Gamma Irradiated Pasteurella multocida Vaccine induces strong humoral immunity and protects rabbits from disease.

Pasteurella multocida is affecting a multitude of animals and severely affects livestock production. Existing vaccines are mostly chemically inactivated and do not lead to wide protection. Irradiated vaccines are enjoying a renaissance and the concept of "replication defficient but metabolically active" vaccines was recently evaluated in several vaccine trials. P. multocida was isolated from the nasal swab, blood, and lung swab samples from infected rabbits. Gamma irradiation of P. multocida for inhibition of replication was evaluated at an optimized irradiation dose of 10 Kgy established. Four groups of rabbits were (mock) vaccinated with a commercial P. multocida vaccine and three irradiated formulations as liquid, lyophilized formulations with added Trehalose and lyophilized-Trehalose with an "activation" culturing the irradiated bacteria for 24 in broth. Evaluation of humoral immune response by ELISA showed that all three irradiated vaccines produced an effective, protective, and continued IgG serum level after vaccination and bacterial challenge. The IFN-γ expression is maintained at a normal level, within each individual group however, the lyophilized trehalose irradiated vaccine showed peak mean of IFN-γ titer at one week after booster dose (day 21) which was statistically significant. Cumulatively, the results of this study show that gamma-irradiated P. multocida vaccines are safe and protect rabbits against disease. Moreover, Rabbits' immunization with the three irradiated formulations avoided adverse side effects as compared to commercial polyvalent vaccine, the body weight gain for the irradiated vaccine groups indicates less stress compared to the commercial polyvalent vaccine.

Development of a multiplex polymerase chain reaction assay for detection of hepatitis C virus, hepatitis B virus, and human immunodeficiency virus 1.

BACKGROUND: Hepatitis C virus (HCV), hepatitis B virus (HBV), and human immunodeficiency virus 1 (HIV-1) are the most epidemic blood-borne viruses, posing threats to human health and causing economic losses to nations for combating the infection transmission. The diagnostic methodologies that depend on the detection of viral nucleic acids are much more expensive, but they are more accurate than serological testing. AIM: To develop a rapid, cost-effective, and accurate diagnostic multiplex polymerase chain reaction (PCR) assay for simultaneous detection of HCV, HBV, and HIV-1. METHODS: The design of the proposed PCR assay targets the amplification of a short conserved region featured with a distinguishable melting profile and electrophoretic molecular weight inside each viral genome. Therefore, this diagnostic method will be appropriate for application in both conventional (combined with electrophoresis) and real-time PCR facilities. Confirmatory in silico investigations were conducted to prove the capability of the approached PCR assay to detect variants of each virus. Then, Egyptian isolates of each virus were subjected to the wet lab examination using the given diagnostic assay. RESULTS: The in silico investigations confirmed that the PCR primers can match many viral variants in a multiplex PCR assay. The wet lab experiment proved the efficiency of the assay in distinguishing each viral type through high-resolution melting analysis. Compared to related published assays, the proposed assay in the current study is more sensitive and competitive with many expensive PCR assays. CONCLUSION: This study provides a simple, cost-effective, and sensitive diagnostic PCR assay facilitating the detection of the most epidemic blood-borne viruses; this makes the proposed assay promising to be substitutive for the mistakable and cheap serological-based assays.

Detection of immune effects of the Mannheimia haemolytica gamma irradiated vaccine in sheep.

Exposure to gamma rays from cobalt 60 (Co60) can induce a complete inactivation of Mannheimia haemolytica. The inactivated bacterial pathogen is a potential vaccine candidate for immunization of ruminants such as sheep. The subcutaneous administration of irradiated vaccine in a two-dose regimen (4.0 × 109 colony forming unit (CFU) per dose) results in no mortality in any of the vaccinated sheep during immunization and after subsequent challenge of the live bacteria of the same strain of M. haemolytica. A significant rise in serum IgG titer, detected through ELISA, is observed after the passage of two weeks from the inoculation of the first dose whereas, the peak of the mean serum antibody titer occurred after two weeks of booster dose. The vaccination does not bring significant change to the IFN-γ levels in serum. The bacterial challenge of the vaccinated sheep does not induce a further seroconversion relative to serum antibody titer. In conclusion, the vaccinated sheep are protected by the elevated IgG titer and increased levels of IL-4 (Th-2 response) compared to the non-vaccinated sheep. Radiation technology can provide the opportunity for mass production of immunologically safe vaccines against animal and zoonotic diseases. Ethics Approval by the National Research Center Ethics Committee (Trial Registration Number (TRN) no 13,602,023, 13/5/2023) was obtained.

