Genska karakterizacija, kloniranje i ekspresija Toll-like receptora 1 mRNA nilske tilapije (Oreochromis niloticus)

Toll-like receptors (TLRs) are the most studied group of pathogen recognition receptor categories that detects infectious agents in vertebrates. Fish TLRs exhibit clear, distinct features, structure and a larger diversity than in other vertebrates. This study focused on identifying and detecting the structure of Oreochromis niloticus (Nile tilapia) Toll- like receptor-1 (TLR1|) as a model in freshwater bony fish. The full-length cDNA sequence of Oreochromis niloticus TLR1 mRNA was cloned. Cloning and sequence analysis revealed that the complete cDNA sequence of Oreochromis niloticus TLR1 consists of 2355 base pairs and encodes a polypeptide of 785 amino acids. The molecular analysis of the amino acid sequence indicated that Oreochromis niloticus TLR1 has the standard structural features and major components of amino acids of TLR family members, and is considered an orthologue to the vertebrate TLR1, not a paralogue. The translated amino acid analysis showed 96%, 88%, 85%, and 85% degrees of identity with Zebra Mbuna, Sea bass, Damsel fish, and Clownfish, respectively; and showed 66% identity t with electric eels and 61% with starlets. Phylogenetic analysis revealed that the Nile tilapia TLR1 is closely related to Larimichthys crocea, Epinephelus coioides, and Takifugu rubripes TLR1. Oreochromis niloticus TLR1 was expressed in the kidneys, brain, spleen, intestines, muscle, liver, gills, heart and skin. Quantitative RT-PCR showed differences in the expression levels between the tested tissues. In conclusion, this study is the first report (according to our knowledge) and provides a complete molecular and functional characterization of Oreochromis niloticus toll-like receptor 1, which is considered to be functionally orthologous to TLR1 in other species models.Toll-like receptori (TLR) najviše su istraživana skupina receptora za prepoznavanje uzročnika bolesti u kralježnjaka. TLR u riba pokazuju jasna razlikovna svojstva, strukturu i veliku raznolikost u odnosu na druge kralježnjake. Ovo je istraživanje usredotočeno na identifikaciju i otkrivanje Toll-like receptora 1 (TLR1) u nilske tilapije (Oreochromis niloticus) kao predstavnika slatkovodnih riba. Klonirana je puna sekvencija cDNA TLR1 mRNA. Utvrđeno je da se kompletna sekvencija cDNA TLR1 nilske tilapije sastoji od 2355 baznih parova i kodira polipeptid od 785 aminokiselina. Molekularna analiza sekvencija aminokiselina upućuje na to da TLR1 nilske tilapije ima standardna strukturna svojstva i glavne komponente porodice TLR receptora i smatra se ortologom, ne paralogom TLR1 kralježnjaka. Analiza prevedenih aminokiselina pokazala je stupanj identičnost od 96 % s mbuna zebrom, 88 % s lubinom, 85 % s damsel ribom i 85 % s ribom klaun, dok je stupanj identičnosti s električnom jeguljom bio 66 %, a s ribom starlet 61 %. Filogenetska analiza pokazala je da je TLR1 nilske tilapije usko povezan s TLR1 vrsta Larimichthys crocea, Epinephelus coioides i Takifugu rubripes. TLR1 nilske tilapije bio je izražen u bubrezima, mozgu, slezeni, crijevima, mišiću, jetri, škrgama, srcu i na koži. Kvantitativni RT-PCR pokazao je razlike u razini ekspresije među testiranim tkivima. Prema našim podacima ovo je istraživanje prvo koje donosi kompletnu molekularnu i funkcionalnu karakterizaciju Toll-like receptora 1 nilske tilapije, te se smatra funkcionalnim ortologom TLR1 u drugih vrsta

Prevalence, molecular typing, and antimicrobial resistance of bacterial pathogens isolated from ducks

