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The spread of duck hepatitis virus (DHV) in Egypt with its’ most prevalent genotypes (1 and 3) causes vast economic losses in the duck industry despite the regular vaccination with live attenuated vaccines. However, the use of live-vaccines is considered a potential risk for the non-vaccinated birds due to the viral shedding from vaccinated populations. The current study aimed to evaluate the protective efficacy and safety of two inactivated monovalent and one bivalent vaccines against DHV 1 and 3 genotypes. The inactivated monovalent (DHAV-1, DHAV-3) and bivalent (DHAV-1+3) vaccines were produced by using Montanide ISA 70 oil as an adjuvant. Three groups of 4-week-old ducklings (n=50) were vaccinated with one of the three vaccines, respectively. One group of ducklings was used as negative control (n=25). The immune-response of the vaccinated groups was measured by Virus Neutralization Test and expressed with Neutralizing Indices (NIs). The NIs for the bivalent vaccine group (5.6 and 5.4) were higher compared to the monovalent vaccine groups (5.0 and 4.7). In conclusion, the use of inactivated bivalent DHV vaccine could produce higher protective efficacy compared to the monovalent DHAV-1 and DHAV-3 vaccines.

ABSTRACT: 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.

Avian influenza (AI) viruses pose a risk to the worldwide poultry industry. Ultimately, improving the efficiency of the H9N2 vaccine is necessary to better control low-pathogenic avian influenza-H9N2 by using natural immunostimulant. Therefore, the goal of the present study was to examine varying doses of the cyanobacterium Spirulina extract on the effectiveness of H9N2 vaccine. Thus, a total of 150 specific pathogen-free (SPF) chickens were allocated into 6 groups, 25 birds each, as follow: G1, G2, and G6 were supplemented with 200, 400, and 400 mg Spirulina extract/kg feed, respectively, whilst the feed in G3, G4, and G5 were not supplemented with Spirulina extract. At 21-days-old, only the chickens in G1, G2, and G3 were vaccinated with the H9N2 AI vaccine. After 4 wk postvaccination, the chickens in G1, G2, G3, G4, and G6 were challenged with H9N2 AI Egyptian strain. The challenged virus was selected from a recent circulating Egyptian strain during 2022, and it was related to A/quail/Hong Kong/G1/97-like virus lineage and clustered with G1-B sub-lineage EGY-2 group. It had a high amino acids identity percentage of 92.6% with the A/chicken/Iran/av1221/1998 (Boehringer Ingelheim) vaccine. The results of real-time reverse-transcriptase polymerase-chain-reaction (rRT-PCR) revealed that no shedding of the virus was reported in G1, G2, G3, and G5. The supplementation of Spirulina extract in low (200 mg/kg of feed) and high (400 mg/kg of feed) concentration with the birds vaccinated with H9N2 AI vaccine (G1 and G2) induced prominent immuno-stimulatory effect in a dose dependent manner where it strongly enhanced the phagocytic activities of broilers' peripheral blood monocytes, and lysozyme at all days postvaccination (dpv) and days postchallenge (dpc) compared to other groups with significant differences at all day of experiment and 21st dpv, 28th dpv, 7th dpc, and 14th dpc, respectively. The supplementation with Spirulina extract in G1 and G2 induced the highest hemagglutination inhibition antibody titer in a dose-dependent manner at all-time intervals. The antibody titer postvaccination was significantly increased in G1 and G2 at 14th, and 21st dpv, in comparison with G3. Furthermore, G1 and G2 showed higher significant antibody titers at 7th and 14th dpc, compared to other groups. Furthermore, Spirulina extract (200 and 400 mg/kg feed) in G1 and G2 showed anti-inflammatory effect in a dose dependant manner by downregulating nitric oxide levels at all times postchallenge with a significant difference at 3 to 7 dpc compared to G3, G4, and G6, with improved histopathological alterations in the trachea, lung, kidney, spleen, and bursa of Fabricius. G6 supplied with 400 mg/kg Spirulina extract feed only without vaccination had a similar effect as vaccinated groups on innate immunity. However, it delayed the production of antibodies and did not prevent viral shedding as in vaccinated groups. In conclusion, vaccination in conjunction with either dose of Spirulina extract (G1, and G2) prevents viral shedding, increases the immune response, and reduces inflammation and histopathological change caused by H9N2 AI infection in a dose dependent manner. We recommend the use of 400 mg Spirulina extract/kg feed as a natural immunostimulant in conjunction with the H9N2 vaccine to achieve the highest possible level of protection against H9N2 AI infection.

