDV isolates. Methods: BVDV antibodies... more
Purpose: This study was focused on (i) detection of BVDV specific antibodies in selected cattle farms, (ii) identification of persistently infected (PI) animals, and (iii) genetic typing of selected BVDV isolates. Methods: BVDV antibodies were detected in 1023 blood samples collected from three cattle farms in the Kharkiv region during 2011-2012. The total number of animals was 815 on the first farm, 900 and 5500 animals on the second and third farms, respectively. PI animals were identified by the RT-PCR method utilizing the pan-pestivirus 324/326 primers for BVDV detection in samples from antibody-negative cattle. Selected PCR amplicons were sequenced. Phylogenetic analysis of 5’-UTR (245 bp fragment) was used for the genetic typing of BVDV isolates into subgenotypes. Results: BVDV specific antibodies were detected in 694 out of 1023 samples analyzed (67.8%). This number is in agreement with findings in many cattle herds around the world. However, the number of positive samples differed in the herds. While 43 samples out of 250 (17.2%) were identified in the first herd, 398 out of 473 (84.1%), and 253 out of 300 (84.3%) animals were seropositive in the second and third herds, respectively. The BVDV's RNA was detected in a high number of animals in all herds. The RT-PCR assay detected 143 out of 1023 samples analyzed (14.1%). 32 samples out of 250 (12.8%) were detected in the first herd, 79 out of 473 (16.7%), and 32 out of 300 (10.7%) were found in the second and third herds, respectively. The total number of PI animals detected in the cattle herds was in accordance with our previous serological findings. Genetic typing of 12 isolates indicated that all viruses belonged to the BVDV-1b subgenotype and all of them contained an identical 5’-UTR region. It is not excluded that identical isolates may be the result of a live BVDV vaccine usage on those farms but this hypothesis has yet to be verified. Conclusion: Our results indicated that the BVDV infection is widespread in cattle herds in the Kharkiv region. Better characterization of viral isolates, as well as the introduction of the biosecurity programs on farms, are in progress under the ongoing Swiss-Ukraine-Slovak SCOPES project.
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Pestiviruses and Mycoplasmas are potential contaminants of biological products, which are manufactured using animal raw materials, such as bovine or porcine sera. These materials are widely used in diagnostic laboratories and as a cell... more
Pestiviruses and Mycoplasmas are potential contaminants of biological products, which are manufactured using animal raw materials, such as bovine or porcine sera. These materials are widely used in diagnostic laboratories and as a cell growth-promoting factor in cell culture and vaccine productions. Therefore, any viral contaminants or antibodies present in serum may hamper proper diagnosis and efficient application of the vaccines. Vaccine contaminations may not only influence vaccination results but also lead to new infections, causing serious economic problems within a herd. In the present work, we developed and optimized the PCR detection protocol of Bovine Viral Diarrhea Virus (BVDV) and mycoplasma in semen samples, cell cultures, bovine sera, and immunobiological products. The 5’ UTR gene and 16S-23S intergenic spacer regions were used for the amplification of 287 and 450-480 bp specific amplicons of BVDV and mycoplasmal genomes, respectively. DNA from M. orale N-I, M. hyorhinis BTS-7, M. bovis PG45T, and cDNA from BVDV strain Oregon were used to detect the level of the reaction sensitivity, which were determined as 100 cells/ml for mycoplasmas and 1 lg/ml for BVDV. In total, 62 samples of semen, 90 samples of veterinary immunobiological preparations, 1084 samples of raw materials for production of native bovine blood serum, and 37 cell cultures FLK were investigated by these methods. As a result, 8 samples of native blood sera before filtration were contaminated with Mycoplasma and 6 samples among them contained the genetic material of Bovine Viral Diarrhea Virus.
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The recombinant plasmid pTZR/T-VD carrying the insertion of 5’ UTR untranslated region of BVDV (267 b.p. long) was constructed. The orientation of the insert was confirmed by PCR-screening. In summary, effective control for PCR was... more
The recombinant plasmid pTZR/T-VD carrying the insertion of 5’ UTR untranslated region of BVDV (267 b.p. long) was constructed. The orientation of the insert was confirmed by PCR-screening. In summary, effective control for PCR was obtained.
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An improved sorbent nucleic acids extraction method was developed. Its efficacy was demonstrated on DNA and RNA templates (Gallid herpesvirus 2 and Bovine viral diarrhoea virus, respectively) by polymerase chain reaction amplification.
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The potential of polymerase chain reaction to be used as a part of the general diagnostics system for detection of the Porcine Reproductive Respiratory Syndrome Virus (PRRSV) in pigs was studied. Additionally, different methods for... more
The potential of polymerase chain reaction to be used as a part of the general diagnostics system for detection of the Porcine Reproductive Respiratory Syndrome Virus (PRRSV) in pigs was studied. Additionally, different methods for nucleic acid extraction from biological materials were compared and optimal amplification parameters were determined. The main objectives of this study were: (a) to compare two primer sets for the genetic material of the PRRSV detection in biological materials; (b) to optimize the PRRSV amplification parameters. During this study, new experimental data were obtained; the amplification conditions for highly specific and sensitive detection of PRRSV allocation were determined; and statistical analysis of the acquired results was carried out.
