Specific Oligonucleotide Primers Research Articles

Molecular identification of Baylisascaris melis (Gedoelst, 1920) from the Eurasian badger (Meles meles) and ascarids from other wild carnivores in Kazakhstan

IntroductionThe presence of gastrointestinal nematodes, including zoonotic ascarids, in wild canids, felids and mustelids as definitive hosts in Central Asian countries has been documented in many studies based on traditional morphological methods. In contrast, relevant data for the badger are scarce. The aim of this study was the molecular identification of ascarid nematodes from five wild carnivore species in different regions of Kazakhstan.MethodsA total of 211 adult ascarids were collected from gray wolves (Canis lupus, 8 of 83 infected with 2–6 Toxascaris leonina), red foxes (Vulpes vulpes, 26 of 53, with 2–8 Toxascaris leonina), corsac foxes (Vulpes corsac, 6 of 11, 3–6 Toxascaris leonina), lynx (Lynx lynx, 2 of 3, with 2–5 Toxocara cati) and badgers (Meles meles, 2 of 4, with 2–7 Baylisascaris melis). Genomic DNA was extracted from the worms and ribosomal DNA, including the first and second internal transcribed spacer genes, was amplified by polymerase chain reaction using specific oligonucleotide primers and then sequenced.ResultsToxascaris leonina, but not Toxocara canis, was molecularly identified in the wild canids, Toxocara cati in the lynx and Baylisascaris melis in the badger. The maximum likelihood phylogenetic tree showed three distinct clades: the canid Toxascaris leonina was placed in one clade, Toxocara cati in another and Baylisascaris melis in a third.DiscussionThe study provides the world’s first molecular data and phylogenetic analysis of Baylisascaris melis, identified for the second time since its description over 100 years ago. This species was shown to be genetically distinct from other Baylisascaris spp. (B. columnaris, B. procyonis, B. transfuga, B. devosi). The possible zoonotic significance of ascarids from wild carnivores is discussed in the light of conditions in Central Asia.

Association of Telomere Length in T Lymphocytes, B Lymphocytes, NK Cells and Monocytes with Different Forms of Age-Related Macular Degeneration.

Age plays a primary role in the development of age-related macular degeneration (AMD). Telomere length (TL) is one of the most relevant biomarkers of aging. In our study, we aimed to determine the association of TL with T lymphocytes, B lymphocytes, NK cells or monocytes with different forms of AMD. Our study included 62 patients with AMD: geographic atrophy (GA), neovascular AMD (NVAMD) with and without macular atrophy and 22 healthy controls. Each leukocyte subtype was isolated from peripheral blood by immunomagnetic separation, and the DNA was purified. The TL in the genomic DNA was determined using qPCR by amplifying the telomere region with specific oligonucleotide primers and normalizing to the control gene. Statistical analysis was performed using R version 4.5.1. We observed a statistically significant increase in TL in the T cells between the control and NVAMD groups but not for the GA group. The B cells and monocytes showed a significant decrease in TL in all AMD groups. The TL in the NK cells did not decrease in any of the AMD groups. The TL in the monocytes had the strongest association with AMD. It reflects a person's "telomeric status" and may become a diagnostic hallmark of these degenerative processes.