Photoprotection Against UV-Induced Skin Damage Using Hyaluronic Acid Produced by Lactiplantibacillus plantarum and Enterococcus durans.

Exposure to ultraviolet (UV) radiation is one of the major factors that causes skin aging, erythema, sunburns, and skin cancer. This study aimed to select probiotic bacterial isolates able to produce high yield of hyaluronic acid (HA) to be employed for skin photoprotection and other possible biological applications. The selected isolates K11 and St3 were able to produce the highest yields of HA 4.8 and 4.4 mg/ml, respectively. Both isolates were identified as Enterococcus durans strain K11 and Lactiplantibacillus plantarum strain St3 using 16S rRNA gene sequencing. The antioxidant activity of HA produced by E. durans strain K11 and L. plantarum strain St3 was (65.4 0.2%) and (66.6 0.1%), respectively. The viability of UVB-irradiated keratinocytes pre-treated with HA produced by E. durans strain K11 and L. plantarum strain St3 was 91.3 and 91.4%, respectively, compared with the control. While the viability of UVB-irradiated keratinocytes post-treated with HA produced by E. durans strain K11 and L. plantarum strain St3 was 86 and 88.5%, respectively. To the best of our knowledge, this is the first recordation of HA production by Enterococcus durans and Lactiplantibacillus plantarum which revealed a significant radioprotection of the human keratinocytes against UVB radiation.

Evaluation of room temperature (30°C to 35°C) lyophilized vaccine with radio inactivated Mannheimia haemolytica whole cells isolated from infected sheep.

Background and Aim: Vaccines are one of the important tools for fighting diseases and limiting their spread. The development of vaccines with high efficacy against diseases is essential. Ionizing radiation is the method used for the preparation of the irradiated gamma Mannheimia haemolytica vaccine. The study aimed to measure the metabolic activity and electron microscopic examination of the irradiated bacterial cells and immunological efficiency of different preparations of the irradiated M. haemolytica vaccine. Materials and Methods: The irradiated vaccines were prepared in three forms at a dose of 2×109 colony-forming unit (CFU) (irradiated M. haemolytica, trehalose irradiated M. haemolytica, and trehalose lyophilized irradiated M. haemolytica). The formalin-killed vaccine was prepared at a dose of 2×109 CFU. Scanning electron microscopy was used to determine the difference between the non-irradiated bacterial cells and the bacterial cells exposed to gamma radiation. The metabolic activity of the irradiated bacterial cells was measured using the Alamar blue technique. Rabbits were divided into five groups (control, vaccinated groups with the formalin-killed vaccine, irradiated bacterial cells without trehalose, trehalose irradiated bacteria, and trehalose lyophilized irradiated bacterial cells). The rabbits were subcutaneously inoculated twice in 2-week intervals. Enzyme-linked immunosorbent assay, interferon-gamma (IFNγ), and interleukin 4 (IL4) assays were used to evaluate the vaccines' immunological efficiency in rabbits. Results: The metabolic activity tests showed that the bacterial cells exposed to gamma radiation at the lowest lethal dose have metabolic activity. The difference in the metabolic activity between preparations of the irradiated bacterial cells varied according to the cell concentration and incubation time. The highest level of metabolic activity was 8 h after incubation in the nutrient broth medium compared with 4 and 18 h. The scanning electron microscopy of irradiated bacterial cells showed a cavity at the bacterial cell center without rupture of the surrounding cell membrane compared to the non-irradiated bacterial cells. The antibody level in the groups vaccinated with the different preparations of the irradiated bacterial cells was high compared with the control and formalin-killed vaccine groups. The level of the IFNγ showed an increase after the second dose in the group vaccinated with irradiated bacterial cells without trehalose compared with the other groups. The IL4 level in the vaccinated groups with the irradiated bacterial cells without trehalose, irradiated bacterial cells with trehalose, and trehalose lyophilized irradiated bacterial cells were at a high level when compared with the formalin-killed vaccinated group and control group after the second inoculation. Conclusion: The irradiated M. haemolytica vaccine provides a wide range of humoral and cellular immunity. This study showed high immunological efficiency in rabbits inoculated with the irradiated M. haemolytica vaccine that was shown in the high levels of antibodies (IFNγ and IL4) compared with the group treated with the formalin-killed vaccine. The second dose of irradiated M. haemolytica vaccine is an immune booster that gives the irradiated vaccine a long-acting immunological efficiency.

