Microscopic and molecular detection of piroplasms among sheep in Upper Egypt.

Introduction: Blood parasites pose a significant threat to livestock production in southern Egypt, yet there is a scarcity of information regarding their circulation and epidemiology in sheep in this region. This study aimed to investigate the seroprevalence of blood parasite infections in sheep in Assiut governorate, Upper Egypt. Methods: A total of 400 blood samples were collected from sheep of varying ages and genders. The preliminary screening for the presence of piroplasms, mainly Babesia and Theileria spp., via microscopic examination, followed by investigation of the potential risk factors linked with the exposure to infection. Moreover, molecular identification of both parasites on some of positive samples was performed using PCR targeting Babesia 18S rRNA and Theileria annulata Tams1 gene. Results: The microscopic examination revealed that among the examined sheep, there was an overall prevalence of blood parasites at 44% (176 out of 400), with Babesia spp. observed in 14% (56 out of 400) and Theileria spp. in 30% (120 out of 400). Furthermore, the infection rate was non-significantly higher in young animals (50%) compared to adults (38.5%) (P = 0.246). Male sheep exhibited a significantly higher vulnerability to both parasites' infection (63.3%) compared to females (35.7%) (P = 0.011). Interestingly, the prevalence of both blood parasites was significantly higher during the cold season (66.1%) compared to the hot season (15.9%) (P = < 0.001). The molecular analysis identified the presence of Babesia ovis and Theileria annulata among a subsample of the positive sheep's bloods films. The identified species were recorded in the GenBank™ databases and assigned specific accession numbers (OQ360720 and OQ360719 for B. ovis), and (OP991838 for T. annulata). Conclusions: Taken together, this study confirms a high prevalence of piroplasmosis and offers epidemiological and molecular insights into blood parasites in sheep from Upper Egypt, highlighting the importance of detecting these parasites in various hosts and their competent vectors (ticks).

Combating Cariogenic Streptococcus mutans Biofilm Formation and Disruption with Coumaric Acid on Dentin Surface.

Streptococcus mutans, the primary cause of dental caries, relies on its ability to create and sustain a biofilm (dental plaque) for survival and pathogenicity in the oral cavity. This study was focused on the antimicrobial biofilm formation control and biofilm dispersal potential of Coumaric acid (CA) against Streptococcus mutans on the dentin surface. The biofilm was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) viability assay, microtiter plate assay, production of extracellular polymeric substances (EPSs), florescence microscopy (surface coverage and biomass µm2) and three-dimensional (3D) surface plots. It was observed that CA at 0.01 mg/mL reduced bacterial growth by 5.51%, whereases at 1 mg/mL, a significant (p < 0.05) reduction (98.37%) was observed. However, at 1 mg/mL of CA, a 95.48% biofilm formation reduction was achieved, while a 73.45% biofilm dispersal (after 24 h. treatment) was achieved against the preformed biofilm. The MTT assay showed that at 1 mg/mL of CA, the viability of bacteria in the biofilm was markedly (p < 0.05) reduced to 73.44%. Moreover, polysaccharide (EPS) was reduced to 24.80 µg/mL and protein (EPS) to 41.47 µg/mL. ImageJ software (version 1.54 g) was used to process florescence images, and it was observed that the biofilm mass was reduced to 213 (µm2); the surface coverage was reduced to 0.079%. Furthermore, the 3D surface plots showed that the untreated biofilm was highly dense, with more fibril-like projections. Additionally, molecular docking predicted a possible interaction pattern of CA (ligand) with the receptor Competence Stimulating Peptide (UA159sp, PDB ID: 2I2J). Our findings suggest that CA has antibacterial and biofilm control efficacy against S. mutans associated with dental plaque under tested conditions.

Whole-Genome Sequence Insight into the Plant-Growth-Promoting Bacterium Priestia filamentosa Strain AZC66 Obtained from Zygophyllum coccineum Rhizosphere.

