Continuous clinicopathological and molecular recognition of very virulent infectious bursal disease virus in commercial broiler chickens.

Gumboro virus is one of the most dangerous immunosuppressant viruses that infect chickens and causes massive financial losses worldwide. The current study aims to conduct a molecular characterization of chicken farms for the infectious bursal disease virus (IBDV). Based on postmortem (PM) lesions, 125 bursal samples from 25 farms were collected from clinically diseased commercial chicken farms with increased mortality and suspected Gumboro virus infection. Pooled bursal samples from suspected IBD-vaccinated flocks were tested for IBDV by reverse transcriptase polymerase chain reaction (RT-PCR). Fifteen out of 25 pooled specimens were found positive for IBDV, with a 60% detection rate, and confirmed positive for very virulent IBDV (vvIBDV) by sequence analysis. Nucleotide phylogenetic analysis of VP1 and VP2 genes was employed to compare the 5 chosen isolates with strains representing different governorates in Egypt during 2022. All strains were clustered with vvIBDV with no evidence of reassortment in the VP1 gene. The VP1 and VP2 genes are divided into groups (I, II). The strains in our study were related to group II, and it acquired a new mutation in the VP2 gene that clustered it into new subgroup B. By mutation analysis, the VP2 gene of all strains had a characteristic mutation to vvIBDV. It acquired new mutations in HVRs compared with HK46 in Y220F, A222T/V in all strains in our study, and Q221K that was found in IBD-EGY-AH5 and AH2 in the loop PBC in addition to G254S in all strains in our study and Q249k that found in IBD-EGY-AH1 and AH3 in the loop PDE. These mutations are important in the virulency and antigenicity of the virus. The VP1 had 242E, 390M, and 393D which were characteristic of vvIBDV and KpnI restriction enzyme (777GGTAC/C782) in addition to a new mutation (F243Y and N383H) in IBD-EGY-AH1 and AH4 strains. According to the current study, the strains were distinct from the vaccinal strain; they could be responsible for the most recent IBDV outbreaks observed in flocks instead of received vaccinations. The current study highlighted the importance of molecular monitoring to keep up to date on the circulating IBDV for regular evaluation of commercial vaccination programs against circulating field viruses.

Current situation and genomic characterization of fowlpox virus in lower Egypt during 2022.

A total of 45 samples of vaccinated and non-vaccinated layer chickens were collected from farms in the Egyptian governorates of Sharqia, Ismailia, Menofia, Gharbia, Kafr El Sheikh, Qalyubia, and Dakahlia in the year 2022. They exhibited nodular lesions on their combs, mouth corners, and eyelids, suggesting they were infected with pox disease, which was associated with a 3 to 5% mortality rate. The samples were grown on the chorioallantoic-membrane of embryonated chicken eggs to ensure their viability. In both vaccinated and non-vaccinated farms, 35 of 45 virus isolates were confirmed positive via polymerase chain reaction (PCR) of fpv167 (P4b), based on the amplicon length of the fpv167 gene locus. The 6 strains from various Egyptian governorates were chosen for sequencing and genetic characterization. Phylogenetic investigation of the fpv167 (P4b) gene of sequenced strains clustered within sub clade A1 showed 100% correlation between FWPVD, TKPV13401 and fowlpox-AN2, fowlpox-AN3, and fowlpox-AN6, but only a 98.6% correlation between fowlpox-AN1, fowlpox-AN4, and fowlpox-AN5. Comparing the fowlpox-AN1, fowlpox-AN4, and fowlpox-AN5 strains with commercial vaccine strains (HP1-444-(FP9), vaccine-VSVRI), they had 98.6% identity, while other strains had 100% identity. The results of this study's mutation research showed that fowlpox-AN1, fowlpox-AN4, and fowlpox-AN5 had acquired novel mutations; fowlpox-AN1 had R201G and T204A; fowlpox-AN4 and fowlpox-AN5 had L141F and H157P. Further research is required to determine the effectiveness of the current vaccine in order to develop a new vaccine.

Selenium nanoparticles enhance the efficacy of homologous vaccine against the highly pathogenic avian influenza H5N1 virus in chickens.

