Nucleic Acid Isolation and Downstream Applications

Nucleic acids are not only a source of life but also a means of observing, understanding, and regulating it. Nucleic acids, DNA and RNA, and their characteristics are discussed in other chapters of the book. This chapter describes the fundamental principles of different methods for nucleic acid sample preparation / nucleic acid extraction, such as column-based methods using silica membranes and traditional ones without a column purification procedure (commercially available or homemade). Other topics discussed here include comparative analysis of the use of these methods in DNA and RNA extraction from a variety of biological and clinical samples, as well as the relationship between the type of sample, the method used and the quality and amount of extracted DNA or RNA. Finally, the chapter outlines the application of nucleic acids in the diagnosis of various diseases, in scientific research, and bird sex determination by downstream applications such as restriction enzyme analysis, polymerase chain reactions (PCR, reverse transcription-PCR, real-time PCR), and different sequencing methods (Sanger, cycling sequencing, and next-generation sequencing)

Comparison between Some Phenotypic and Genotypic Methods for Assessment of Antimicrobial Resistance Trend of Bovine Mastitis Staphylococcus aureus Isolates from Bulgaria

The aim of this study was to assess the resistance of bovine mastitis S. aureus isolates from farms in Bulgaria to different classes of chemotherapeutic drugs by comparison of some phenotypic and genotypic methods by means of Cohen’s kappa statistics. The study comprised 546 milk samples from subclinical and clinical mastitis at 14 farms from 9 districts in the country. A total of 92 Staphylococcus aureus strains were isolated from tested samples and identified by nuc PCR. The results demonstrated high levels of resistance to sulfadimethoxine (87%), followed by resistance to penicillin (33.7%), erythromycin (13%), streptomycin (8.7%), tetracycline (6.5%) and gentamicin (1.1%). The comparison of both phenotypic tests with respect to 9 antimicrobials revealed strong agreement with kappa coefficient 0.836. An almost complete agreement was evidenced between phenotypic resistance to penicillin and blaZ gene presence, to methicillin with mecA gene, to tetracycline with tet genes, but the agreement between erythromycin resistance and erm genes presence was moderate. This study was the first to demonstrate discrepancy between the behaviour to cefoxitin in the disk diffusion test and oxacillin in the MIC test for an isolate shown to carry the mecA gene in the subsequent genetic analysis. Considering the detected discrepancies for some of isolates, an integral evaluation through phenotypic and molecular methods for monitoring of antimicrobial resistance of Staphylococcus aureus is recommended

Human ELISA Detects anti-SARS-CoV-2 Antibodies in Cats: Seroprevalence and Risk Factors for Virus Spread in Domestic and Stray Cats in Bulgaria

The aim of this study was to verify whether the human DR-ELISA for the detection of anti-SARS-CoV-2 antibodies can be applied in cats, and to assess the risk factors that determine the spread of the virus among the cat population in Bulgaria. The study included 92 serum samples collected from 68 domestic and 24 stray cats aged from 3 months to 20 years of age in the period of January–June 2021. The samples originated from three regions in Bulgaria and from three places of inhabitance. DR-ELISA based on peroxidase-labeled SARS-CoV-2 N protein was employed to detect IgA, IgG and IgM antibodies in the samples. Subsequently, the results were compared with a commercially available multi-species ELISA kit. There was high seroprevalence (83.33%) in stray cats and 41.18% in domestic cats, confirmed by the human and veterinary ELISA kit. The positive cases in the regional cities were 42.86%, in small towns 50% and in villages 78.26%. Cats under 7 years had a five times higher risk than those over 7 years (p = 0.001). The risk was seven times higher for stray cats than for domestic cats (p = 0.001). In addition, the results indicate that the risk was the highest for cats in villages (p = 0.006) compared to cats in other places of inhabitance. This study demonstrates that human DR-ELISA may be successfully applied to monitor the circulation of SARS-CoV-2 in cats and other susceptible species. Cats might serve as sentinel animals for tracking the virus in nature and in inhabited areas (strays) and to discover asymptomatic cases in humans/owners

Spontaneous Direct Carotid-Cavernous Fistula in an Elderly Patient

We describe the case of an 83-year-old woman with left-sided ophthalmoplegia. She had no family history of connective tissue disease. The computed tomography study found a dilated left cavernous sinus. The conventional cerebral panangiography confirmed the diagnosis - a direct carotid-cavernous fistula (CCF), with no evidence of ruptured aneurysm. The woman underwent endovascular treatment with coiling of the cavernous sinus in combination with application of the Onyx embolic agent in the fistula. During the first 48 hours after the embolization the local pain, exophthalmos and conjunctival injection of the left eye were significantly ameliorated. The pulsatile tinnitus on the left disappeared and the ptosis of the left eyelid partially recovered. Selective angiography is the best method for the diagnosis and classification of CCF. Currently, treatment is possible with low mortality and morbidity rates. The endovascular intervention is able to completely occlude the fistula and maintain adequate blood fl ow through the carotid artery

