Doszpoly, A., M. Shaalan, and M. El-Matbouli, "Silver Nanoparticles Proved to Be Efficient Antivirals In Vitro against Three Highly Pathogenic Fish Viruses.", Viruses, vol. 15, issue 8, 2023. Abstract

The efficacy of silver nanoparticles (AgNPs) was tested in vitro against three different fish viruses, causing significant economic damage in aquaculture. These viruses were the spring viraemia of carp virus (SVCV), European catfish virus (ECV), and Ictalurid herpesvirus 2 (IcHV-2). The safe concentration of AgNPs that did not cause cytotoxic effects in EPC cells proved to be 25 ng/mL. This dose of AgNPs decreased significantly (5-330×) the viral load of all three viruses in three different types of treatments (virus pre-treatment, cell pre-treatment, and cell post-treatment with the AgNPs). In a higher concentration, the AgNPs proved to be efficient against ECV and IcHV-2 even in a delayed post-cell-treatment experiment (AgNP treatment was applied 24 h after the virus inoculation). These first in vitro results against three devastating fish viruses are encouraging to continue the study of the applicability of AgNPs in aquaculture in the future.

EL-NAHASS, E. L. - S. H. A. Y. M. A. A., M. K. Abdelhamid, A. Ali, A. A. Shalaby, and M. Shaalan, "Pathological assessment and tissue tropism of two different Egyptian infectious bronchitis strains.", Virusdisease, vol. 34, issue 3, pp. 410-420, 2023. Abstract

Avian infectious bronchitis is one of the most common viral infections in chickens affecting all ages. The tropism of infectious bronchitis virus (IBV) strains became broader and more variable posing major implications for the effective control of IBV infection. In this study, two IBV viruses representing classic and variant strains were inoculated intranasally into day-old SPF chicks (10 EID/0.2 ml/bird). Clinical signs were observed for 15 days post-infection (DPI). Five chicks from each group were euthanized at 2, 4, 6, 8, 10, 12, and 15 DPI for histopathology and virus antigen detection by IHC and quantitative rRT-PCR. Results revealed that both classic and variant IBV strains induced mild clinical signs with no mortalities and fewer various histopathological lesions in infected SPF chickens. Although the viruses were detected by rRT-PCR up to 12 DPI, the affected tissues showed regeneration after 10 DPI with IHC revealing no IBV antigen. In summary, no differences were found in the behaviour of both IBV isolates in chickens. The broad tissue tropism for both IBV strains as indicated by viral antigen detection in various organs with no clinical or gross lesion suggest that the main cause of death in IBV infection under field conditions occurs as a result of complication with secondary infections rather single IBV infection. Due to positive immunostaining in the bursa, it is thought that IBV infection has immunosuppressive consequences, hence further study is required to validate this impact.

Saleh, M., E. Essawy, M. Shaalan, S. Osman, F. Ahmed, and M. El-Matbouli, "Therapeutic Intervention with Dietary Chitosan Nanoparticles Alleviates Fish Pathological and Molecular Systemic Inflammatory Responses against Infections.", Marine drugs, vol. 20, issue 7, 2022. Abstract

Marine bio-sourced chitosan nanoparticles (CSNP) are antimicrobial and immunomodulatory agents beneficial for fish medicine. Herein, dietary CSNP was investigated for the amelioration of the systemic inflammatory responses of an induced fish model. One hundred and forty-four rainbow trout were assigned to one pathogen-free and non-supplemented group (negative control), and three challenged groups: non-supplemented (positive control), CSNP-preventive, and CSNP-therapeutic. After a feeding experiment extended for 21 days, the organosomatic indices (OSI) and molecular aspects were assessed. After a challenge experiment extended for further 28 days, CSNP-therapeutic intervention was assessed on fish survival and systemic inflammatory responses on pathology, histo-morphology, and molecular aspects. With CSNP administration, OSI nonsignificantly decreased and the relative expression of targeted inflammatory-mediator genes was significantly increased. The CSNP-therapeutic fish showed an RPS of 80% as compared to the positive control group, and CSNP-therapeutic administration retained the highest gene expression augmentation up to 28 days after the challenge. Notably, the splenic reticulin fibers framework of the CSNP-therapeutic group retained the highest integrity among the groups during the infection. After recovery, reticulin fibers density in the CSNP-therapeutic samples was significantly higher than in the negative control group, which indicates high innate immunity. Thus, CSNP showed promising biotherapeutic features enhancing fish resistance against infections.