A proper vaccination against avian influenza viruses in chicken can significantly reduce the risk of human infection. Egypt has the highest number of recorded humans highly pathogenic avian influenza (HPAI)-H5N1 infections worldwide despite the widespread use of homologous vaccines in poultry. Enhancing H5N1 vaccine efficacy is ultimately required to better control HPAI-H5N1. The aim of this study is to boost chicken immunity by combined with inactivated HPAI-H5N1 with selenium nanoparticles (SeNPs). The chickens groups 1-3 were fed diets supplemented with SeNPs concentrations (0.25, 0.5, and 1 mg/kg) for 3 weeks and then vaccinated (inactivated HPAI-H5N1). while groups 4,5 and 6 were fed with SeNPs free diets and administered with 0.5 ml of the vaccine combined with 0.02, 0.06, and 0.1 mg/dose of SeNPs and then all groups were challenged with homologous virus 3 weeks post-vaccination (WPV). Group 7, 8 were used as control positive and negative respectively. At 4, 5, and 6 WPV, antibody titer was considerably higher in the group fed a meal supplemented with 1 mg SeNPs/kg. In contrast, both methods of SeNPs supplementation significantly increased the Interleukin 2 (IL2), Interleukin 6 (IL6), and Interferon γ (IFNγ) expressions in the blood cells in a dose-dependent manner, with a higher expression observed in the group that was vaccinated with 0.1 mg/dose. After the challenge, all groups that received SeNPs via diet or vaccines dose showed significant reduction in viral shedding and milder inflammation in lung, trachea, spleen, and liver in addition to higher expression of IL2, IL6, and IFNγ, with the highest expression observed in the group that was vaccinated with 0.1 mg/dose compared the plain vaccinated group. The groups of 1 mg SeNPs/kg and combined vaccinated with 0.1 mg/dose showed the best vaccine efficacy. However, the group vaccinated with 0.1 mg/dose showed the earliest reduction in viral shedding. Overall, SeNPs supplementation in the diet and the administration of the vaccine formula with SeNPs could enhance vaccine efficacy and provide better protection against HPAI-H5N1 in chickens by enhancing cellular immunity and reducing inflammation. We recommend using SeNPs as a vaccine combination or feeding with diet to increase the immunity and vaccine efficacy against H5N1.

Evaluation of the antiparasitic activity of the chitosan-silver nanocomposites in the treatment of experimentally infested pigeons with Pseudolynchia canariensis.

This study aimed to evaluate the efficacy of chitosan-silver nanocomposites in the treatment of experimentally infested pigeons with Pseudolynchia canariensis (P. canariensis) with evaluation of different immunological parameters before and after treatment. Therefore, fourteen birds were divided into 2 groups; group1(infested group including 12 birds) which subdivided into 6 sub-groups experimentally infested pigeons 2 pigeons each, and five group of them were treated with chitosan-silver nanocomposites and sub-group number 6 was treated with deltamethrin while, group 2 including two pigeons were kept as control negative ones. P. canariensis flies distributed under the wing and /or under the tail in infested group and these pigeons showed significantly lower RBCs and higher WBCs than that in non-infested pigeons. The cell mediated immune response against experimentally infested pigeons with P. canariensis was studied. P. canariensis infestation in pigeons have a negative impact on pigeon's blood parameters, increase TNF-α and IL-1ß cytokines levels. This study cleared out the role of P. canariensis in the induction of a case of oxidative stress indicated by high level of nitric oxide and malondialdehyde (MDA) with low antioxidant capacity in shape of reduced zinc concentration in the sera of experimentally infested pigeon. Chitosan-silver nanocomposite has a promising effect in the elimination of P. canariensis infestation in pigeons.

The natural antiviral and immune stimulant effects of Allium cepa essential oil onion extract against virulent Newcastle disease virus.