Typing of alpha and beta coronaviruses by DNA barcoding of NSP12 gene.

Since the spread of the COVID-19 pandemic, the world paid attention to coronaviruses (CoVs) evolution and their diverged lineages because many researches studies supposed that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is evolutionarily developed from a lineage of bats CoVs. This is due to the ability of some mutant CoVs to transmit from a host to different hosts. For this reason, there are many fears about the pathogenicity of the upcoming variants of CoVs. Thus, it is important to get a rapid and economic technique for typing a wide range of human and animal CoVs species for following up their mutant transmission. Therefore, the present study aims at approaching a simple design of DNA barcoding of a wide range of mammals' CoVs (including alpha and beta CoVs), by universal amplification of a species-specific sequence inside a conserved gene (NSP12) followed by amplicon sequencing. The in silico evaluation involved 96 nucleotide sequences of different CoVs (18 alpha CoVs and 78 beta CoVs), and was applied experimentally into the lab on 5 human CoVs isolates; 3 of them belong to beta CoVs (OC43, MERS, and SARS-CoV-2) and 2 are alpha CoVs (229E and NL63). The results indicated that the designed universal primers are able to amplify 332 bp of a taxonomic region inside the NSP12 coding sequence that facilitates the identification and classification of mammals' CoVs upon the resulting phylogenetic tree.

The use of irradiated amnion dressing for the treatment of antibiotic-disinfected skin ulcer.

Skin ulcers are non-healed wounds caused by inflammation of epidermis up to the dermis, which causes pain and limits body movements, significantly reducing quality of life. Amniotic membrane is a placental collagenous biomaterial with many biological and mechanical properties important for tissue engineering and regenerative medicine. The aim of this work is to evaluate the efficacy of topical antibiotic washing followed with irradiated human amniotic membrane (iHAM) dressing for treating five different types of ulcers. The current study included 15 patients who were recruited from the outpatient clinic of the Egyptian Atomic Energy Authority. Follow up of all treated cases that completed the regimen was up to 3 months. The clinical progression of all treated ulcers was quantitatively evaluated by computerized estimation of the wound size reduction based on 3D image analysis. All cases in this study showed great outcomes within several weeks of treatment depending on wound infection, ulcer depth and size, period of healing disorder, age, blood glycemia, and other clinical criteria. Patients' questionnaires revealed that pain was controlled by the first time of treatment. After 1 week post-treatment, granulation tissue was generated and observed in all patients, and all microbial colonies have been eliminated from wounds with previous infection. The current study indicated that the dressing of ulcers with iHAM induces fast healing without complication.

Histologic Evaluations of Xenotransplanted Rabbit Knees by In Vitro-Propagated Human Amniotic Epithelial Cells: A Preclinical Study.

OBJECTIVES: Human amniotic epithelial cells have multipotent differentiation capacity and are considered as potential therapeutic cells for clinical use. This study represents the first published report on the evaluation of the safety and clinical feasibility of human amniotic epithelial cells for transplant into knee joints, serving as an initial step for subsequent therapeutic evaluations within arthritis clinics. MATERIALS AND METHODS: Our experimental design was based on subjecting groups of rabbits as a recipient model for human amniotic epithelial cell transplant into knee joints. Twenty rabbits received 200 µL sterile 0.9% sodium chloride solution containing 1 × 109 human amniotic epithelial cells/knee joint by intra-articular injection. Control groups received cell-free saline into knees, and some animals were not treated. After 10 days of xenotransplant, radiology scans and histologic sections of transplanted and nontrans planted knees were examined and compared. Immunohistochemistry staining was also applied to detect tumor necrosis factor-alpha and interleukin 17 (as inflammatory and immuno-rejection markers) in knee sections. RESULTS: Similar to results shown in noninjected and saline-injected knees, all treated knees appeared normal, with no signs of acute immunorejection, no microbial colonization, no pain, no allergic reactions, no inflammation, and normal motion. Use of human amniotic epithelial cells appeared safe without risk of immunorejection or tumor formation in the transplanted knee joint. CONCLUSIONS: Human amniotic epithelial cells can be safely transplanted into knee joints, encouraging a need for complementary research for further therapeutic evaluations of human amniotic epithelial cells for curing arthritis.

