Experimental infection of Clades 2.2.1.2 (H5N1) and 2.3.4.4b (H5N8) of highly pathogenic avian influenza virus infection in commercial broilers.

In this study the pathogenicity, infectivity, and transmissibility of H5N1 highly pathogenic avian influenza (HPAI) clade 2.2.1.2 and H5N8 HPAI clade 2.3.4.4b viruses were evaluated in commercial broilers on days 24 and 31. The mortality rate was 100 % in both challenge viruses and in contact birds either on day 24 or day 31 which confirmed the highly pathogenicity of both clades (2.2.1.2/ 2.3.4.4b) in commercial broilers. Both clades (H5N8 clade 2.3.4.4b/ H5N1 clade 2.2.1.2 viruses) were efficiently replicate within and transmitted between commercial broilers. The H5N8-infected birds shed high titer of viruses from oropharynx and cloaca, which associated with the field spread of AIV-H5N8 in commercial broilers. Mean lesion score in both challenged clades showed similar scores, which confirmed the pathogenicity of both clades in commercial broilers' organs (mainly spleen, cerebellum, thymus, Bursa, Lung) which confirm the neurogenic affinity of the virus. In the central nervous system, non-suppurative encephalitis consisting in multifocal areas of necrosis in cerebral hemispheres, intense spongiosis, neuronal chromatolysis and gliosis were commonly observed. In cerebrum, chromatolysis of Purkinje neurons was a common finding. In the lung, interstitial pneumonia consisting of moderate to severe increase of the cellularity (macrophages and lymphoid cells) in air capillaries and focal areas of necrosis associated with intense viral replication was commonly observed. In lymphoid tissues, including spleen, thymus, and bursa of Fabricius, multifocal areas of necrosis/apoptosis of variable intensity in mononuclear cells were present. Particularly, diffuse necrotic areas were present in the spleen. In the liver, we detected focal areas of necrosis with mild distention of hepatic sinusoids. To conclude the AIV either H5N1 or H5N8 have neurological affinity with immune suppression effect based on necrosis and apoptosis of lymphoid tissues.

Comparison Between Erector Spinae Plane Block versus Serratus Anterior Plane Block Regarding Analgesia and Stress Response After Modified Radical Mastectomy: Randomized Controlled Trial.

Background: Modified radical mastectomy (MRM) is the primary surgical treatment for breast cancer, yet it leads to significant postoperative pain. Objectives: This randomized controlled trial evaluates the effects of an erector spinae plane block (ESPB) versus a serratus anterior plane block (SAPB) on post-MRM pain management and stress response reduction. Methods: Sixty individuals scheduled for unilateral MRM under general anesthesia from October 2021 to October 2022 were divided into three groups. Group A comprised 20 patients who received ultrasound-guided ESPB (20 mL of 0.25% bupivacaine). Group B included 20 patients who received ultrasound-guided SAPB (20 mL of 0.25% bupivacaine). Group C was treated with intravenous morphine based on pain scores. Anesthesia was induced using 2 µg/kg of fentanyl and 2 - 3 mg/kg of propofol. The study compared the three groups regarding pain scores using a numerical rating scale, serum cortisol levels, total fentanyl, and morphine consumption, changes in mean arterial blood pressure (MAP) and heart rate (HR) during surgery, and the occurrence of postoperative complications. Results: Statistically significant reductions in pain scores were observed in group A compared to groups B and C. Moreover, group A exhibited a significant decrease in postoperative morphine consumption, serum cortisol levels 1 hour post-surgery (P = 0.021), MAP, and postoperative vomiting and nausea compared to group B. Furthermore, groups A and B showed statistically significant improvements in all parameters compared to group C. Conclusions: The study demonstrates that ESPB provides superior analgesic effects compared to SAPB in patients undergoing MRM, with reduced morphine use and lower postoperative cortisol levels. Both blocks offer more effective pain control than intravenous morphine alone.

Emergence of the novel infectious bursal disease virus variant in vaccinated poultry flocks in Egypt.