MICRОCYSTIS AЕRUGINОSA AS A SОURCЕ ОF BIОLОGICAL HAZARDS IN WATЕR RЕSЕRVОIRS ОF UKRAINЕ

Introduction. Phytoplankton, including cyanobacteria, is ubiquitous in various types of reservoirs, and it plays a crucial role in aquatic ecosystems. Among phytoplankton, cyanobacteria, considered one of the most primitive chlorophyll-containing organisms, have a significant presence in both standing freshwater lakes and seawater. Microcystis aeruginosa (M. aeruginosa), a common cyanobacterial species, is known for forming harmful “water blooms” that pose serious ecological and health risks. The rise of urbanization in Ukraine and the consequences of the war has intensified the eutrophication and cyanobacterial blooms in drinking water sources, necessitating improved detection and prevention strategies.
 The aim of this study was to develop modern and effective methods for diagnosing microcystin-containing cyanobacteria, particularly M. aeruginosa, to address biological hazards in Ukrainian water reservoirs.
 Materials and methods. Water samples were collected from a stagnant town lake in the Ivano-Frankivsk region during both summer and winter seasons to account for seasonal fluctuations in cyanobacteria content. Cyanobacteria were isolated from water samples and subjected to DNA extraction. PCR analysis using specific oligonucleotide primers targeted the 16S rRNA gene of M. aeruginosa.
 The results revealed the presence of 16S rRNA products in all water samples, irrespective of the season, indicating a consistent distribution of M. aeruginosa in the lake throughout the year.
 Discussion. We found cyanobacteria in all water samples regardless of the season. This indirectly suggests the presence of anthropogenic pollution, which helps maintain a consistent concentration of cyanobacteria throughout the year.
 In conclusion, the developed PCR-based method offers a sensitive and specific means of detecting M. aeruginosa, enabling comprehensive investigations across various water reservoirs. The presence of cyanobacteria in water bodies, while natural, can serve as an indicator of anthropogenic pollution, particularly nitrogen and phosphorus. This methodology holds promise for safeguarding Ukraine's drinking water sources and managing the impact of anthropogenic pollution, ultimately ensuring public health through effective planning and preventive measures.

Viruses Demonstrate Selective Survival During Simulated Anaerobic Digestion of Plant Biomass

Objective. This research aimed at laboratory simulation of anaerobic digestion of plant biomass contaminated by a mixture of viruses with a wide host range to evaluate if viruses can retain their infectivity and pose a biohazard during the use of plant-based reusable resources such as digestate. Methods. Anaerobic digestion was simulated in air-tight desiccators using anaerobic bags and indicator strips to ensure proper conditions, and using fresh biomass of naturallyvirus-infected plants. In one of the variants, a commercial mixture of anaerobic bacteria and enzymes was also added to see if it would be more efficient in terms of plant digestion and virus inactivation. For serological detection of the tomato mosaic virus, cucumber mosaic virus, and potato virus Y, DAS-ELISA was used with commercial diagnostic kits and positive controls. Absorbance values were measured at 405 nm. Total RNA was extracted using a RNeasy Plant Mini kit following the manufacturer’s instructions. Molecular detection of viruses was carried out by two-step RT-PCR using specific oligonucleotide primers available from the literature. To check if the viruses can retain their infectivity after the anaerobic digestion of plant biomass waste, a bioassay was used where Nicotiana rustica plants producing necrotic lesions upon infection with many viruses were mechanically inoculated at the stage of 2 true leaves. For statistical processing of data, the mean and standard error were calculated using Microsoft Excel software. Results. After prolonged anaerobic digestion, tomato mosaic virus was still detected in plant residues, but it is important to note that it was also found in high concentration in the liquid phase. Hence, during the anaerobic digestion, a significant proportion of the virus was successfully eluted from plant residues into the digestate. Interestingly and contrary to tomato mosaic virus, cucumber mosaic virus has not practically eluted to the liquid phase. On the other hand, potato virus Y was supposedly completely decomposed during the anaerobic digestion of plant biomass waste, as it was not detected either by ELISA or by PCR. It is likely that not all of the investigated viruses represent a potential biohazard in the context of the use of renewable resources. RT-PCR outcomes showed the presence of tomato mosaic virus and cucumber mosaic virus both in the solid and the liquid phases, augmenting the ELISA data. Lastly, bioassay results have confirmed that at least tomato mosaic tobamovirus may escape the aggressive process of waste decomposition and remain able to infect host plants raising issues about the safety of reusable plant resources. Conclusions. The survival of plant viruses during plant biomass waste processing was studied in the context of biological safety. The preservation of serological markers and genetic signatures of model viruses in the process of anaerobic digestion has been confirmed. For the first time, it was proven that tomato mosaic virus may preserve its infectivity during long-term incubation of plant biomass waste in anaerobic conditions, which requires further attention when using renewable resources. On the example of PVY, it has been shown that potyviruses do not survive during anaerobic digestion and therefore may not pose an epidemic risk in using processed renewable resources. It has been demonstrated that artificial introduction of anaerobic microorganisms does not have a decisive effect on the stability of the studied model viruses.