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This handbook is devoted to the description of a polymerase chain reaction - the main molecular method of diagnostics for infectious, invasive, and hereditary diseases; in-depth studies on genotyping, pathotyping, and differentiation of... more
This handbook is devoted to the description of a polymerase chain reaction - the main molecular method of diagnostics for infectious, invasive, and hereditary diseases; in-depth studies on genotyping, pathotyping, and differentiation of microorganisms and macroorganisms. Basic information on the structure of nucleic acids, genes, genomes, biophysical mechanisms of the polymerase chain reaction, theoretical and practical aspects of the development and use of methods for the indication and identification of pathogenic bacteria and viruses, creation and control of recombinant constructs, and the analysis of variable fragments of genes. This handbook is of theoretical and practical interest to a wide range of specialists, especially in veterinary and human medicine, specialists in general biology and biotechnology, scientists, and undergraduate and graduate students in these fields. In addition to the theoretical part, this handbook contains many practical guidelines, that were developed at the NSC "IECVM", for the detection and differentiation of animal pathogens.
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This handbook is devoted to the description of a polymerase chain reaction - the main molecular method of diagnostics for infectious, invasive, and hereditary diseases; in-depth studies on genotyping, pathotyping, and differentiation of... more
This handbook is devoted to the description of a polymerase chain reaction - the main molecular method of diagnostics for infectious, invasive, and hereditary diseases; in-depth studies on genotyping, pathotyping, and differentiation of microorganisms and macroorganisms. Basic information on the structure of nucleic acids, genes, genomes, biophysical mechanisms of the polymerase chain reaction, theoretical and practical aspects of the development and use of methods for the indication and identification of pathogenic bacteria and viruses, creation and control of recombinant constructs, and the analysis of variable fragments of genes. This handbook is of theoretical and practical interest to a wide range of specialists, especially in veterinary and human medicine, specialists in general biology and biotechnology, scientists, and undergraduate and graduate students in these fields. In addition to the theoretical part, this handbook contains many practical guidelines, that were developed at the NSC "IECVM", for the detection and differentiation of animal pathogens.
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Purpose: Screening of bull semen in order to find its contamination with Chlamydia, Bovine viral diarrhea virus (BVDV), infectious rhinotracheitis virus (IRTV), and rotaviral infection (RI) is the essential step to define the role of... more
Purpose: Screening of bull semen in order to find its contamination with Chlamydia, Bovine viral diarrhea virus (BVDV), infectious rhinotracheitis virus (IRTV), and rotaviral infection (RI) is the essential step to define the role of semen in the transmission of these infections and their outbreaks on farms in Southern and Eastern Ukraine. Methods: Screening was conducted with the conventional PCR technique using CHOMP, BVDV, IRTV, and rotab primers. In total 252 bull semen samples were investigated. Samples were collected from bulls of different breeds from 8 farms with a high burden of respiratory, reproductive, and gastrointestinal infections in Kharkiv, Lugansk, Donetsk, Dnipropetrovsk, Odessa, and Mykolayiv regions. Vaginal swabs and pathological material from misbirths were investigated in separate cases as well. Results: Chlamydial DNA was found in 15 semen samples collected at 2 farms. In one of these farms, a mixed infection of IRV and chlamydiosis was found. These cases were accompanied by abortions, the birth of nonviable calves, and affection of joints during the first year of life. The infectious agent was detected in both, semen and clinical material, and identified as Chlamydophila abortus. In semen samples from another farm, only chlamydial DNA was found. Bovine viral diarrhoea virus was found in 22% of investigated samples, and in most cases, these samples were also contaminated with rotavirus. DNA of herpesvirus was detected in 12% of samples and was further differentiated as IRTV type I. In farms, where IRTV contaminated semen was used, reproduction system disorders, accompanied with a decreasing percentage of calf births caused by abortions and infertility were observed. Investigations of vaginal swabs and pathological material from misbirths also revealed the presence of IRTV DNA in 14% of cases. Conclusions: The results of PCR screening showed a high percentage of viral contamination (33% of total samples) in bull semen. PCR proved to be one of the most rapid and sensitive instruments for large-scale screening.
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The recombinant plasmid pTZR/T-VD carrying the insertion of 5’ UTR untranslated region of BVDV (267 b.p. long) was constructed. The orientation of the insert was confirmed by PCR-screening. In summary, effective control for PCR was... more
The recombinant plasmid pTZR/T-VD carrying the insertion of 5’ UTR untranslated region of BVDV (267 b.p. long) was constructed. The orientation of the insert was confirmed by PCR-screening. In summary, effective control for PCR was obtained.
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Research Interests:
In ovo vaccination has been employed by the poultry industry for over 20 years to control numerous avian diseases. Unfortunately, in ovo live vaccines against Newcastle disease have significant limitations, including high embryo mortality... more
In ovo vaccination has been employed by the poultry industry for over 20 years to control numerous avian diseases. Unfortunately, in ovo live vaccines against Newcastle disease have significant limitations, including high embryo mortality and the inability to induce full protection during the first two weeks of life. In this study, a recombinant live attenuated Newcastle disease virus vaccine containing the antisense sequence of chicken interleukin 4 (IL-4), rZJ1*L-IL4R, was used. The rZJ1*L-IL4R vaccine was administered in ovo to naïve specific pathogen free embryonated chicken eggs (ECEs) and evaluated against a homologous challenge. Controls included a live attenuated recombinant genotype VII vaccine based on the virus ZJ1 (rZJ1*L) backbone, the LaSota vaccine and diluent alone. In the first of two experiments, ECEs were vaccinated at 18 days of embryonation (DOE) with either 104.5 or 103.5 50% embryo infectious dose (EID50/egg) and chickens were challenged at 21 days post-hatch ...
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We report the complete genome sequences of 11 virulent Newcastle disease viruses. The isolates were obtained from vaccinated broiler and layer chickens in three different provinces of Indonesia in 2013 and 2014. Phylogenetic analysis... more
We report the complete genome sequences of 11 virulent Newcastle disease viruses. The isolates were obtained from vaccinated broiler and layer chickens in three different provinces of Indonesia in 2013 and 2014. Phylogenetic analysis revealed that all isolates belong to subgenotype VII.2 in the class II cluster.