Fifty broiler chicks were divided into five groups to study the antiviral and immune-stimulant effect of Allium cepa essential oils (ACEO). The effect of Allium cepa essential oils administration single or combined with NVD vaccine in broilers, more than one parameter was studied in this study i.e., the clinical symptoms that appeared on the chicks after the experimental infection with velogenic Newcastle disease virus, postmortem lesions, pathological lesions scoring, mortality rate (MR), and viral shedding, birds immunity was assessed by HI test and protection percent post-challenge with vNDV. Our result showed that mild clinical signs, lesion scoring, decreased viral shedding in ACEO treated groups 3 (G 3) more than control groups post-challenge with vNDV. Delayed onset of mild clinical signs in G3 followed by complete recovery 7th-day post-challenge (DPC). Low MR (40 and 0%) and high protection percent (100 and 60%) in ACEO treated G3 and G5, respectively. spleen, thymus, cecal tonsil, proventriculus, and cerebrum lesions scoring in G3 and G5 were significantly ((p ≤ 0.05).) lower than the control group, proving a decrease in NDV replication and effective antiviral activity of ACEO. HI titer significantly increased (p ≤ 0.05) In G3, G4 and G5 compared with control groups. There is no significant difference in HI titer in ACEO treated groups and vaccinated groups. In conclusion, oral administration of ACEO combined with NDV vaccines significantly reduces or eliminates lethal clinical signs, lesions, viral shedding, and enhances immune response and protection percent after vNDV challenge proving the natural antiviral and immune stimulant effect of ACEO onion extract. Implementing such a regime might aid NDV control in broiler flocks in endemic areas and reduce the epidemiological load of NDV in the environment.

Morphological and molecular characterization of Ascaridia columbae in the domestic pigeon (Columba livia domestica) and the assessment of its immunological responses.

Parasitism is a divesting problem that is frequently overlooked and may result in severe prominent clinical manifestation. This study aimed to investigate the seasonal and sexual prevalence of the gastrointestinal nematode Ascaridia columbae (A. columbae) infection among domestic pigeons in Giza governorate, Egypt, during the period from 2020 to 2021. One hundred and sixty suspected pigeons were clinically investigated. Blood & tissue samples were collected from infected birds to estimate serum zinc concentration, malondialdehyde (MDA), and nitric oxide levels. As well as tumor necrosis factor-alpha (TNF-α), interleukin 1ß (IL1ß) activity, and histopathological examination were estimated; also, worms were collected for morphological identification using electron microscope (SEM) and molecularly identified using polymerase chain reaction (PCR), further sequenced, and submitted in GenBank with accession number MZ343369. The average ascarid (length × breadth) were 72.4 ± 3.3 µm (70.5 - 79.9 µm) × 39.9 ± 2.5 µm (37.6 - 42.3 µm). The distinguishing morphological characteristics that have been noticed in ascarid worms were creamy white, cylindrical worm with triradiate lips with wide cephalic alae extending on both the lateral sides and filariform esophagus. In males, spicules were almost equal with the presence of precloacal chitinous-rimmed sucker. The prevalence of A. columbae infection was (63.1%) with a higher incidence in females (79.2%) than males (46.1%). The highest seasonal prevalence was observed in winter (92.5%), followed by summer and spring (87.5% and 55%), respectively while, the lowest prevalence was observed in autumn (17.5%). The intensity of worms in the infected intestine varied from 5 to 120 adult worms. The histopathological examination revealed the presence of chronic diffuse moderate catarrhal enteritis with roundworms in the lumen. Infected birds showed a significant increase in nitric oxide and MDA levels while serum zinc levels were lowered in infected pigeons. Infected pigeons revealed a marked increase in IL1-ß and TNFα than apparently healthy ones.

Genetic evolution of Marek's disease virus in vaccinated poultry farms.