Investigation of anthraquinone-producing Trichoderma reesei by highperformance liquid chromatography (HPLC)

Objective: To investigate the isolates of Trichoderma that produce anthraquinones by High-performance LiquidChromatography (HPLC).Methods: Trichoderma specimen were collected from Wassit Soil and identified dependent on morphological featuresand tested for production of anthraquinones by HPLC, Trichoderma isolate which produced a high concentration ofanthraquinones diagnosed for the species using PCR-ITS region.Results: Ten isolates were identified as Trichoderma according to morphological and microscopic features. Resultsthree Trichoderma isolates show differences between the concentrations of anthraquinones among the ten isolates.The total concentration of this compound in the extracts of specimens 1, 2 and 3 were (7.765ug/ml), (2.308ug/ml),and (4.977ug/ml) respectively. At the final concentration of Trichoderma isolates, genomic DNA have been extracted(400 to 600 ug) / (2 to 3g) fresh mycelium, and with a concentration of (1.6 to1.8), and the results of amplifyingTrichoderma DNA samples by using ITS-1 and ITS-4 showed a single unique band consistent with T. reesei F48-03, Whichwith other isolates of the Trichoderma were missing, were identified successfully.Conclusion: For identification and phylogenetic classification of Trichoderma, DNA-based methods that provide usefulclassification information are presently used. For several years, most T. spp. is regarded as a single species due to theirmorphological similarity. This research used ITS markers to distinguish genotypes within T. spp. because of amplifyinga distinct, naturally determined locus with a couple of T.reesei -specific oligonucleotide primers. This research wascarried out to provide supporting evidence for the long-standing antimicrobial use of anthraquinone.Keywords: DNA, Primers, Trichoderma, Phylogeny, Soil, Chromatography, Liquid, Polymerase, Reaction,Anthraquinones, Mycelium, Plant, Genomics.

AVIAN INFLUENZA, SPREADING OF THE AIV AND DEVELOPMENT OF TEST-SYSTEMS USING SAMPLES COLLECTED FROM FOCI POINTS

To date, pathogenic strains of the influenza A virus have caused several dangerous pandemics and caused high mortality. Viral diseases cause irreversible pathology, which leads to a significant decrease in productivity and death of the infected bird. The only way to protect birds from the avian influenza virus is to keep birds in isolation and develop immunity in birds to the pathogen through vaccines. However, despite the above possibilities of protection against the above-mentioned viral pathogens, the epizootic situation among birds in many countries, including the Republic of Kazakhstan, remains tense. There are many publications on the development of test systems, but the variability of the AIV genome and its susceptibility to mutations creates serious problems in laboratory evaluation. In this regard, the development of effective domestic methods for the rapid diagnosis of avian influenza, such as PCR test systems, is relevant. Thus, this is the only express method necessary to further strengthen the complex of veterinary-sanitary and special veterinary measures to prevent the spread of infection in the country. For this purpose, more than 30 samples were selected in the North Kazakhstan region for the selection and design of specific oligonucleotide primers for the detection of AIV.

Optimization of polymerase chain reaction for monitoring of Borrelia burgdorferi infection by ixodid ticks

Determination of the infection rate of ixodid ticks with tick-borne borreliosis pathogens and determination of belonging to the pathogenic genotype by PCR is an important component for monitoring, risk assessment and control of the epizootic situation of Lyme borreliosis in different territories. The results of testing and optimization of the internal laboratory protocol of the classical polymerase chain reaction for the identification of Lyme disease pathogens are presented. Eight samples of extracted DNA from ixodid ticks collected from vegetation in the forest park tract "Golendernya", Bila Tserkva, Kyiv region, were examined by classical PCR. Samples were formed from pools of ten tick specimens: seven pools - ticks of the genus I. ricinus and one pool - ticks of the genus D. reticulatus. For detection of borrelia DNA, primer sets were used to detect DNA of Borrelia burgdorferi sensu lato complex; Borrelia burgdorferi and pathogenic borrelia: Borrelia burgdorferi sensu stricto, Borrelia garinii and Borrelia afzelii. The protocol for nucleic acid extraction from ticks was modified using the commercial IndiSpin Pathogen Kit. Optimization of amplification temperature conditions was carried out by the annealing temperature gradient method for each primer pair. Based on the results of the study, internal laboratory protocols for classical PCR using specific oligonucleotide primers were developed. It was found that in each of the pools of I. ricinus and D. reticulatus there were infected tick specimens with the Borrelia burgdorferi sensu lato complex and Borrelia afzelii genus, and also identified the Borrelia burgdorferi sensu stricto genus in one of the pools of I. ricinus and D. reticulatus, DNA of the Borrelia garinii genus was not detected. The developed internal laboratory protocols of classical PCR will be further used to study the infection of ixodid ticks with tick-borne borreliosis pathogens: Borrelia burgdorferi sensu lato, Borrelia burgdorferi sensu stricto and Borrelia afzelii. Key words: Lyme borreliosis, Ixodes ticks, polymerase chain reaction, Borrelia burgdorferi sensu lato, Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii.