This study aimed to isolate, screen the plant-growth-enhancing features, and explore the whole-genome sequence of AZC66 isolated from the rhizosphere of Zygophyllum coccineum and determine its biostimulating effects on the growth of cowpea under greenhouse conditions. Salkowski reagent was used to measure AZC66's indole acetic acid production. AZC66's inorganic phosphate solubility on Pikovskaya agar was evaluated using tricalcium phosphate. The results indicated the ability of AZC66 to fix nitrogen, produce IAA (66.33 ± 0.44 µg mL-1), solubilize inorganic phosphate, and exhibit the activity of ACC deaminase (278.40 ± 21 mol -ketobutyrate mg-1 h-1). Cowpea's root and shoot dry weights were also significantly increased after in vitro inoculation with AZC66. The identity of AZC66 was confirmed as Priestia filamentosa, and 4840 genes were predicted in its genome. The gene sequences were compared against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and the results showed that the top three pathways wherein the maximum number of genes are involved are signaling and cellular processes, genetic information processing, and carbohydrate metabolism. The genome sequencing of the strain AZC66 revealed a number of genes implicated in plant biostimulation activities such as nitrogen fixation (nifU), phytohormone synthesis (trpAB genes), phosphate solubilization (PhbCEF, pstABCS, and phoU), and siderophore formation (FbpA, feoAB, and fetB). The AZC66 genome contained numerous genes involved in nitrogen metabolism, nitrogen regulation, and the nitrate reduction pathway. The phenazine biosynthetic gene in AZC66 demonstrated biocontrol and soil survival properties. The trehalose synthesis genes in AZC66 may help plants resist osmotic and salt stress. The discovery of glycine betaine, cold shock, and heat shock protein genes demonstrated that AZC66 could withstand harsh conditions. AZC66 might be used to create robust, sustainable biological fertilizers for future agricultural use in Saudi Arabia. Furthermore, the predicted adaptable metabolic pathways might serve as the basis for potential biotechnological applications in agriculture and industry.

Circulation of thermophilic Campylobacter in pigeons, turkeys, and humans at live bird markets in Egypt.

Live bird markets increase the risk of transmission of zoonotic diseases. Few studies have investigated the potential zoonotic transmission of Campylobacter in Egypt. Therefore, our study was carried out to investigate the presence of Campylobacter species, mainly Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli), in pigeons and turkeys sold at poultry shops. Furthermore, the study aimed to explore the potential occupational risk of Campylobacter infection, mainly among workers at poultry shops. Six hundred (n = 600) samples from various organs were obtained from pigeons and turkeys from live bird shops in the Giza and Asyut provinces in Egypt. Additionally, 100 stool samples were collected from persons working at poultry shops. Circulation of thermophilic Campylobacter in pigeons, turkeys, and humans was investigated based on culture and molecular methods. The rate of detection of Campylobacter species from the samples was significant when the culture method was used alone in comparison to when it was used in combination with mPCR. The prevalence rates of Campylobacter species detected by mPCR were 36% (C. jejuni 20%; C. coli 16%), 28% (C. jejuni 12%; C. coli16%), and 29% (C. jejuni 15%; C. coli 14%) in pigeons, turkeys, and workers, respectively. In pigeons, significant variations in the C. jejuni and C. coli occurrence rates were reported in terms of the intestinal content (15, 4%), liver (4, 13%), and skin (9, 7%), respectively. In turkeys, Campylobacter species were mostly detected in liver samples with a percentage of 19%, followed by the skin (12%), and the intestinal content (8%). In conclusion, Campylobacter species are circulating in poultry farms in Egypt and could represent a hazard for humans. It is recommended that biosecurity measures should be applied to mitigate the occurrence of Campylobacter in poultry farms. Moreover, there is an urgent need to transform live bird markets into chilled poultry markets.

Immunological and Oxidative Biomarkers in Bovine Serum from Healthy, Clinical, and Sub-Clinical Mastitis Caused by Escherichia coli and Staphylococcus aureus Infection.

The study aimed to investigate the mastitis' emerging causative agents and their antimicrobial sensitivity, in addition to the hematological, biochemical indicators, oxidative biomarkers, acute phase protein (APP), and inflammatory cytokine changes in dairy farms in Gamasa, Dakahlia Governorate, Egypt. One hundred Holstein Friesian dairy cattle with clinical and subclinical mastitis were investigated and were allocated into three groups based on a thorough clinical examination. Escherichia coli and Staphylococcus aureus were found responsible for the clinical and subclinical mastitis in dairy farms, respectively. Multiple drug resistance (MDR) was detected in 100%, and 94.74% of E. coli and S. aureus isolates, respectively. Significantly low RBCs count, Hb, and PCV values were detected in mastitic cows compared with both subclinical mastitic and control groups; moreover, WBCs, lymphocytes, and neutrophil counts were significantly diminished in mastitic cows compared to the controls. Significantly higher levels of AST, LDH, total protein, and globulin were noticed in both mastitic and subclinical mastitic cows. The haptoglobin, fibrinogen, amyloid A, ceruloplasmin, TNF-α, IL-1ß, and IL-6 levels were statistically increased in mastitic cows compared to the controls. Higher MDA levels and reduction of TAC and catalase were identified in all the mastitic cases compared to the controls. Overall, the findings suggested potential public health hazards due to antimicrobial resistance emergence. Meanwhile, the APP and cytokines, along with antioxidant markers can be used as early indicators of mastitis.

