Detection and Molecular Characterization of Canine Babesiosis Causative Agent Babesia canis in Naturally Infected Dogs in the Dobrogea Area (Southeastern Romania).

Canine babesiosis is an emerging tick-borne disease of major veterinary concern in Europe. Its prevalence has increased in the last two decades and is spreading rapidly toward the north. The aim of this study was to investigate the genetic diversity of Babesia spp. strains isolated from naturally infected dogs in a tick-endemic area (Dobrogea) in southeastern Romania. For this purpose, a total of twenty-three samples from dogs diagnosed with various clinical forms of babesiosis, evaluated by means of clinical history, physical examination, and hematological tests, were subjected to a molecular investigation using PCR, sequencing analysis, and genetic characterization. A microscopic examination of thin Diff-quick-stained blood smears revealed large intra-erythrocytic Babesia piroplasms in all dogs. The PCR and sequencing analysis results indicated the presence of Babesia canis in 22 dogs (95.7%) and Babesia vogeli in 1 dog (4.3%). Among the B. canis isolates, two genotypes were distinguished based on two nucleotide substitutions (GA→AG) observed in the 18S rRNA gene sequences (at positions 609 and 610), with the AG genotype predominating (54.5% of samples), while the GA variant was identified in 9.1% of samples. In the remaining isolates (36.4%), both variants were identified. The B. vogeli-positive dog also tested positive for antibodies against Ehrlichia canis and displayed severe disease. This study reports, for the first time, the presence of genetically heterogenic B. canis strains in dogs with clinical babesiosis in Romania. These findings provide a basis for future studies on the relationship between the genetic structure of the causative agents of canine babesiosis in Romania and the course of the disease.

Characterization of Staphylococcus aureus strains and evidence for the involvement of non-classical enterotoxin genes in food poisoning outbreaks.

To an increasing extent, molecular and genetic characterization is now used to investigate foodborne outbreaks. The aim of this study was to seek molecular links among coagulase-positive staphylococci (CPS) isolated from three recent food poisoning outbreaks in Romania using polymerase chain reaction and pulsed-field gel electrophoresis techniques. The 19 CPS isolates were identified as Staphylococcus aureus by detection of the 23S rDNA gene. Among them, 15 carried at least one staphylococcal enterotoxin-encoding gene. The Calarasi outbreak strains grouped in pulsotype 2 and were sed/sej/ser-positive, whereas the Arad outbreak strains clustered in pulsotype 17 and were either sed/seg/sei/sej/ser- or seg/sei-positive. The Pitesti outbreak strains clustered in pulsotype 1 and, surprisingly, possessed only one enterotoxin gene, i.e. seh. Similar to other European countries, the seh gene has been identified with increasing frequency in Romanian outbreaks; this highlights the importance of considering the application of methods recommended for staphylococcal enterotoxin regulation in Europe.

Parasitological and molecular diagnostic of a clinical Babesia caballi outbreak in Southern Romania.

Equine piroplasmosis (EP) is a tick-borne disease of equids caused by Babesia caballi and/or Theileria equi, which is endemic in many tropical and temperate areas of the world. However, clinical outbreaks of EP in Romania during the last decades have not been reported Therefore, the aim of this paper is (i) to describe a clinical B. caballi outbreak in horses on several farms in Southern Romania using a diagnostic and therapeutic approach and (ii) the molecular diagnostic of EP in an endemic area of Romania. In the first case, a 10-month-old stallion male was presented with lethargy, anorexia, fever (40.9 °C), pale mucosal/mucous/membranes and a marked anemia. In the subsequent weeks, three horses from other farms located in the same area, displayed similar clinical signs. B. caballi was diagnosed in all the horses based on Giemsa-stained blood smears and the diagnosis was further confirmed by polymerase chain reaction (PCR), using a single-round and multiplex PCR and sequencing. All four horses were treated with imidocarb dipropionate, at a dose rate of 2.2 mg/kg body weight (two injections at 48 h apart), and all horses clinically recovered within 24-48 h, post-treatment. This report presents the first molecularly characterized B. caballi outbreak in Romania in clinically affected horses, confirmed by DNA sequencing.

A bioanalytical approach of chemical composition, bioactivity and cytotoxicity of Berteroa incana L. herb.