Marouf, S., X. Li, H. M. Salem, Z. S. Ahmed, S. M. Nader, M. Shaalan, F. H. Awad, H. Zhou, and T. Cheang, "Molecular detection of multidrug-resistant Pseudomonas aeruginosa of different avian sources with pathogenicity testing and in vitro evaluation of antibacterial efficacy of silver nanoparticles against multidrug-resistant P. aeruginosa.", Poultry science, vol. 102, issue 10, pp. 102995, 2023. Abstract

Pseudomonas aeruginosa (P. aeruginosa) is a serious zoonotic pathogen threaten the poultry industry causing severe economic losses therefor, this study aimed to isolation, phenotypic, molecular identification of P. aeruginosa from different avian sources (chickens, turkey, pigeons, table eggs, and dead in shell chicken embryos), from different Egyptian governorates (Giza, Qalubia, Beheira, El-Minya, and Al-Sharqia) with applying of antibiotic sensitivity test on all P. aeruginosa isolates. Highly resistant isolates (n = 49) were subjected to molecular identification of P. aeruginosa with detection of resistant genes including carbapenemase-encoding genes blaKPC, blaOXA-48, and blaNDM. On the base of molecular results, a highly resistant P. aeruginosa strain was tested for its pathogenicity on day old specific pathogen free (SPF) chicks. Also, in vitro experiment was adopted to evaluate the efficacy of silver nanoparticles (Ag-NPs) against highly antibiotic-resistant P. aeruginosa strains. The overall isolation percentage was from all examined samples were 36.2% (571/1,576) representing 45.2% (532/1,176) from different birds' tissues and 39/400 (9.7%) from total egg samples. Some of isolated strains showed multidrug resistance (MDR) against kanamycin, amoxicillin, amoxicillin-clavulanic acid, neomycin, chloramphenicol, vancomycin, cefotaxime clavulanic acid, lincomycin-spectinomycin, co-trimoxazole, cefoxitin, gentamycin, and doxycycline. These MDR strains were also molecularly positive for ESBL and carbapenemase-encoding genes. MDR strain showed high pathogenicity with histopathological alterations in different organs in challenged birds. Main histopathological lesions were necrosis of hepatocytes, renal tubular epithelium, and heart muscle bundles. The MDR strain showed in vitro sensitivity to Ag-NPs. In conclusion, MDR P. aeruginosa is a serious pathogen causing high morbidity, mortality, and pathological tissue alterations. Ag NPs revealed a promising in vitro antimicrobial sensitivity against MDR P. aeruginosa and further in vivo studies were recommended.

Ahmed, N. H. M., A. Ghallab, M. Shaalan, M. Saied, and E. S. Mohammed, "First molecular identification and phylogenetic illustration of Sarcocystis species infection in Red Sea shortfin mako shark (Isurus oxyrinchus Rafinesque, 1810).", BMC veterinary research, vol. 20, issue 1, pp. 104, 2024. Abstract

BACKGROUND: members of the genus Sarcocystis are intracellular obligate protozoan parasites classified within the phylum Apicomplexa and have an obligate heteroxenous life cycle involving two hosts. A more comprehensive understanding of the prevalence and geographic range of different Sarcocystis species in marine ecosystems is needed globally and nationally. Hence, the objective of this study was to document the incidence of Sarcocystis infection in sharks within the aquarium ecosystem of Egypt and to identify the species through the characterization of the SSU rDNA gene.

METHODS: All organs of the mako shark specimen underwent macroscopic screening to detect the existence of a Sarcocystis cyst. Ten cysts were collected from the intestine and processed separately to extract the genomic DNA. The polymerase chain reaction (PCR) was accomplished by amplifying a specific 18S ribosomal RNA (rRNA) gene fragment. Subsequently, the resulting amplicons were subjected to purification and sequencing processes.

RESULTS: Macroscopic examination of the mako shark intestinal wall sample revealed the presence of Sarcocystis cysts of various sizes and shapes, and sequencing of the amplicons from Sarcocystis DNA revealed a 100% nucleotide identity with the sequence of Sarcocystis tenella recorded from sheep in Iran; The mako shark sequence has been deposited in the GeneBank with the accession number OQ721979. This study presents the first scientific evidence demonstrating the presence of the Sarcocystis parasite in sharks, thereby documenting this specific marine species as a novel intermediate host in the Sarcocystis life cycle.

CONCLUSIONS: This is the first identification of Sarcocystis infection in sharks, and we anticipate it will be an essential study for future screenings and establishing effective management measures for this disease in aquatic ecosystems.