Characterisation of P2Y receptor subtypes mediating vasodilation and vasoconstriction of rat pulmonary artery using selective antagonists

Pulmonary vascular tone is modulated by nucleotides, but which P2 receptors mediate these actions is largely unclear. The aim of this study, therefore, was to use subtype-selective antagonists to determine the roles of individual P2Y receptor subtypes in nucleotide-evoked pulmonary vasodilation and vasoconstriction. Isometric tension was recorded from rat intrapulmonary artery rings (i.d. 200–500 µm) mounted on a wire myograph. Nucleotides evoked concentration- and endothelium-dependent vasodilation of precontracted tissues, but the concentration–response curves were shallow and did not reach a plateau. The selective P2Y2 antagonist, AR-C118925XX, inhibited uridine 5′-triphosphate (UTP)- but not adenosine 5′-triphosphate (ATP)-evoked relaxation, whereas the P2Y6 receptor antagonist, MRS2578, had no effect on UTP but inhibited relaxation elicited by uridine 5′-diphosphate (UDP). ATP-evoked relaxations were unaffected by the P2Y1 receptor antagonist, MRS2179, which substantially inhibited responses to adenosine 5′-diphosphate (ADP), and by the P2Y12/13 receptor antagonist, cangrelor, which potentiated responses to ADP. Both agonists were unaffected by CGS1593, an adenosine receptor antagonist. Finally, AR-C118925XX had no effect on vasoconstriction elicited by UTP or ATP at resting tone, although P2Y2 receptor mRNA was extracted from endothelium-denuded tissues using reverse transcription polymerase chain reaction with specific oligonucleotide primers. In conclusion, UTP elicits pulmonary vasodilation via P2Y2 receptors, whereas UDP acts at P2Y6 and ADP at P2Y1 receptors, respectively. How ATP induces vasodilation is unclear, but it does not involve P2Y1, P2Y2, P2Y12, P2Y13, or adenosine receptors. UTP- and ATP-evoked vasoconstriction was not mediated by P2Y2 receptors. Thus, this study advances our understanding of how nucleotides modulate pulmonary vascular tone.

Discrimination of Bacillus subtilis from Other Bacillus Species Using Specific Oligonucleotide Primers for the Pyruvate Carboxylase and Shikimate Dehydrogenase Genes.

Bacillus subtilis is a useful bacterium in the food industry with applications as a starter strain for fermented food and as a probiotic. However, it is difficult to discriminate B. subtilis from other Bacillus species because of high phenotypic and genetic similarity. In this study, we employed five previously constructed multilocus sequence typing (MLST) methods for the discrimination of B. subtilis from other Bacillus species and all five MLST assays clearly distinguished B. subtilis. Additionally, the 17 housekeeping genes used in the five MLST assays also clearly distinguished B. subtilis. The pyruvate carboxylase (pyrA) and shikimate dehydrogenase (aroE) genes were selected for the discrimination of B. subtilis because of their high number of polymorphic sites and the fact that they displayed the lowest homology among the 17 housekeeping genes. Specific primer sets for the pyrA and aroE genes were designed and PCR products were specifically amplified from B. subtilis, demonstrating the high specificity of the two housekeeping genes for B. subtilis. This species-specific PCR method provides a quick, simple, powerful, and reliable alternative to conventional methods in the detection and identification of B. subtilis.