ABSTRACT: Avian influenza virus (AIV) and Newcastle disease virus (NDV) are respiratory illness syndromes that have recently been detected in vaccinated flocks and are causing major financial losses in the chicken farming industry. The objective was to evaluate the efficacy of Valley Vac H5Plus NDVg7 vaccine in protecting chickens against the H5N8 and NDV strains that have recently been circulating in comparison with the efficacy of the commercially available bivalent H5+ND7 vaccine. In contrast to the H5+ND7 vaccine, which was made of genetically distinct H5N8/2018 clade 2.3.4.4b genotype group (G5), H9N2/2016, H5N1/2017, and genetically comparable NDV genotype VII 1.1/2019 of the recently circulating challenge viruses, the Valley Vac H5Plus NDVg7 vaccine consisted of the recently isolated (RG HPAI H5N1 AIV/2015 Clade 2.2.1.2, RG HPAIV H5N8/2020 Clade 2.3.4.4b genotype group 6 (G6), and NDV genotype VII 1.1/2012) which were genetically similar to challenged strains. To determine the effectiveness of the Valley Vac H5Plus NDVg7 vaccine, a total of 70-day-old commercial chicks were divided into 7 groups of 10 birds each. Groups (G1 and G4) received Valley Vac H5Plus NDVg7 vaccine. Groups (G2 and G5) groups received commercial H5+ND7 vaccine. While groups (G3 and G6) were kept nonvaccinated, and group (G7) was kept as a nonchallenged and nonvaccinated. After 3-wk post vaccination (WPV), groups G1, G2, and G3 were challenged with A/Duck/ Egypt/SMG4/2019(H5N8) genotype G6. On the other hand, groups G4, G5, G6 were challenged with NDV/EGYPT/18629F/2018 genotype VII 1.1 with an intranasal injection of 0.1 mL. Antibody titer was calculated at the first 3 wk after vaccination, and the viral shedding titer was calculated at 3-, 5-, and 7-days post challenge. Mortality and morbidity rates were monitored daily during the experiment, and for the first 10 d after the challenge, to provide an estimate of the protection rate. The results showed that a single dosage of 0.5 mL per bird of Valley Vac H5Plus NDVg7 vaccine provides 80% protection against both H5N8 and NDV, compared to the bivalent H5+ND7 vaccine, which provided 20 and 80% protection against H5N8 and NDV, respectively. In addition, 0.5 mL per bird of Valley Vac H5Plus NDVg7 vaccine produced a greater immune response against both viruses than commercial vaccination at 1 to 3 WPV with a significant difference at 1 WPV for H5N8 and a comparatively higher immune response for NDV. Furthermore, it reduced virus shedding of H5N8 on the third, fifth, seventh, and tenth days lower than H5+ND7 vaccine with a significant difference on the third day for H5N8 and relatively lower than bivalent H5+ND7 vaccine for NDV with a significant difference on the fifth day. The Valley vaccinated group demonstrated more tissue intactness compared to the commercially vaccinated group against the H5N8 challenge, however the bivalent commercially vaccinated group showed the similar level of tissue integrity against NDV. In conclusion, Valley Vac H5Plus NDVg7 that contains the genetically similar strain to recently circulating challenged virus (H5N8 genotype G6) provided better protection with greater immune response and decreased the amount of virus shed against H5N8 genotype G6 and showed less histopathological alteration than the commercial bivalent H5+ND7 vaccine that contain genetically distinct (H5N8 genotype G5). However the Valley Vac H5Plus NDVg7 provided the same protection with relatively high immune response and relatively decreased the amount of virus shed and showed equal tissue integrity than the commercial bivalent H5+ND7 vaccine against NDV genotype VII 1.1 that contain the same genotype of NDV genotype VII 1.1.

ABSTRACT: 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.