Marouf, S., X. Li, H. M. Salem, Z. S. Ahmed, S. M. Nader, M. Shaalan, F. H. Awad, H. Zhou, and T. Cheang, "Molecular detection of multidrug-resistant Pseudomonas aeruginosa of different avian sources with pathogenicity testing and in vitro evaluation of antibacterial efficacy of silver nanoparticles against multidrug-resistant P. aeruginosa.", Poultry science, vol. 102, issue 10, pp. 102995, 2023. Abstract

Pseudomonas aeruginosa (P. aeruginosa) is a serious zoonotic pathogen threaten the poultry industry causing severe economic losses therefor, this study aimed to isolation, phenotypic, molecular identification of P. aeruginosa from different avian sources (chickens, turkey, pigeons, table eggs, and dead in shell chicken embryos), from different Egyptian governorates (Giza, Qalubia, Beheira, El-Minya, and Al-Sharqia) with applying of antibiotic sensitivity test on all P. aeruginosa isolates. Highly resistant isolates (n = 49) were subjected to molecular identification of P. aeruginosa with detection of resistant genes including carbapenemase-encoding genes blaKPC, blaOXA-48, and blaNDM. On the base of molecular results, a highly resistant P. aeruginosa strain was tested for its pathogenicity on day old specific pathogen free (SPF) chicks. Also, in vitro experiment was adopted to evaluate the efficacy of silver nanoparticles (Ag-NPs) against highly antibiotic-resistant P. aeruginosa strains. The overall isolation percentage was from all examined samples were 36.2% (571/1,576) representing 45.2% (532/1,176) from different birds' tissues and 39/400 (9.7%) from total egg samples. Some of isolated strains showed multidrug resistance (MDR) against kanamycin, amoxicillin, amoxicillin-clavulanic acid, neomycin, chloramphenicol, vancomycin, cefotaxime clavulanic acid, lincomycin-spectinomycin, co-trimoxazole, cefoxitin, gentamycin, and doxycycline. These MDR strains were also molecularly positive for ESBL and carbapenemase-encoding genes. MDR strain showed high pathogenicity with histopathological alterations in different organs in challenged birds. Main histopathological lesions were necrosis of hepatocytes, renal tubular epithelium, and heart muscle bundles. The MDR strain showed in vitro sensitivity to Ag-NPs. In conclusion, MDR P. aeruginosa is a serious pathogen causing high morbidity, mortality, and pathological tissue alterations. Ag NPs revealed a promising in vitro antimicrobial sensitivity against MDR P. aeruginosa and further in vivo studies were recommended.

Mahboub, H. H., K. Shahin, S. M. Mahmoud, D. E. Altohamy, W. A. Husseiny, D. A. Mansour, S. I. Shalaby, M. M. S. Gaballa, M. Shaalan, M. Alkafafy, et al., "Silica nanoparticles are novel aqueous additive mitigating heavy metals toxicity and improving the health of African catfish, Clarias gariepinus.", Aquatic toxicology (Amsterdam, Netherlands), vol. 249, pp. 106238, 2022. Abstract

Silica nanoparticles (SiNPs) are among the non-toxic nanoparticles (NPs) that have magnetic capabilities. It is hypothesized that SiNPs may be able to reduce toxic effects exerted by a mixture of lead (Pb) and mercury (Hg) in African catfish Clarias gariepinus. The in vitro magnetic potential of SiNPs to absorb Pb and Hg was tested. Fish (N = 240) were divided into four groups in triplicates for 30 days. The first group served as control and the second group (SiNPs) was exposed to 1/10 of 96 h LC50 of SiNPs (14.45 mg/L). The third group (HMM) was exposed to 1/10 of 96-h LC50 of a mixture of mercury chloride (HgCl) and lead chloride (PbCl) equal to 0.04 mg/ L and 23.1 mg/L. The fourth group (SiNPs+ HMM) was exposed to a suspension composed of SiNPs, HgCl, and PbCl at the same concentrations as the third group. Results showed that fish exposed to heavy metals revealed the following consequences; a significant decrease in hematological, immunological (complement-3 and nitric oxide), and antioxidants (total antioxidant capacity, glutathione peroxidase, superoxide dismutase, and catalase) indices, down-regulation of IL-1β, IL-8, TGF-β, NF-κβ, HSP70, and Hepcidin genes, the highest mortality rate (48.33%), higher values of alkaline phosphatase, alanine, and aspartate aminotransferases, urea, creatinine, and branchial malondialdehyde, marked up-regulation of CC chemokine and CXC chemokines, and high HMs residues levels in muscles. Extensive pathology showed degeneration with diffuse vacuolation of hepatopancreatic cells and hemorrhage in the HMM group. Interestingly, the exposed group to SiNPs and HMM demonstrated a decline of HMs concentration in fish muscles and modulated the abovementioned parameters with the regeneration of histological alterations of liver and gills. Based on the study outcomes, we highlight the importance and the safety of SiNPs as a novel aqueous additive to alleviate HMs toxicity and recommend using SiNPs for enhancing fish performance for sustaining aquaculture without adverting safety of human health by their little accumulation in muscular tissue.