Application of eDNA for monitoring freshwater bivalve Nodularia nipponensis and its glochidium larvae

AbstractHabitat loss, the introduction of invasive species, and climate change due to human activity have threatened many freshwater unionid bivalves worldwide. Unionid bivalves represent important members of freshwater ecosystems, providing bitterling fish with spawning grounds and contributing to water clarification via the filtration of suspended solids. This study examined an environmental DNA (eDNA) approach for monitoring the unionid bivalve Nodularia nipponensis in a lake environment. We developed oligonucleotide primers for the specific detection of mitochondrial DNA from N. nipponensis and evaluated the decay and release rates of eDNA in a laboratory tank experiment under two bivalve densities (49 and 164 individuals m−2) selected to ensure sufficient eDNA yields. The eDNA release rate was weakly and positively correlated with bivalve density but strongly and positively correlated with inorganic nitrogen and phosphorus excretion from the bivalve. In a eutrophic lake, Lake Hachiro, 67% of the samples (16 out of 24) collected from June to July yielded detectable amounts of eDNA and were not detected at all in May and August (0 out of 20). This difference was probably due to the release of N. nipponensis glochidium larvae. During these months, unionid glochidia adhered to bundles of polyvinylidene chloride fibers placed on the lake bottom for 8 h, confirmed by microscopic inspection. We successfully obtained N. nipponensis eDNA from all bundles examined over the months, demonstrating the efficacy of bundle capture in quantifying unionid bivalve eDNA. Our eDNA‐based technique with specific oligonucleotide primers and fiber bundles may be a promising tool for monitoring N. nipponensis in freshwater environments. Fiber bundles are useful for tracing eDNA derived from glochidium larvae, even at low unionid bivalve densities, which may be conducive to conservation efforts.

Species-specific PCR-based marker for rapid detection of Aspidiotus rigidus Reyne (Hemiptera: Diaspididae)

• A marker was designed from InDels of Aspidiotus rigidus 28 s rDNA sequences. • Species-specific delineation of A. rigidus works in singleplex and multiplex PCR. • A. rigidus multiplex-based detection still works with parasitized samples. The Philippine coconut production has been greatly affected by the recent devastating infestation of Aspidiotus spp. However, identification of the outbreak species, Aspidotus rigidus , has been a challenge using morphological approaches. Molecular identification via PCR sequencing of insect barcoding genes has been implemented, but the overall process is time-consuming and costly. Thus, we developed and optimized a species-specific PCR-based molecular marker for rapid, efficient and cost-effective molecular identification of A. rigidus . The molecular marker was designed based on the sequences of the partial 28S ribosomal RNA gene from species of Aspidiotus that feed on coconut in the Philippines, A. rigidus, A. destructor and A. excisus . Multiple alignment of nucleotide sequences revealed a conserved 16-bp insertion-deletion (InDel) site common to all A. rigidus specimens identified from which the A. rigidus- specific oligonucleotide (RIG1) primer targeting an approximately 570 bp fragment size was designed. Results showed that the species-specific DNA marker technology consistently delineated laboratory-reared and field-collected A. rigidus samples from A. destructor and A. excisus . The protocol offers a rapid and reliable method for the early detection of A. rigidus infestation in high-risk areas planted with coconut in the country.

Molecular fish sexing on Kohaku Koi (Cyprinus carpio) based on ArS.9-15 gene amplification by PCR method

This study aims to sex determine on Kohaku Koi fish rapidly, precisely, accurately, and as early as possible based on the amplification of the ArS.9-15 gene using the PCR method. This study began with the maintenance of 10 Kohaku Koi fish, obtained from Koi fish farmers in the Sleman area in Yogyakarta. The 10 Kohaku Koi were adapted in a filtered aquarium with O2 aeration and fed ad libitum by fish pellets for 1-3 days. Then, a collection of 0.5 ml peripheral blood samples per fish from the caudalis vein was performed under anesthesia conditions using special anesthetic preparation for Koi fish, namely Koi Anesthesia mixed with fresh water. Those samples were then collected in tubes containing the Ca-EDTA anticoagulant. The DNA then was extracted from peripheral blood samples and used as a template for PCR amplification by specific oligonucleotide primer pairs to amplify the ArS.9-15 gene. PCR products in the form of DNA fragments were visualized with 1.5% agarose gel electrophoresis and SybrSafe staining using Transilluminator-UV in a dark room. The DNA fragments were analyzed descriptively to sex determination between male and female Kohaku Koi. Electrophoresis results of PCR products showed two DNA bands of 850 bp and 1,100 bp in male Kohaku Koi, while in female Kohaku Koi showed only a band DNA in size of 850 bp. The results of the molecular fish sexing of the 10 Kohaku Koi showed that 70% are female and 30% are male.