Late in 2016, multiple reassortant highly pathogenic (HP) avian influenza virus (AIVs) H5N8 was detected. AIVs infect different isolated hosts with a specific viral tropism. In the current study, the whole genome of the Egyptian A/chicken/NZ/2022 was genetically characterized. The H5N8-A/Common-coot/Egypt/CA285/2016, A/duck/Egypt/SS19/2017 previously isolated in Egypt, and the recently circulating A/chicken/Egypt/NZ/2022 reassortant viruses' replication, pathogenicity, and viral load in comparison to the H5N1-Clade 2.2.1.2 were investigated on Madin-Darby canine kidney cell (MDCK), by using the cytopathic effect (CPE) percent and matrix-gene reverse transcription quantitative real-time polymerase chain reaction to compute the virus titer at various points in time. The A/chicken/Egypt/NZ/2022 virus was similar to the reassortant strain clade 2.3.4.4b discovered in farms in 2016. The 2 sub-groupings of hemagglutinin (HA) and neuraminidase (NA) genes were identified (I and II); the A/chicken/Egypt/NZ/2022 HA and NA genes were associated with subgroup II. The subgroup II of the HA gene was further divided into A and B owing to acquired specific mutations. The A/chicken/Egypt/NZ/2022 in our study was associated with subgroup B. The M, NS, PB1, and PB2 genes were shown to be clustered into clade 2.3.4.4b by full genome sequence analysis; however, the PA and NP genes were found to be associated with H6N2 viruses, which had particular mutations that improved viral virulence and mammalian transmission. The current results showed that the circulating H5N8 viruses were more variable than previous viruses analyzed in 2016 and 2017. Compared to other reassortant HPAI H5N8, and HPAI H5N1, the growth kinetics of A/chicken/Egypt/NZ/2022 had a high CPE without the addition of trypsin and the most viral copies with a significant difference (P < 0.01) in comparison to HPAI H5N8 and HPAI H5N1 reassortant viruses. Accordingly, the effective viral replication of A/chicken/Egypt/NZ/2022 in the MDCK than other viruses may play a factor in the spread and maintenance of specific reassortant H5N8 influenza virus in the field.

ABSTRACT: Highly pathogenic avian influenza (HPAI) is a serious viral infection that causes massive economic losses in poultry. The current study investigated the HPAI virus prevalence in commercial broiler chicken flocks from 2019 to 2022. Organ samples, including trachea, cecal tonsils, spleen, brain, as well as tracheal and cloacal swabs, were harvested from 111 problematic broiler chicken flocks that suffered from variable mortalities accompanied with respiratory signs (103 H5-vaccinated and 8 nonvaccinated flocks) in Egypt during the observation duration. Molecular tools were used to analyze the samples, including real-time reverse transcription-polymerase chain reaction (rRT-PCR) and sequence analysis of some PCR positive strains. The results indicated that 24 flocks were positive for HPAI H5N8, representing 21.6%, with 22.3% (23/103) prevalence and 12.5% (1/8) detection in vaccinated and nonvaccinated flocks, respectively, and they were almost detected in the autumn and winter seasons. Phylogenetic evaluation of the hemagglutinin (HA) gene showed that the 6 Egyptian strains were clustered in clade 2.3.4.4b and allocated into 2 groups (I and II). The samples recovered in 2019 were clustered in new subgroup A, and samples recovered in 2020 to 2022 were clustered in new subgroup B with 10 nucleotide mutations (R72S, A83D, T140A). In conclusion, HPAI H5N8 is a serious threat even in vaccinated birds; to control such problems, periodic molecular monitoring with vaccine efficacy evaluation and the use of preventive strategies are recommended.

ABSTRACT: Many pathogens that cause chronic diseases in birds use the respiratory tract as a primary route of infection, and respiratory disorders are the main leading source of financial losses in the poultry business. Respiratory infections are a serious problem facing the poultry sector, causing severe economic losses. Avian influenza virus, Newcastle disease virus, infectious bronchitis virus, and avian pneumovirus are particularly serious viral respiratory pathogens. Mycoplasma gallisepticum, Staphylococcus, Bordetella avium, Pasteurella multocida, Riemerella anatipestifer, Chlamydophila psittaci, and Escherichia coli have been identified as the most serious bacterial respiratory pathogens in poultry. This review gives an updated summary, incorporating the latest data, about the evidence for the circulation of widespread, economically important poultry respiratory pathogens, with special reference to possible methods for the control and prevention of these pathogens.