BACKGROUND AND AIM: The Marek's disease virus (MDV) is a neoplastic disease causing serious economic losses in poultry production. This study aimed to investigate MDV occurrence in poultry flocks in the Lower Egypt during the 2020 breakout and genetically characterized Meq, gL, and ICP4 genes in field strains of MDV. MATERIALS AND METHODS: Forty samples were collected from different breeds from eight Egyptian governorates in 2020. All flocks had received a bivalent vaccine (herpesvirus of turkey FC-126 + Rispens CVI988). However, weight loss, emaciation, reduced egg production, paralysis, and rough/raised feather follicles occurred. Samples were collected from feather follicles, liver, spleen, and nerve tissue for diagnosis by polymerase chain reaction. MDV genetic characterization was then performed by sequencing the Meq, gL, and ICP4 genes of five positive samples representing different governorates and breeds. RESULTS: A total of 28 samples were positive for MDV field strains, while two were related to MDV vaccinal strains. All samples tested negative for ALV (A, B, C, D, and J) and REV. Phylogenetic analysis of the Meq gene of sequenced samples revealed that all MDVs were related to the highly virulent European viruses (Gallid herpesvirus 2 ATE and PC12/30) with high amino acid (A.A.) identity 99.2-100%. Alternatively, there was low A.A. identity with the vaccine strains CVI988 and 3004 (up to 82.5%). These results indicate that further investigation of the efficacy of current Egyptian vaccines is required. The Egyptian strains also harbor a specific mutation, allowing clustering into two subgroups (A and B). By mutation analysis of the Meq gene, the Egyptian viruses in our study had R101K, P217A, and E263D mutations present in all Egyptian viruses. Furthermore, R176A and T180A mutations specific to our strains contributed to the high virulence of highly virulent strains. There were no mutations of the gL or ICP4 genes. CONCLUSION: Further studies should evaluate the protection contributed by current vaccines used in Egypt.

Eficacia protectora de una vacuna inactivada contra el virus de la enfermedad hemorrágica del conejo tipo 2 preparada a partir de un aislado local en Egipto / Protective efficacy of an inactivated vaccine against Rabbit hemorrhagic disease virus 2 prepared from a local isolate in Egypt

Rabbit hemorrhagic disease is a contagious viral disease of rabbits controlled by vaccination. The present study was aimed to diagnose rabbit hemorrhagic disease from 11 infected farms from Qalubia governorate during 2019 and to prepare homologous vaccine against rabbit hemorrhagic disease virus 2. For this purpose, 11 liver samples were collected from suspected cases and subjected to detection and identification of circulating rabbit hemorrhagic disease virus. Ten samples were confirmed to be rabbit hemorrhagic disease virus using hemagglutination test, animal inoculation and reverse transcriptase polymerase chain reaction. Sequencing and phylogenetic analysis of two isolates (R5&R6) revealed the presence of rabbit hemorrhagic disease virus 2 (A/Qalubia/2019 and B/Qalubia/2019) under accession number MT07629 and MT067630 respectively. The inactivated rabbit hemorrhagic disease virus vaccines were prepared using Montanide ISA 206 oil or aluminum hydroxide gel adjuvants. Prepared vaccines were inoculated subcutaneously in susceptible rabbits and submitted to sterility, safety and potency tests. Obtained results showed that mean hemagglutination inhibition titer for aluminum hydroxide gel vaccine was 6,7.7,8.9 and 9.1 log2 while, Montanide vaccine reached to 6.7,8.7,9.2 and 9.5 log2 at 1st, 2nd, 3rd, and 4th weeks post vaccination, respectively. Immunized rabbits with Montanide vaccine showed better protection reach to 70 percent, 90 percent percent, 100 percent and 100 percent when compared to aluminum hydroxide gel vaccine 60 percent, 70 percent, 90 percent and 90 percent at 1st, 2nd, 3rd and 4th weeks post vaccination respectively. It was concluded that newly emerged rabbit hemorrhagic disease virus 2 was isolated from suspected cases. The two prepared vaccines were sterile, safe and potent. The oily adjuvanted rabbit hemorrhagic disease virus 2 vaccine stimulated an earlier and higher humoral immune response than the aluminum hydroxide gel adjuvanted vaccine. This humoral immune response achieved significant level of protection(AU)