Investigation of many bacterial and viral infections circulating in pigeons showing nervous symptoms

Pigeon’s flocks have shown several neurological symptoms including circling, torticollis, tremors, paralysis, which caused suspicion for viral or bacterial natural infections. Pigeon paramyxovirus type-1 (PPMV-1) is a notifiable disease-causing high morbidity and mortality with severe nervous symptoms. Clinical represented tissue specimens were collected from 50 infected pigeon flocks in eight governorates. All samples were examined bacteriologically (isolation, identification and serotyping) for E. coli, Salmonella spp., S. aureus and pseudomonas aeruginosa. Antimicrobial susceptibility test (AST) was accomplished for all isolates using a disk-diffusion test. For viral identification, RT-PCR specific oligonucleotide primers were used for distinguishing of Avian influenza virus, PPMV-1 and PPMV-3. Neurological manifestations were observed in pigeon’s flocks mainly in winter and autumn. The mortality rate in eight governorates was about 50% in 10 flocks and other houses mortality rate was ranged from 10 to 20%. Post mortem examination have shown hemorrhagic enteritis, soft and friable brain tissues and/or hemorrhages. The percentage of isolated bacteria E. coli, Salmonella spp., S. aureus and Pseudomonas aeruginosa were 75%, 75%, 50% and 18.75%; respectively. The antibiotic resistance pattern for bacterial isolates showed resist to ampicillin, amoxicillin- clavulinic acid, teteracyclin, ceftriaxone, doxycycline, sulfamethoxazole-trimethoprim and ceftazidine with different result for each type of bacteria, while Salmonella spp., isolates showed only a highly intermediate result for ciprofloxacin. Eight samples are positive with 16% to PPMV-1. Also, sample No.5,6,9 was co-infected with different types of bacterial isolates in addition to NDV. In conclusion, we reported several neurological symptoms in pigeon’s flocks mainly of bacterial infections (E. coli, Salmonella spp., S. aureus and Pseudomonas aeruginosa).

CATALASE CRS-262T GENE POLYMORPHISM AND CHANGES IN VENTILATION LUNG CAPACITY IN CHILDREN-RESIDENTS OF RADIOACTIVELY CONTAMINATED TERRITORIES.

to determine the association of catalase С-262Т gene polymorphism with the presence of bronchial hyper-reactivity in children living in radioactively contaminated territories. There were examined school-age children-residents of radioactively contaminated territories (RCT), who did not have clinical signs of respiratory pathology. Catalase (CAT) С-262Т gene deletion polymorphism was studied in the molecular genetic laboratory of the State Institution «Reference Center for Molecular Diagnostic of Public Health Ministry of Ukraine». Determination of the polymorphic variant by the catalase С-262Т gene was performed by Polymerase Chain Reaction (PCR) using specific oligonucleotide primers, followed by Restriction Fragment Length Polymorphism (RFLP) analysis. The CAT С-262Т gene polymorphism in children living in RCT was compared with that in the reference group of practically healthy individuals. Ventilation lung capacity was performed by computer spirometry according to the analysis of the loop «the flow-volume». A pharmacological inhalation test with a bronchodilator that acts on β2-adrenergic receptors of the lungs was used to detect early changes in the ventilatory capacity of the lungs - bronchial hyperreactivity. Comparative analysis showed that in the presence of bronchial hyperreactivity in children living in RCT, the CT genotype was more common than in children without bronchial hyperreactivity, and the frequency of the CC genotype was correspondingly reduced. There was a trend towards a decrease in the frequency of the TT genotype. An analysis of the frequency distribution of allelic variants of the CAT С-262Т gene polymorphism in children living in the RCT revealed a tendency to increase in the frequency of the T-allele and according to the decrease in the frequency of C-allele in the presence of bronchial hyperreactivity.Сonclusions. Thus, among children living in RCT, CT-homozygotes of CAT С-262Т gene polymorphism had bronchial hyperreactivity probably more often than CC-heterozygotes. In the presence of bronchial hyperreactivity, there was a trend towards an increase in the frequency of the T-allele and, accordingly, a decrease in the frequency of the C-allele.

Successful treatment of severe form of bovine tropical theileriosis in dairy cattle and genotyping of Theileria annulata isolates of Tamil Nadu, India.