Since the detection of antigenically atypical very virulent Infectious bursal disease viruses (vvIBDV) in Egypt in 1999, the country has been experiencing recurrent outbreaks with high mortality rates and typical gross lesions associated with typical vvIBDV. However, a significant change occurred in 2023, marked by a notable increase in reported subclinical IBDV cases. To evaluate the field situation, samples from 21 farms in 2023 and 18 farms from 2021 and 2022, all of which had experienced IBD outbreaks based on clinical diagnosis, were collected, and subjected to VP2-HVR sequencing. Phylogenetic analysis revealed that all samples collected in 2021 and 2022 clustered with classical virulent strains and vvIBDV. In 2023, one sample clustered with the Egyptian vvIBDV, another with classical virulent IBDV, and the rest with the novel variant IBDV (nVarIBDV) circulating in China. The alignment of deduced amino acid sequences for VP2 showed that all Egyptian classic virulent strains were identical to the Winterfield or Lukert strains, while vvIBDV strains exhibited two out of the three typical residues found in Egyptian vvIBDV, namely Y220F and G254S, but not A321T. Meanwhile, all Egyptian variant strains exhibited typical residues found in nVarIBDV. However, all Egyptian variants showed a mutation at position 321 (321V), which represents the most exposed part of the capsid and is known to have a massive impact on IBDV antigenicity, except for one sample that had 318G instead. This report highlights the emergence of a new variant IBDV in Egypt, clustered with the Chinese new variants, spreading subclinically in broiler farms across a wide geographic area.RESEARCH HIGHLIGHTS New variant IBDV which emerged in Egypt clustered with Chinese nVarIBDV.nVarIBDV spread subclinically across a wide geographic area.Mutation at 321 represents capsid's most exposed part, a defining feature.Antigenically modified vvIBDV still circulating in Egypt with typical lesions.

First Detection and Molecular Characterization of Novel Variant Infectious Bursal Disease Virus (Genotype A2dB1b) in Egypt.

Infectious bursal disease (IBD) is an immunosuppressive disease causing significant damage to the poultry industry worldwide. Its etiological agent is infectious bursal disease virus (IBDV), a highly resistant RNA virus whose genetic variability considerably affects disease manifestation, diagnosis and control, primarily pursued by vaccination. In Egypt, very virulent strains (genotype A3B2), responsible for typical IBD signs and lesions and high mortality, have historically prevailed. The present molecular survey, however, suggests that a major epidemiological shift might be occurring in the country. Out of twenty-four samples collected in twelve governorates in 2022-2023, seven tested positive for IBDV. Two of them were A3B2 strains related to other very virulent Egyptian isolates, whereas the remaining five were novel variant IBDVs (A2dB1b), reported for the first time outside of Eastern and Southern Asia. This emerging genotype spawned a large-scale epidemic in China during the 2010s, characterized by subclinical IBD with severe bursal atrophy and immunosuppression. Its spread to Egypt is even more alarming considering that, contrary to circulating IBDVs, the protection conferred by available commercial vaccines appears suboptimal. These findings are therefore crucial for guiding monitoring and control efforts and helping to track the spread of novel variant IBDVs, possibly limiting their impact.

Isolation of Genetically Diverse H5N8 Avian Influenza Viruses in Poultry in Egypt, 2019-2021.

The global spread of avian influenza virus (AIV) of clade 2.3.4.4b since 2016 has caused severe losses in wild birds and poultry and has posed a risk for the infection of mammals including humans. The vaccination of poultry has been used to limit the spread of the virus and mitigate its socioeconomic impact. Here, we describe H5N8 epidemics in chickens, turkeys and ducks from different localities in Egypt from 2019 to 2021. About 41.7% (n = 88/211) flocks were tested positive by RT-qPCR for H5N8 viruses with prevalence rates of 45.1% (n = 65/144) and 34.3% (n = 23/67) in vaccinated and non-vaccinated flocks, respectively. A sequence analysis of the hemagglutinin and neuraminidase genes indicated not only the multiple introduction events of H5N8 viruses in Egypt but also the establishment of endemic viruses in commercial poultry in 2020/2021. The recent H5N8 viruses in poultry in Egypt are genetically distinct from the majority of licensed vaccines used in the field. Together, our findings indicate that poultry in Egypt is an endemic center for clade 2.3.4.4b in the Middle East. The efficiency of current vaccines should be regularly evaluated and updated to fully protect poultry flocks in Egypt against H5N8 viruses.