Designing a novel chimeric multi-epitope vaccine against Burkholderia pseudomallei, a causative agent of melioidosis.

Burkholderia pseudomallei, a gram-negative soil-dwelling bacterium, is primarily considered a causative agent of melioidosis infection in both animals and humans. Despite the severity of the disease, there is currently no licensed vaccine on the market. The development of an effective vaccine against B. pseudomallei could help prevent the spread of infection. The purpose of this study was to develop a multi-epitope-based vaccine against B. pseudomallei using advanced bacterial pan-genome analysis. A total of four proteins were prioritized for epitope prediction by using multiple subtractive proteomics filters. Following that, a multi-epitopes based chimeric vaccine construct was modeled and joined with an adjuvant to improve the potency of the designed vaccine construct. The structure of the construct was predicted and analyzed for flexibility. A population coverage analysis was performed to evaluate the broad-spectrum applicability of B. pseudomallei. The computed combined world population coverage was 99.74%. Molecular docking analysis was applied further to evaluate the binding efficacy of the designed vaccine construct with the human toll-like receptors-5 (TLR-5). Furthermore, the dynamic behavior and stability of the docked complexes were investigated using molecular dynamics simulation, and the binding free energy determined for Vaccine-TLR-5 was delta total -168.3588. The docking result revealed that the vaccine construct may elicit a suitable immunological response within the host body. Hence, we believe that the designed in-silico vaccine could be helpful for experimentalists in the formulation of a highly effective vaccine for B. pseudomallei.

Pan-Genome-Assisted Computational Design of a Multi-Epitopes-Based Vaccine Candidate against Helicobacter cinaedi.

Helicobacter cinaedi is a Gram-negative bacterium from the family Helicobacteraceae and genus Helicobacter. The pathogen is a causative agent of gastroenteritis, cellulitis, and bacteremia. The increasing antibiotic resistance pattern of the pathogen prompts the efforts to develop a vaccine to prevent dissemination of the bacteria and stop the spread of antibiotic resistance (AR) determinants. Herein, a pan-genome analysis of the pathogen strains was performed to shed light on its core genome and its exploration for potential vaccine targets. In total, four vaccine candidates (TonB dependent receptor, flagellar hook protein FlgE, Hcp family type VI secretion system effector, flagellar motor protein MotB) were identified as promising vaccine candidates and subsequently subjected to an epitopes' mapping phase. These vaccine candidates are part of the pathogen core genome: they are essential, localized at the pathogen surface, and are antigenic. Immunoinformatics was further applied on the selected vaccine proteins to predict potential antigenic, non-allergic, non-toxic, virulent, and DRB*0101 epitopes. The selected epitopes were then fused using linkers to structure a multi-epitopes' vaccine construct. Molecular docking simulations were conducted to determine a designed vaccine binding stability with TLR5 innate immune receptor. Further, binding free energy by MMGB/PBSA and WaterSwap was employed to examine atomic level interaction energies. The designed vaccine also stimulated strong humoral and cellular immune responses as well as interferon and cytokines' production. In a nutshell, the designed vaccine is promising in terms of immune responses' stimulation and could be an ideal candidate for experimental analysis due to favorable physicochemical properties.

Reverse Vaccinology and Immunoinformatic Assisted Designing of a Multi-Epitopes Based Vaccine Against Nosocomial Burkholderia cepacia.

Burkholderia cepacia is a Gram-negative nosocomial pathogen and is considered as a troublesome bacterium due to its resistance to many common antibiotics. There is no licensed vaccine available to prevent the pathogen infections, thus making the condition more alarming and warrant the search for novel therapeutic and prophylactic approaches. In order to identify protective antigens from pathogen proteome, substantial efforts are put forth to prioritized potential vaccine targets and antigens that can be easily evaluated experimentally. In this vaccine design investigation, it was found that B. cepacia completely sequenced proteomes available in NCBI genome database has a total of 28,966 core proteins. Out of total, 25,282 proteins were found redundant while 3,684 were non-redundant. Subcellular localization revealed that 18 proteins were extracellular, 31 were part of the outer membrane, 75 proteins were localized in the periplasm, and 23 were virulent proteins. Five proteins namely flagellar hook protein (FlgE), fimbria biogenesis outer membrane usher protein, Type IV pilus secretin (PilQ), cytochrome c4, flagellar hook basal body complex protein (FliE) were tested for positive for antigenic, non-toxic, and soluble epitopes during predication of B-cell derived T-cell epitopes. A vaccine peptide of 14 epitopes (joined together via GPGPG linkers) and cholera toxin B subunit (CTBS) adjuvant (joined to epitopes peptide via EAAAK linker) was constructed. Binding interaction of the modeled vaccine with MHC-I, MHC-II, and Toll-like receptor 4 (TLR-4) immune receptors was studied using molecular docking studies and further analyzed in molecular dynamics simulations that affirms strong intermolecular binding and stable dynamics. The maximum root mean square deviation (RMSD) score of complexes in the simulation time touches to 2 Å. Additionally, complexes binding free energies were determined that concluded robust interaction energies dominated by van der Waals. The total energy of each complex is < -190 kcal/mol. In summary, the designed vaccine showed promising protective immunity against B. cepacia and needs to be examined in experiments.