Bovine tropical theileriosis (BTT) is a tick-borne protozoan disease of cattle and responsible for major economic losses to the dairy farmers in India. This report describes diagnosis, genotyping and successful treatment of heavy infection of Theileria annulata in an organized dairy farm at Kattupakkam, Chennai. Four cross bred cows of 2 to 5years of age showed clinical signs i.e., anorexia, salivation and panting. Clinical examination revealed pyrexia (40.0°C to 40.1°C), pale mucus membranes, enlarged prescapular lymph nodes and haemoglobinuria. The peripheral blood smear examination of infected cows revealed presence of piroplasm within the RBCs indicating high parasitemia. Haematology results suggested that decreased levels of Hb, RBC, WBC and PCV in the infected cows when compared with normal reference values. There were increased serum ALT and AST values and reduced serum total protein, albumin, calcium and phosphorous values in the infected cows. Semi-nested PCR using T. annulata specific oligonucleotide primers amplified 199bp of the partial T. annulata 18S rRNA gene. Presence of four satellite markers TS6, TS8, TS9, and TS12 in the Theileria annulata isolates 1 and 2 indicating that the isolates were the same haplotype and suggested the infection in the farm was due to a single haplotype of T. annulata parasite. Based on the clinical signs, microscopic examination of blood smear and molecular diagnosis, the condition was diagnosed as tropical theileriosis. Infected cows were successfully treated with a single deep intramuscular injection of buparvaquone (Zubion®, INTAS pharmaceuticals LTD, Ahmedabad, India) along with supportive medication.

РАЗРАБОТКА ВЫСОКОСПЕЦИФИЧНЫХ ОЛИГОНУКЛЕОТИДНЫХ ПРАЙМЕРОВ И ЗОНДОВ ДЛЯ ИДЕНТИФИКАЦИИ НЕ ТУБЕРКУЛЕЗНЫХ МИКОБАКТЕРИЙ

The purpose of this work was to develop highly specific oligonucleotide primers and probes for the identification of non-tuberculosis mycobacteria in pathological material taken from tuberculin-responsive cattle. The article presents the result of the development and design of a set of oligonucleotide primers and probes, research of their specificity and sensitivity. The primers and probes developed by us for the identification of non-tuberculosis mycobacteria in order to differentiate nonspecific reactions to tuberculin in cattle have high specificity and sensitivity.

A multiplex PCR assay for the simultaneous detection of five potexviruses infecting cactus plants using dual-priming oligonucleotides (DPOs) primers

Five potexviruses, namely, cactus virus X (CVX), opuntia virus X, pitaya virus X (PiVX), schlumbergera virus X (SchVX) and zygocactus virus X (ZyVX), have been reported in cactus plants. In this report, a multiplex RT-PCR assay, based on specific dual-priming oligonucleotide (DPO) primers, was developed to detect these five viruses simultaneously in field samples. Using 18 field plants comprising 16 cactus species, these viruses were detected among nine of the 18 plants, including the simultaneous detection of CVX, PiVX, SchVX and ZyVX co-infecting an Aporocactus flagelliformis and a Notocactus leninghausii f. cristatus plant. The multiplex PCR assay was thus applied successfully in the field plants and it would be useful in the diagnosis of viral infections in cactus plants.

Bacillus cereus saba.zh, a novel bacterial strain for the production of bioplastic (polyhydroxybutyrate).

The identification of novel bacterial strains with a high production potential of polyhydroxybutyrate (PHB) to substitute the bioplastics with non-biodegradable plastics and reducing environmental pollution is really effective. The present study was done with the purpose of PHB bioplastic production using a novel bacterial strain. Twenty-one different bacterial isolates were obtained from petrochemical wastewater, which among them, 10 isolates were PHB positive. The presence of PHB granules was detected in the isolates using Sudan Black B staining. The most excellent PHB-accumulating bacterium with a maximum yield of PHB (61.53%) was selected and identified as Bacillus cereus saba.zh, based on morphological, biochemical, and molecular techniques. 16S rRNA nucleotide sequence of the bacterium was assigned accession number: MT975245 in the NCBI database. The phylogenetic tree data showed that the closest type strain to the Bacillus cereus saba.zh is the Bacillus cereus SDB4 (91%). The three genes (phaA, phaB, and phaC) responsible for the PHB biosynthesis were amplified using the specific oligonucleotide primers by PCR technique. The highest PHB yield was achieved when the culture medium was supplemented with 3% sugarcane molasses as a carbon source, ammonium sulfate as the nitrogen source, at pH 7, and temperature of 30°C. The characterization of the extracted polymer by FTIR and 1H NMR spectroscopy proves the presence of methyl, methylene, methine, hydroxyl, and ester carbonyl groups and confirmed the structure of biopolymer as PHB. The novel strain Bacillus cereus saba.zh has good potential as an appropriate candidate for low-cost industrial production of bioplastic.