Berteroa incana is a wild herb widespread in temperate zones which was practically not studied for its biological effects. Methanolic and aqueous extracts of B. incana were assessed for the content in polyphenols and the related antioxidant and antimicrobial activities and the polysaccharide extract for the content in saccharides and the associated cytostatic effect. The results obtained highlighted that the methanolic extracts of B. incana contain moderate amounts of polyphenols, the most representative been isoquercitrin 4.41 ± 0.02 mg100 g-1dry weight plant material (DW), quercetin 4.21 ± 0.05, sinapic acid 5.23 ± 0.12 and ferulic acid 5.05 ± 0.12 mg 100 g-1DW, with correlated moderate antioxidant activities (IC50 13.40 ± 0.01 µg mL-1) and absent antibacterial activity. The polysaccharide fraction showed high content in saccharides, especially in arabinose (312.22 ± 7.54 mg g-1 polysaccharide extract) and glucose (279.22 ± 5.59), and promising cytostatic effect.

Molecular Docking and Screening Studies of New Natural Sortase A Inhibitors.

To date, multi-drug resistant bacteria represent an increasing health threat, with a high impact on mortality, morbidity, and health costs on a global scale. The ability of bacteria to rapidly and permanently acquire new virulence factors and drug-resistance elements requires the development of new antimicrobial agents and selection of new proper targets, such as sortase A. This specific bacterial target plays an important role in the virulence of many Gram-positive pathogens, and its inhibition should produce a mild evolutionary pressure which will not favor the development of resistance. A primary screening using a fluorescence resonance energy transfer assay was used to experimentally evaluate the inhibitory activity of several compounds on sortase A. Using molecular docking and structure-activity relationship analyses, several lead inhibitors were identified, which were further tested for antimicrobial activity using the well diffusion test and minimum inhibitory concentration. The toxicity was assessed using the Daphnia magna test and used as a future screening filter. Three natural compounds were identified in this study as promising candidates for further development into therapeutically useful anti-infective agents that could be used to treat infections caused by multi-drug resistant bacterial pathogens which include sortase A in their enzymatic set.

Structural Analysis of Sortase A Inhibitors.

Bacterial sortases are cysteine transpeptidases that regulate the covalent linkage of several surface protein virulence factors in Gram-positive bacteria. Virulence factors play significant roles in adhesion, invasion of host tissues, biofilm formation and immune evasion, mediating the bacterial pathogenesis and infectivity. Therefore, sortases are emerging as important targets for the design of new anti-infective agents. We employed a computational study, based on structure derived descriptors and molecular fingerprints, in order to develop simple classification methods which could allow predicting low active or high active SrtA inhibitors. Our results indicate that a highly active SrtA inhibitor has a molecular weight ranging between 180 and 600, contains one up to four nitrogen atoms, up to three oxygen atoms and under 18 hydrogen atoms. Also the hydrogen acceptor number and the molecular flexibility, as assessed by the number of rotatable bounds, have emerged as the most relevant descriptors for SrtA affinity. The Bemis-Murcko scaffolding revealed favoured scaffolds as containing at least two ring structures bonded directly or merged in a condensed cycle. This data represent a valuable tool for identifying new potent SrtA inhibitors, potential anti-virulence agents targeted against Gram-positive bacteria, including multiresistant strains.

New insights into the molecular epidemiology of Trichinella infection in domestic pigs, wild boars, and bears in Romania.

Trichinellosis is a food-borne zoonosis caused by the parasitic nematode Trichinella, characterized by an extremely wide host range and geographical distribution. In Romania, it is recognized as one of the most serious zoonotic diseases. A cross-sectional study, covering all regions of Romania, was conducted in 2014 to investigate and update the prevalence of Trichinella infection among domestic pigs, wild boars, and bears. Additional, molecular identification of Trichinella species circulating among these animals was performed in order to establish the biogeography of Trichinella species within the seven geographical regions of Romania. For this, a total of 113,383 pigs raised in non-controlled housing conditions (backyards), 5596 hunted wild boars and 147 hunted bears were subjected to Trichinella analysis. The highest prevalence of Trichinella infections was found in bears (12.93%), followed by wild boars (1.66%) and domestic pigs (0.20%). Of 294 Trichinella isolates that tested positive by multiplex PCR, 219 (74.49%) were identified as Trichinella spiralis, 66 (22.45%) as Trichinella britovi, and 9 isolates (3.06%) as mixed infections of T. spiralis and T. britovi. T. spiralis was more prevalent in domestic pigs (165/228; 72.37%) than in game (63/228; 27.63%), while T. britovi showed a higher prevalence in game (50/75; 66.66%) than in domestic pigs (25/75; 33.33%). Moreover, the present study revealed a significant host- and area- related distribution of Trichinella species within the seven regions of Romania. Therefore, these findings are of epidemiological relevance, updating data on the prevalence and distribution of Trichinella species circulating among domestic and wild animals in South-Eastern Europe.