Aim: This study aimed to investigate the prevalence of different bacterial species affecting ducks as well as demonstrating the antimicrobial susceptibility and molecular typing of the isolated strains. Materials and Methods: A total of 500 samples were randomly collected from different duck farms at Ismailia Governorate, Egypt. The collected samples were subjected to the bacteriological examination. Polymerase chain reaction (PCR) was applied for amplification of Kmt1 gene of Pasteurella multocida and X region of protein-A (spA) gene of the isolated Staphylococcus aureus strains to ensure their virulence. The antibiotic sensitivity test was carried out. Results: The most common pathogens isolated from apparently healthy and diseased ducks were P. multocida (10.4% and 25.2%), Escherichia coli (3.6% and 22.8%), Staphylococcus epidermidis (10% and 8.8%), Pseudomonas aeruginosa (2% and 10%), and Proteus vulgaris (0.8% and 10%), respectively. In addition, S. aureus and Salmonella spp. were isolated only from the diseased ducks with prevalence (12.2%) and (2.8%), respectively. Serotyping of the isolated E. coli strains revealed that 25 E. coli strains were belonged to five different serovars O1, O18, O111, O78, and O26, whereas three strains were untypable. Salmonella serotyping showed that all the isolated strains were Salmonella Typhimurium. PCR revealed that four tested P. multocida strains were positive for Kmt1 gene with specific amplicon size 460 bp, while three strains were negative. In addition, all the tested S. aureus strains were positive for spA gene with specific amplicon size 226 bp. The antibiotic sensitivity test revealed that most of the isolated strains were sensitive to enrofloxacin, norfloxacin, and ciprofloxacin. Conclusion: P. multocida is the most predominant microorganism isolated from apparently healthy and diseased ducks followed by E. coli and Staphylococci. The combination of both phenotypic and genotypic characterization is more reliable an epidemiological tool for identification of bacterial pathogens affecting ducks

Current Genomic Characterization of Circulating Chicken Infectious Anemia Virus in Backyard and Commercial Chicken Flocks in Ismailia and Sharkia Provinces, Egypt

This study aimed to evaluate the occurrence, molecular characterization, partial sequencing, and phylogenetic analysis of the chicken infectious anemia virus (CIAV) circulating in chicken flocks in Ismailia and Sharkia Provinces, Egypt. Tissue pool samples (liver, thymus, spleen, and bone marrow) were collected from commercial and backyard flocks with anemia, uneven growth, and vaccinal failure history. The occurrence of CIAV was 51% (51/100) using specific primers through the polymerase chain reaction test, which was higher in the backyard (26/50) 52% than that in commercial flocks (25/50) 50%. The highest rate of CIAV detection was 77% (13/17) and 75% (9/12) in Saso and Arbor Acer breeds respectively, followed by the Baladi 52% (26/50) and Cobb 27 % (4/15). The histopathological study reflected severe lymphocytic depletions in lymphoid organs with the presence of apoptotic cells and eosinophilic intranuclear inclusion bodies. Partial sequence analysis of six selected field circulating CIAVs showed changes in VP1 at position H 22 Q, VP2 at position A 153 V, T 180 S and VP3 at position R 118 C indicating low affinity of the obtained viruses to grow in the cell line. Some obtained viruses showed mutations in the epitopic site which may develop escape mutation virus from the currently used vaccines. Phylogenetically, the six selected fields CIAVs were classified into two distinct groups. The continuous surveillance activities and epidemiological mapping for CIAV among Egyptian governorates using updating primers are essential to facilitate control program strategies

Isolation and Genotypic Characterization of New Emerging Avian Reovirus Genetic Variants in Egypt

Avian reovirus (ARV) strains cause a variety of symptoms in chickens, including viral arthritis/tenosynovitis, a disease that has emerged as a significant cause of economic losses in commercial chicken flocks in recent years in various countries, including Egypt. Furthermore, ARV strains are frequently isolated from birds suffering from malabsorption. In the actual study, seventy-five samples were collected in 2021 and 2022 from broiler and vaccinated broiler breeder flocks at different farms in Giza Province, Egypt, with reovirus-like symptoms such as significant weight fluctuation and arthritis/malabsorption. ARV was screened using real-time PCR, and fifteen positive samples were detected (20%), which were then subjected to embryonated chicken egg (ECE) isolation and molecular characterization (11/15 sample) of a partial segment of the sigma (σ)C gene (S1-gene). Phylogenetically, nine strains were found to belong to genotypic cluster IV, with 82–89% identity with Israeli ARV 2018, and two strains belong to genotypic cluster V with a 78% nucleotide identity with Japan ARV 2021. No correlation between lesions and genotype was found. The strains under study had a low sequence identity (43–55%) when compared with various commercial vaccines belonging to genotypic cluster I (e.g., strain S1133). These findings imply that novel ARV genotypes representing clusters IV and V have recently been introduced to Egyptian poultry farms. A homologous vaccine is suggested; because this variation raises the possibility that commercial vaccines may not offer protection against circulating ARVs

Efficacy of the Newcastle Disease Virus Genotype VII.1.1-Matched Vaccines in Commercial Broilers