Aquaponic systems: biological and technological parameters

AbstractAquaponics is a specific ecosystem that combines aquaculture, hydroponics and beneficial bacteria in a symbiotic relationship. This integrated approach is emerging as a sustainable method of organic food production. This review analyzes the biological and technological parameters of aquaponic systems based on a survey of over 200 scientific papers from 1974 to 2021. The biological parameters include the characteristics of the species in the aquaponic system, their relationship with nutrition and environmental conditions, the content of nutrients and their impact on productivity. The technological parameters focus on the structural and functional components of the aquaponic system, environmental management techniques and economic feasibility. The advances in aquaponics technology contribute to more sustainable food systems. This calls for further research on the biological and technological characteristics of aquaponic systems, as well as on the environmental, operational and socio-economic aspects and their interrelationship, of interest to both researchers and stakeholders

Development of Nested PCR for SARS-CoV-2 Detection and Its Application for Diagnosis of Active Infection in Cats

SARS-CoV-2 emerged in 2019 and found diagnostic laboratories unprepared worldwide. To meet the need for timely and accurate virus detection, laboratories used rapid Ag tests and PCR kits based on costly multi-channel real-time techniques. This study aimed to develop a conventional nested PCR based on the SARS-CoV-2 N gene, validate it against some approved assays, and apply it to samples from six cats with respiratory symptoms obtained in early 2020 during the first COVID-19 wave in humans in Bulgaria. The nested PCR technique showed 100% sensitivity and specificity; it could detect extracted SARS-CoV-2 RNA at concentrations as low as 0.015 ng/μL. The results identified the six tested cat samples as positive. Sequence analysis performed in two of them confirmed this. The presented technique is reliable, easy to implement and inexpensive, and can be successful in strategies for the prevention and control of SARS-CoV-2 in humans, cats and other susceptible species

Analysis of biofilm formation in nosocomial Stenotrophomonas maltophilia isolates collected in Bulgaria: An 11-year study (2011-2022)

The present study aimed to explore the genotypic and phenotypic characteristics of biofilm formation in Bulgarian nosocomial Stenotrophomonas maltophilia isolates (n 5 221) during the period 2011-2022, by screening for the presence of biofilm-associated genes (BAG) (spgM, rmlA and rpfF), their mutational variability, and assessment of the adherent growth on a polystyrene surface. The methodology included: PCR amplification, whole-genome sequencing (WGS) and crystal violet microtiter plate assay for biofilm quantification. The overall incidence of BAG was: spgM 98.6%, rmlA 86%, and rpfF 66.5%. The most prevalent genotype was spgM+/rmlA+/rpfF+ (56.1%), followed by spgM+/rmlA+/rpfF-(28.5%), and spgM+/rmlA-/rpfF+ (9.5%), with their significant predominance in lower respiratory tract isolates compared to those with other origin (P < 0.001). All strains examined were characterized as strong biofilm producers (OD550 from 0.224 +/- 0.049 to 2.065 +/- 0.023) with a single exception that showed a weak biofilm-forming ability (0.177 +/- 0.024). No significant differences were observed in the biofilm formation according to the isolation source, as well as among COVID-19 and non-COVID-19 isolates (1.256 +/- 0.028 vs. 1.348 +/- 0.128, respectively). Also, no correlation was found between the biofilm amounts and the corresponding genotypes. WGS showed that the rmlA accumulated a larger number of variants (0.0086 per base) compared to the other BAG, suggesting no critical role of its product to the biofilm formation. Additionally, two of the isolates were found to harbour class 1 integrons (7-kb and 2.6-kb sized, respectively) containing stil1 in their 3' conservative ends, which confers sulfonamide resistance. To the best of our knowledge, this is the first study on S. maltophilia biofilm formation in Bulgaria, which also identifies novel sequence types (ST819, ST820 and ST826). It demonstrates the complex nature of this adaptive mechanism in the multifactorial pathogenesis of biofilm-associated infections

Molecular epidemiology and antimicrobial resistance of Haemophilus influenzae isolates from otitis media in Bulgaria

Haemophilus influenzae is one of the main bacteria responsible for otitis media (OM) among children worldwide. We aimed to estimate the distribution of encapsulated and non-capsulated variants (NTHi), biotypes, antibiotic susceptibility, and molecular epidemiology of H. influenzae isolates recovered from pediatric OM cases in Bulgaria. Capsule detection was done by PCR for bexB gene, absent in NTHi. All encapsulated strains were subjected to PCR serotyping. MIC susceptibility testing was performed according to the criteria of EUCAST. MLST was conducted for all 71 OM isolates. The capsule detection and PCR - serotyping disclosed a predominance of NTHi (90.1%) and a few "a", "f", and "c" types. Biotype I was the most widespread (42.3%). beta-lactam resistance was found in 35.2% of the isolates. MLST represented heterogenic population structure, whereas the most represented clonal complexes belonged to ST-3, ST-57, ST-105, and ST-1426. 42.3% of the STs showed relatedness to globally represented clones, and 11.3% displayed affiliation to international type 2. Most of the H. influenzae isolates recovered from children with otitis media were non-typable strains from biotype I. The examined population structure was genetically diverse, with a predominance of international type 2 isolates