In-silico evidence for enhancement of avian influenza virus H9N2 virulence by modulation of its hemagglutinin (HA) antigen function and stability during co-infection with infectious bronchitis virus in chickens.

In the last few decades, frequent incidences of avian influenza (AI) H9N2 outbreaks have caused high mortality in poultry farms resulting in colossal economic losses in several countries. In Egypt, the co-infection of H9N2 with the infectious bronchitis virus (IBV) has been observed extensively during these outbreaks. However, the pathogenicity of H9N2 in these outbreaks remained controversial. The current study reports isolation and characterization of the H9N2 virus recovered from a concurrent IBV infected broiler chicken flock in Egypt during 2011. The genomic RNA was subjected to RT-PCR amplification followed by sequencing and analysis. The deduced amino acid sequences of the eight segments of the current study H9N2 isolate were compared with those of Egyptian H9N2 viruses isolated from healthy and diseased chicken flocks from 2011 to 2013. In the phylogenetic analysis, the current study isolate was found to be closely related to the other Egyptian H9N2 viruses. Notably, no particular molecular characteristic difference was noticed among all the Egyptian H9N2 isolates from apparently healthy, diseased or co-infected with IBV chicken flocks. Nevertheless, in-silico analysis, we noted modulation of stability and motifs structure of Hemagglutinin (HA) antigen among the co-infecting H9N2 AI and the IBV and isolates from the diseased flocks. The findings suggest that the putative factor for enhancement of the H9N2 pathogenicity could be co-infection with other respiratory pathogens such as IBV that might change the HA stability and function. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13337-021-00688-1.

Molecular Detection of a Novel Fowl Adenovirus Serotype-4 (FadV-4) from an Outbreak of Hepatitis Hydropericardium Syndrome in Commercial Broiler Chickens in Egypt.

Hepatitis hydropericardium syndrome (HHS) is an acute infectious disease caused by fowl adenovirus serotype-4 (FAdV-4), which mainly affects broilers aged 4-5 wk. During the winter of January 2021, a 32-day-old broiler flock (Cobb-500) suffered from unusually high mortality (15%) in the Alexandria Governorate, Egypt. The chickens showed depression, ruffled feathers, and greenish diarrhea besides the typical pathologic features of suspected HHS involving flabby hearts, accumulation of a straw-colored fluid in the pericardial sacs, and pale, enlarged hemorrhagic and friable livers with necrotic foci. The kidneys exhibited edema with uric acid depositions. Histopathologic examination of bird livers naturally infected with HHS showed multifocal areas of necrosis, vascular changes, and basophilic intranuclear inclusion bodies (INIB) in the hepatocytes. Molecular identification of the causative agent was accomplished by PCR and sequence analysis of the hyper-variable regions of loop 1 of the hexon gene of fowl aviadenovirus. A pathogenic strain of the novel genotype-4 (FAdV-4) was demonstrated, closely similar to the Israeli strain IS/1905/2019, with an identity of 98%. This is the first report to identify FADV-4 in Egypt, prompting further studies to elucidate its epidemiologic role in all poultry sectors and associated economic losses to provide insights to its control and prevention.