Comparative study between topically applied irradiated human amniotic membrane in combination with tea tree oil versus topical tioconazole in pityraisis versicolor treatment.

Pityriasis versicolor (PV) is a chronic skin disease caused by virulence activities of Malassezia, a genus of skin-associated yeasts. Traditionally, Tioconazole is used as a topical antifungal for curing PV. Previous investigations cited that human amniotic membrane (HAM), a placental tissue, has antimicrobial and anti-inflammatory activities and is useful as a dressing for healing skin lesions. Moreover, tea tree oil (TTO) has a potent antifungal efficacy. This clinical trial aims to achieve an alternative therapeutic treatment able to kill Malassezia and heal PV lesions using TTO-saturated HAM (TOSHAM), with little application times. This study subjected 120 patients with hypopigmented or hyperpigmented PV lesions; half patients were treated weekly with TOSHAM compared with the others who applying 1% Tioconazole cream daily as a traditional treatment. Microbiological evaluation of in vitro fungicidal activity of TOSHAM versus Tioconazole was carried out against Malassezia furfur culture. The clinical outcomes of this study proved the superior activity of TOSHAM to heal PV lesions than Tioconazole; this was in harmony with microbiological findings. This study approached a novel therapeutic treatment of PV with great outcomes by using TOSHAM.

Molecular displaying of differential immunoresponse to various infections of amniotic epithelia.

PROBLEM: Molecular displaying for the interaction of innate immune cells against pathogen is important for knowing their defense mechanism. This study aimed to visualize the differential gene expression profiles of amniotic epithelium, in response to various infections, using simple reverse transcription polymerase chain reaction (RT-PCR) techniques. METHOD OF STUDY: Antimicrobial activity of human amniotic epithelial cells (HAECs) was verified against three different models of pathogenic microorganisms. RNA was extracted from infected and non-infected cells. Transcripts were visualized by two methods of RT-PCR; one of them targets ß-defensin 2 (BDEF2). The other is a novel design method of RNA fingerprinting based on differential length amplification of transcripts (DifLAT) to polymorphism more than 69 coding sequences for antimicrobial proteins. RESULTS: The semiquantitative gel analysis indicated that BDEF2 was upregulated during all infections. DifLAT experiment visualized different patterns of HAECs transcripts for each case of infection. CONCLUSION: This study proved that HAECs uses alternative gene expression profiles for fighting different pathogens.

Biological Activity Alterations of Human Amniotic Membrane Pre and Post Irradiation Tissue Banking.

BACKGROUND AND OBJECTIVE: Innate immunity of Human Amniotic Membrane (HAM) and its highly active secretome that rich with various types of growth factors and anti-inflammatory substances proposed it as a promising material for many medical studies and applications. METHODOLOGY: This study evaluate the biological activity of cultivated HAM pre and post tissue banking process in which freeze-dried HAM was sterilized by 25 KGray (kGy) dose of γ radiation. The HAM's antimicrobial activity, viability, growth of isolated human amniotic epithelial cells (HAECs), hematopoietic stimulation of co-cultivated murine bone marrow cells (mammalian model), scaffold efficiency for fish brain building up (non-mammalian model) and self re-epithelialization after trypsin denuding treatment were examined as supposed biological activity features. RESULTS: Native HAM revealed viability indications and was active to kill all tested microorganisms; 6 bacterial species (3 Gram-positive and 3 Gram-negative) and Candida albicans as a pathogenic fungus. Also, HAM activity promoted colony formation of murine hematopoietic cells, Tilapia nilotica brain fragment building-up and self re-epithelialization after trypsin treatment. In contrary, radiation-based tissue banking of HAM caused HAM cellular death and consequently lacked almost all of examined biological activity features. CONCLUSION: Viable HAM was featured with biological activity than fixed HAM prepared by irradiation tissue banking.