Isolation and Identification of Natural Colorant Producing Soil-Borne Aspergillus niger from Bangladesh and Extraction of the Pigment.

Natural colorants have been used in several ways throughout human history, such as in food, dyes, pharmaceuticals, cosmetics, and many other products. The study aimed to isolate the natural colorant-producing filamentous fungi Aspergillus niger from soil and extract pigments for its potential use specially for food production. Fourteen soil samples were collected from Madhupur National Park at Madhupur Upazila in the Mymensingh district, Bangladesh. The Aspergillus niger was isolated and identified from the soil samples by following conventional mycological methods (cultural and morphological characteristics), followed by confirmatory identification by a polymerase chain reaction (PCR) of conserved sequences of ITS1 ribosomal DNA using specific oligonucleotide primers. This was followed by genus- and species-specific primers targeting Aspergillus niger with an amplicon size of 521 and 310 bp, respectively. For pigment production, a mass culture of Aspergillus niger was conducted in Sabouraud dextrose broth in shaking conditions for seven days. The biomass was subjected to extraction of the pigments following an ethanol-based extraction method and concentrated using a rotary evaporator. Aspergillus niger could be isolated from three samples. The yield of extracted brown pigment from Aspergillus niger was 0.75% (w/v). Spectroscopic analysis of the pigments was carried out using a UV–VIS spectrophotometer. An in vivo experiment was conducted with mice to assess the toxicity of the pigments. From the colorimetric and sensory evaluations, pigment-supplemented products (cookies and lemon juice) were found to be more acceptable than the control products. This could be the first attempt to use Aspergillus niger extracted pigment from soil samples in food products in Bangladesh, but for successful food production, the food colorants must be approved by a responsible authority, e.g., the FDA or the BSTI. Moreover, fungal pigments could be used in the emerging fields of the food and textile industries in Bangladesh.

Isolation and detection of antibiotics resistance genes of Escherichia coli from broiler farms in Sukabumi, Indonesia.

Objective:This study aimed to isolate and identify Escherichia coli from broiler samples from Sukabumi, Indonesia. Also, antibiogram studies of the isolated bacteria were carried out considering the detection of the antibiotic resistance genes.Materials and Methods:Cloaca swabs (n = 45) were collected from broilers in Sukabumi, Indonesia. Isolation and identification of E. coli were carried out according to standard bacteriological techniques and biochemical tests, followed by confirmation of the polymerase chain reaction targeting the uspA gene. Antibiotic sensitivity test, using several antibiotics [tetracycline (TE), oxytetracycline (OT), ampicillin (AMP), gentamicin (CN), nalidixic acid (NA), ciprofloxacin (CIP), enrofloxacin (ENR), chloramphenicol, and erythromycin] was carried out following the Kirby–Bauer disk diffusion method. Detection of antibiotic resistance coding genes was carried out by PCR using specific oligonucleotide primers. Statistical analysis was carried out with one-way analysis of variance.Results:The results showed that 55.6% (25/45) of the samples were associated with the presence of E. coli. Antibiotic sensitivity test showed that the E. coli isolates were resistant to TE (88%; 22/25), OT (88%; 22/25), AMP (100%; 25/25), CN (64%; 16/25), NA (100%; 22/25), CIP (88%; 22/25), ENR (72%; 18/25), chloramphenicol (0%; 0/25), and erythromycin (92%; 23/25). On the other hand, the antibiotic resistance coding genes were tetA (86.4%; 19/22), blaTEM (100%; 25/25), aac(3)-IV (0%; 0/16), gyrA (100%; 25/25), and ermB (13%; 3/23). It was found that chloramphenicol is markedly different from other antibiotic treatment groups.Conclusion:Escherichia coli was successfully isolated from cloacal swabs of broiler in Sukabumi, Indonesia. The bacteria were resistant to TE, OT, AMP, CN, NA, CIP, ENR, and erythromycin. Chloramphenicol was more sensitive and effective than other antibiotics in inhibiting the growth of E. coli. The antibiotic resistance genes detected were tetA, blaTEM, gyrA, and ermB.