Development of siderophore-based rhizobacterial consortium for the mitigation of biotic and abiotic environmental stresses in tomatoes: An in vitro and in planta approach.

AIM: Tomato-associated plant-growth-promoting rhizosphere bacteria were screened for effective antagonistic activity against the fungal vascular wilt pathogens; tolerance to heavy metals; and enhancing the bioavailability of iron for tomato plants through in vitro and in vivo approaches. METHODS AND RESULTS: Among the 121 rhizobacteria screened for siderophores, 25 isolates were observed to be siderophore producers and out of these, seven isolates chelate copper and iron thus exhibiting in vitro antagonism against the virulent strains of Fusarium oxysporum f. sp. lycopersici MTCC10270 (Fol), Fusarium equiseti MFol and Sarocladium sp. SWL isolated from infected tomatoes. Pseudomonas stutzeri KRP8 was identified to be the most potent strain among the siderophore producers and its siderophores were chemically characterized by mass spectra as metal bound and metal-free forms. Upon bio-inoculation of fortified bacterial consortium (siderozote) into the rhizosphere of vermiculite pot cultured tomatoes supplied with varying concentrations of iron and copper ions, we observed in planta growth improvements, antagonism, enhancement of bioavailability of iron and heavy metal tolerance using Inductively Coupled Plasma-Optical Emission Spectrometry. CONCLUSION AND SIGNIFICANCE OF THE STUDY: Our rhizobacterial consortium provides an opportunity for soil reclamation through an ecofriendly method for a heavy metal-free agricultural landscape.

Immunoinformatics and Immunogenetics-Based Design of Immunogenic Peptides Vaccine against the Emerging Tick-Borne Encephalitis Virus (TBEV) and Its Validation through In Silico Cloning and Immune Simulation.

Pegivirus, HPgV, earlier known as Gb virus and hepatitis G virus, is an enveloped, positive-stranded RNA and lymphotropic virus classified into the Flaviviridae family. The transmission routes primarily involve blood products, with infections worldwide, leading up to 25% of persistent infections. To date, no effective therapeutic means are available to resolve Pegivirus infections. Effective vaccine therapeutics are the best alternative to manage this disease and any associated potential pandemic. Thus, whole proteome-based mining of immunogenic peptides, i.e., CTL (cytotoxic T lymphocytes), HTL (helper T lymphocytes) and B cell epitopes were mapped to design a vaccine ensemble. Our investigation revealed that 29 different epitopes impart a critical role in immune response induction, which was also validated by exploring its physiochemical properties and experimental feasibility. In silico expression and host immune simulation using an agent-based modeling approach confirmed the induction of both primary and secondary immune factors such as IL, cytokines and antibodies. The current study warrants further lab experiments to demonstrate its efficacy and safety.

Emerging Treatment Strategies for Diabetes Mellitus and Associated Complications: An Update.

The occurrence of diabetes mellitus (DM) is increasing rapidly at an accelerating rate worldwide. The status of diabetes has changed over the last three generations; whereas before it was deemed a minor disease of older people but currently it is now one of the leading causes of morbidity and mortality among middle-aged and young people. High blood glucose-mediated functional loss, insulin sensitivity, and insulin deficiency lead to chronic disorders such as Type 1 and Type 2 DM. Traditional treatments of DM, such as insulin sensitization and insulin secretion cause undesirable side effects, leading to patient incompliance and lack of treatment. Nanotechnology in diabetes studies has encouraged the development of new modalities for measuring glucose and supplying insulin that hold the potential to improve the quality of life of diabetics. Other therapies, such as ß-cells regeneration and gene therapy, in addition to insulin and oral hypoglycemic drugs, are currently used to control diabetes. The present review highlights the nanocarrier-based drug delivery systems and emerging treatment strategies of DM.