Reporte de caso- Detección molecular de un nuevo serotipo 4 de adenovirus del pollo (FadV-4) de un brote del síndrome de hepatitis e hidropericardio en pollos de engorde comerciales en Egipto. El síndrome de hepatitis e hidropericardio (HHS) es una enfermedad infecciosa aguda causada por el adenovirus aviar serotipo-4 (FAdV-4), que afecta principalmente a pollos de engorde de 4 a 5 semanas de edad. Durante el invierno de enero de 2021, una parvada de pollos de engorde de 32 días (Cobb-500) sufrió una mortalidad inusualmente alta (15%) en la gobernación de Alejandría, en Egipto. Los pollos mostraban depresión, plumas erizadas y diarrea verdosa, además de las características lesiones típicas sugestivas del síndrome de hepatitis e hidropericardio, que involucraban corazones flácidos, acumulación de un líquido de color pajizo en los sacos pericárdicos e hígados pálidos, agrandados, hemorrágicos y friables con focos necróticos. Los riñones presentaban edema con depósitos de ácido úrico. El examen histopatológico de hígados de las aves naturalmente infectadas con el síndrome de hepatitis e hidropericardio mostraron áreas multifocales de necrosis, cambios vasculares y cuerpos de inclusión intranucleares basófilos en los hepatocitos. La identificación molecular del agente causal se logró mediante PCR y análisis de las secuencias de las regiones hipervariables del asa 1 del gene del hexón del aviadenovirus aviar. Se demostró la presencia de una cepa patógena del nuevo genotipo 4 (FAdV-4), muy similar a la cepa israelí IS/1905/2019, con una identidad del 98%. Este es el primer reporte que identifica el FADV-4 en Egipto, lo que motivó más estudios para dilucidar su papel epidemiológico en todos los sectores avícolas y las pérdidas económicas asociadas para proporcionar información sobre su control y prevención.

Epidemiology, Genetic Characterization, and Pathogenesis of Avian Influenza H5N8 Viruses Circulating in Northern and Southern Parts of Egypt, 2017-2019.

Highly pathogenic avian influenza (HPAI) viruses of subtype H5N8 continue to circulate, causing huge economic losses and serious impact on poultry production worldwide. Recently, HPAIV H5N8 has been spreading rapidly, and a large number of HPAI H5N8 outbreaks have been reported in Eurasia 2020-2021. In this study, we conducted an epidemiological survey of HPAI H5N8 virus at different geographical locations in Egypt from 2017 to 2019. This was followed by genetic and pathogenic studies. Our findings highlight the wide spread of HPAI H5N8 viruses in Egypt, including in 22 governorates. The genetic analyses of the hemagglutinin (HA) and neuraminidase (NA) gene segments emphasized a phylogenetic relatedness between the Egyptian HPAI H5N8 viruses and viruses of clade 2.3.4.4b recently isolated in Europe. These findings suggest that a potential back transmission of Egyptian HPAI H5N8 virus has occurred from domestic poultry in Egypt to migratory wild birds, followed by further spread to different countries. This highlights the importance of continuous epidemiological and genetic studies of AIVs at the domestic-wild bird interface.

Molecular characterization of Toll-like receptor type-3 in mallard duck and its response to Newcastle disease virus infection.

Toll-like receptors (TLRs), type I transmembrane pattern recognition receptors (PRRs), are composed of the extracellular domain that is implicated in the recognition of microbial products and initiates the innate and adaptive immune response. Previous reports on TLRs in birds showed significant levels of inter- and intraspecific genetic variation. Little is known about the structure and function of the avian immune system, especially waterfowl species. This work aimed to identify and clone Anas platyrhynchos (mallard duck) TLR-3 (dTLR-3) and its expression level following challenge with velogenic Newcastle disease virus (NDV) as a model for waterfowl species. The mallard duck TLR-3 full-length cDNA sequence had been cloned, which consisted of 2457 nucleotides. The translated amino acid sequence showed identity degree as 97% with Muscovy duck, 95% with geese, 89% with helmeted guineafowls, 88% with the chickens TLR-3 gene, 82% with turkey TLR-3, and 79% with zebra finch, while it showed 54% with human one; the analysis data suggested that the new sequence is probably homologous to vertebrates' TLR-3. The predicted protein encoded by the duck dTLR-3 mRNA sequence is composed of 819 amino acids. Analysis of the deduced amino acid sequence indicated that dTLR-3 has typical structural features and contains the main components of proteins in the TLR family. The dTLR-3 expressed in almost all examined tissues of mallard duck following quantitative real-time polymerase chain reaction (qPCR) analysis and using B-actin as a housekeeping gene. To check the functionality of the receptor and its role in viral infection, we evaluate the expression level in different tissues and its changes following NDV infection. The results showed significant (P < 0.05) upregulated in the brain at 24 h (1.84-fold), reached a peak at 48 h (4.82-fold), and recovered to normal levels at 72 h post-infection. These results indicate a complete and functional dTLR-3 that is orthologous to other vertebrate receptors with its potential role in early response against viral infection in mallard duck species.

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.