Class II genotype VII Newcastle disease viruses (NDV) are predominant in the Middle East and Asia despite intensive vaccination programs using conventional live and inactivated NDV vaccines. In this study, the protective efficacies of three commercial vaccine regimes involving genotype II NDV, recombinant genotype VII NDV-matched, and an autogenous velogenic NDV genotype VII vaccine were evaluated against challenge with velogenic NDV genotype VII (accession number MG029120). Three vaccination regimes were applied as follows: group-1 received inactivated genotype II, group-2 received inactivated recombinant genotype VII NDV-matched, and group-3 received velogenic inactivated autogenous NDV genotype VII vaccines given on day 7; for the live vaccine doses, each group received the same live genotype II vaccine. The birds in all of the groups were challenged with NDV genotype VII, which was applied on day 28. Protection by the three regimes was evaluated after infection based on mortality rate, clinical signs, gross lesions, virus shedding, seroconversion, and microscopic changes. The results showed that these three vaccination regimes partially protected commercial broilers (73%, 86%, 97%, respectively, vs. 8.6% in non-vaccinated challenged and 0% in non-vaccinated non-challenged birds) against mortality at 10 days post-challenge (dpc). Using inactivated vaccines significantly reduced the virus shedding at the level of the number of shedders and the amount of virus that was shed in all vaccinated groups (G1-3) compared to in the non-vaccinated group (G-4). In conclusion, using closely genotype-matched vaccines (NDV-GVII) provided higher protection than using vaccines that were not closely genotype-matched and non-genotype-matched. The vaccine seeds that were closely related to genotype VII.1.1 provided higher protection against challenge against this genotype since it circulates in the Middle East region. Updating vaccine seeds with recent and closely related isolates provides higher protection

Validation of polymerase chain reaction assay as an alternative method for detection of chicken anemia virus as a vaccine contaminant.

<p>Freedom of veterinary vaccines from extraneous virus contamination is one of the most important requirements for vaccine before release. Testing of vaccines for detection of extraneous virus contamination is currently achieved in the Central Laboratory for Evaluation of Veterinary Biologics (CLEVB) by conventional in - vitro and / or in - vivo assays as tissue culture inoculation and chicken inoculation, which are time consuming and laborious. In this study, the polymerase chain reaction (PCR) was used as an alternative technique – for detection of chicken anemia virus (CAV) contamination in avian live viral vaccines. Specificity of the assay was verified by detection of CAV specific product only in templates extracted from CAV preparations,  but not detected in  templates of various avian viruses including Newcastle dis-ease virus (NDV), infectious bronchitis virus (IBV), infectious bursal disease virus (IBDV), avian Reovirus (ARV), fowl pox virus (FPV), avian encephalomyelitis virus (AEV) and Marek´s disease virus (MDV). Assay sensitivity was  evaluated by testing serial 10- fold dilutions of CAV stock, and the determined detection limit was high enough to detect 40 TCID50 of CAV per reaction. Another set of CAV dilutions were prepared in three different types of avian live vaccines, namely NDV, ARV , and FPV vaccines( the vaccine matrix was used as diluents), and PCR was performed to study the effect of vaccine matrix on reaction sensitivity as well as to verify the assay efficacy in detection and recovery of CAV in contaminated vaccines.  Results indicated that PCR sensitivity was not affected by the presence of NDV, or ARV matrices. However, the assay sensitivity was decreased by one log in case of Fowlpox vaccine matrix.  This PCR assay could be used as an alternative technique to the time- consuming, tedious, in vivo and in vitro assays, and application of this technique can facilitate the routine testing of vaccines for detection of CAV as an extraneous virus contaminant.</p

Isolation and Genotypic Characterization of New Emerging Avian Reovirus Genetic Variants in Egypt

Avian reovirus (ARV) strains cause a variety of symptoms in chickens, including viral arthritis/tenosynovitis, a disease that has emerged as a significant cause of economic losses in commercial chicken flocks in recent years in various countries, including Egypt. Furthermore, ARV strains are frequently isolated from birds suffering from malabsorption. In the actual study, seventy-five samples were collected in 2021 and 2022 from broiler and vaccinated broiler breeder flocks at different farms in Giza Province, Egypt, with reovirus-like symptoms such as significant weight fluctuation and arthritis/malabsorption. ARV was screened using real-time PCR, and fifteen positive samples were detected (20%), which were then subjected to embryonated chicken egg (ECE) isolation and molecular characterization (11/15 sample) of a partial segment of the sigma (σ)C gene (S1-gene). Phylogenetically, nine strains were found to belong to genotypic cluster IV, with 82–89% identity with Israeli ARV 2018, and two strains belong to genotypic cluster V with a 78% nucleotide identity with Japan ARV 2021. No correlation between lesions and genotype was found. The strains under study had a low sequence identity (43–55%) when compared with various commercial vaccines belonging to genotypic cluster I (e.g., strain S1133). These findings imply that novel ARV genotypes representing clusters IV and V have recently been introduced to Egyptian poultry farms. A homologous vaccine is suggested; because this variation raises the possibility that commercial vaccines may not offer protection against circulating ARVs