EL-NAHASS, E. L. - S. H. A. Y. M. A. A., M. K. Abdelhamid, A. Ali, A. A. Shalaby, and M. Shaalan, "Pathological assessment and tissue tropism of two different Egyptian infectious bronchitis strains.", Virusdisease, vol. 34, issue 3, pp. 410-420, 2023. Abstract

Avian infectious bronchitis is one of the most common viral infections in chickens affecting all ages. The tropism of infectious bronchitis virus (IBV) strains became broader and more variable posing major implications for the effective control of IBV infection. In this study, two IBV viruses representing classic and variant strains were inoculated intranasally into day-old SPF chicks (10 EID/0.2 ml/bird). Clinical signs were observed for 15 days post-infection (DPI). Five chicks from each group were euthanized at 2, 4, 6, 8, 10, 12, and 15 DPI for histopathology and virus antigen detection by IHC and quantitative rRT-PCR. Results revealed that both classic and variant IBV strains induced mild clinical signs with no mortalities and fewer various histopathological lesions in infected SPF chickens. Although the viruses were detected by rRT-PCR up to 12 DPI, the affected tissues showed regeneration after 10 DPI with IHC revealing no IBV antigen. In summary, no differences were found in the behaviour of both IBV isolates in chickens. The broad tissue tropism for both IBV strains as indicated by viral antigen detection in various organs with no clinical or gross lesion suggest that the main cause of death in IBV infection under field conditions occurs as a result of complication with secondary infections rather single IBV infection. Due to positive immunostaining in the bursa, it is thought that IBV infection has immunosuppressive consequences, hence further study is required to validate this impact.

SALEM, M. A. I. A., O. A. Mahdy, M. Shaalan, and R. M. Ramadan, "The phylogenetic position and analysis of Renicola and Apharyngostrigea species isolated from Cattle Egret (Bubulcus ibis).", Scientific reports, vol. 13, issue 1, pp. 16195, 2023. Abstract

Cattle Egret (Bubulcus ibis) is one of the most well-known herons in Egypt. It is called the friend of the farmer because it benefits farmers and helps them get rid of insects and worms. It acts as a reservoir for many diseases. Few researchers have discussed the significance of parasitic diseases that affect this wild bird and may lead to mortalities among the population especially the importance of vital organs such as kidneys. Therefore, this study aimed to spotlight parasitic infection-affected herons in Egypt and consider the risks to this beneficial bird. During this study, 23 Bubulcus ibis were captured after their death from Abou Rewash Giza Governorate, Egypt, during the period from February to September (2022). Renicola species (spp.) and Apharyngostrigea spp. are two important digenean parasites that were recovered from the kidneys, and small intestine of the heron Cattle Egret (Bubulcus ibis) with an infection rate of (17.2%) and (11.8%) respectively. Histopathological techniques were used to assess tissue alterations while light microscopy and molecular assays were used to assess the parasites. The parasites' morphological and morphometrical characteristics, as well as polymerase chain reaction and sequencing assays (mitochondrial sections), were investigated for the first time in Egypt. These parasites were given in-depth illustrations and drawings. The distinctive qualities of the two species were discussed. As the first record from Egypt, the nucleotide sequences discovered in this work have been uploaded into the GenBank database (accession numbers: OR021986 and OQ955829). Microscopically, the renal blood vessels had vasculitis, necrosis, and other degenerative alterations. Further research analyzing the health of various heron spp. and environmental deterioration can help to close information gaps about the interactions between parasites, their hosts, and environmental health.

Doszpoly, A., M. Shaalan, and M. El-Matbouli, "Silver Nanoparticles Proved to Be Efficient Antivirals In Vitro against Three Highly Pathogenic Fish Viruses.", Viruses, vol. 15, issue 8, 2023. Abstract

The efficacy of silver nanoparticles (AgNPs) was tested in vitro against three different fish viruses, causing significant economic damage in aquaculture. These viruses were the spring viraemia of carp virus (SVCV), European catfish virus (ECV), and Ictalurid herpesvirus 2 (IcHV-2). The safe concentration of AgNPs that did not cause cytotoxic effects in EPC cells proved to be 25 ng/mL. This dose of AgNPs decreased significantly (5-330×) the viral load of all three viruses in three different types of treatments (virus pre-treatment, cell pre-treatment, and cell post-treatment with the AgNPs). In a higher concentration, the AgNPs proved to be efficient against ECV and IcHV-2 even in a delayed post-cell-treatment experiment (AgNP treatment was applied 24 h after the virus inoculation). These first in vitro results against three devastating fish viruses are encouraging to continue the study of the applicability of AgNPs in aquaculture in the future.

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