La enfermedad hemorrágica del conejo es una enfermedad viral contagiosa de los conejos que se controla mediante vacunación. El presente estudio tuvo como objetivo diagnosticar la enfermedad hemorrágica del conejo en 11 granjas infectadas de la provincia de Qalubia, durante 2019 y preparar una vacuna homóloga contra el virus de la enfermedad hemorrágica del conejo tipo 2. Para este propósito, se recolectaron 11 muestras de hígado de casos sospechosos y se sometieron a detección e identificación de virus circulante de la enfermedad hemorrágica del conejo. Se confirmó que diez muestras eran positivas al virus de la enfermedad hemorrágica del conejo, utilizando para ello la prueba de hemaglutinación, inoculación en animales y Reacción en cadena de la polimerasa con transcriptasa inversa. La secuenciación y el análisis filogenético de dos aislamientos (R5 y R6) revelaron la presencia del virus de la enfermedad hemorrágica del conejo tipo 2 (A/Qalubia/2019 y B/Qalubia/2019) con los números de acceso MT07629 y MT067630 respectivamente. Las vacunas inactivadas del virus de la enfermedad hemorrágica del conejo se prepararon usando adyuvantes de gel de hidróxido de aluminio o aceite Montanide ISA 206. Las vacunas preparadas se inocularon por vía subcutánea en conejos susceptibles y se sometieron a pruebas de esterilidad, seguridad y potencia. Los resultados obtenidos mostraron que el título medio de inhibición de la hemaglutinación para la vacuna en gel de hidróxido de aluminio fue de 6; 7,7; 8,9 y 9,1 log2, mientras que la vacuna de Montanide alcanzó 6,7; 8,7; 9,2 y 9,5 log2 en la 1ª, 2ª, 3ª y 4ª semanas después de la vacunación, respectivamente. Los conejos inmunizados con la vacuna Montanide tuvieron una mejor protección, alcanzándose niveles de 70 por ciento, 90 por ciento, 100 por ciento y 100 por ciento en comparación con la vacuna en gel de hidróxido de aluminio 60 por ciento, 70 por ciento, 90 por ciento y 90 por ciento en la 1ª, 2ª, 3ª y 4ª semanas después de la vacunación, respectivamente. Se concluyó que el virus de la enfermedad hemorrágica del conejo tipo 2 de reciente aparición se aisló de los casos sospechosos. Las dos vacunas preparadas fueron estériles, seguras y potentes. La vacuna contra el virus de la enfermedad hemorrágica del conejo tipo 2 con adyuvante oleoso estimuló una respuesta inmune humoral más temprana y mayor que la vacuna con adyuvante en gel de hidróxido de aluminio. Esta respuesta inmune humoral confirió un nivel significativo de protección(AU)

Genetic variability of the Avian leukosis virus subgroup J gp85 gene in layer flocks in Lower Egypt.

AIM: This study aimed to determine the prevalence of layer flock tumor disease in Lower Egypt during the period of 2018-2019 and to undertake molecular characterization and determine the genetic diversity of all identified viruses. MATERIALS AND METHODS: Forty samples were collected from layer chicken located in six governorates of Lower Egypt during the period of 2018-2019. Samples were taken from tumors in different organs. Tumor tissues were identified by histopathological sectioning and then further confirmed by a reverse-transcription polymerase chain reaction. Finally, genetic evolution of Avian leukosis virus (ALV-J) gp85 gene was studied. RESULTS: All the study samples were negative for Marek's disease virus, reticuloendotheliosis virus, ALV (A,B,C and D) and 20 samples were positive for ALV-J in backyard in six governrates. Sequencing of ALV-J gp85 gene was performed for six representative samples (one from each governorate), and they were found to be genetically related to prototype virus HPRS-1003 (identity percentage: 91.2-91.8%), but they were from a different group that was similar to the AF88-USA strain (first detected in 2000) with specific mutations, and they differed from a strain that was previously isolated in Egypt in 2005, forming two different subgroups (I and II) that had mutations in the hr1domain (V128F, R136A) and hr2 domain (S197G, E202K). CONCLUSION: The ALV-J virus was the main cause of neoplastic disease in layer chickens from Lower Egypt in the period of 2018-2019. We found that the genetic evolution of ALV-J gp85 gene was related to prototype virus HPRS-1003 but in a different group with a specific mutation. Further studies are needed to evaluate the antigenicity and pathogenicity of recently detected ALV-J strains.