Controlling avian influenza viruses (AIVs) is mainly based on culling of the infected bird flocks or via the implementation of inactivated vaccines in countries where AIVs are considered to be endemic. Over the last decade, several avian influenza virus subtypes, including highly pathogenic avian influenza (HPAI) H5N1 clade 2.2.1.2, H5N8 clade 2.3.4.4b and the recent H5N1 clade 2.3.4.4b, have been reported among poultry populations in Egypt. This demanded the utilization of a nationwide routine vaccination program in the poultry sector. Antigenic differences between available avian influenza vaccines and the currently circulating H5Nx strains were reported, calling for an updated vaccine for homogenous strains. In this study, three H5Nx vaccines were generated by utilizing the reverse genetic system: rgH5N1_2.3.4.4, rgH5N8_2.3.4.4 and rgH5N1_2.2.1.2. Further, the immunogenicity and the cross-reactivity of the generated inactivated vaccines were assessed in the chicken model against a panel of homologous and heterologous H5Nx HPAIVs. Interestingly, the rgH5N1_2.3.4.4 induced high immunogenicity in specific-pathogen-free (SPF) chicken and could efficiently protect immunized chickens against challenge infection with HPAIV H5N1_2.3.4.4, H5N8_2.3.4.4 and H5N1_2.2.1.2. In parallel, the rgH5N1_2.2.1.2 could partially protect SPF chickens against infection with HPAIV H5N1_2.3.4.4 and H5N8_2.3.4.4. Conversely, the raised antibodies to rgH5N1_2.3.4.4 could provide full protection against HPAIV H5N1_2.3.4.4 and HPAIV H5N8_2.3.4.4, and partial protection (60%) against HPAIV H5N1_2.2.1.2. Compared to rgH5N8_2.3.4.4 and rgH5N1_2.2.1.2 vaccines, chickens vaccinated with rgH5N1_2.3.4.4 showed lower viral shedding following challenge infection with the predefined HPAIVs. These data emphasize the superior immunogenicity and cross-protective efficacy of the rgH5N1_2.3.4.4 in comparison to rgH5N8_2.3.4.4 and rgH5N1_2.2.1.2.

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.

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%, 90%, 100% and 100% when compared to aluminum hydroxide gel vaccine 60%, 70%, 90% and 90% 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.

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 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.

ABSTRACT: In late 2016, Egypt encountered multiple cases of the highly pathogenic avian influenza (HPAI) virus of the H5N8 subtype. In a previous study, three distinct genotypes, including A/common-coot/Egypt/CA285/2016 (H5N8) (CA285), A/duck/Egypt/SS19/2017 (H5N8) (SS19), and A/duck/Egypt/F446/2017 (H5N8) (F446), were isolated from wild birds, a backyard, and a commercial farm, respectively, during the first wave of infection. In this current study, we investigated the differences in the pathogenicity, replication and transmissibility of the three genotypes and A/chicken/Egypt/15S75/2015 (H5N1) (S75) was used as the control. The intravenous pathogenicity index was between 2.68 and 2.9. The chicken lethal dose 50 values of F446, SS19 and CA285 were 103.7, 103.7, an 104 with a natural route of infection, respectively. These strains took longer than S75 to cause death when infection was carried out through the natural route (HPAI H5N1). After inoculation with the original concentration of 105 and 106 egg infective dose 50 (EID50), F446 had a higher mortality rate with short mean death times of 4, and 7 days, respectively compared with the other H5N8 viruses. Chickens inoculated with F446 and contacted exposed chickens infected with F446 showed the highest viral titer with remarkable differences in all H5N8 tested swabs at 2-4 days postinfection (dpi) compared to S75 at 2 dpi. This indicates that F446 had a more efficient transmission and spread from contact exposed birds to other birds. All H5N8 viruses were able to replicate systematically in all organs (trachea, brain, lung, and spleen) of the chicken with high viral titer with significantly different and more pathological changes observed in F446 than in other H5N8 viruses at 2 and 4 dpi. Compared with H5N1, we recorded a significantly high viral titer in the samples obtained from the lung, brain and both cloacal and tracheal swabs at 2 and 4 dpi, respectively and in the samples obtained from the spleen at 2 and 4 dpi among the experimental chicken. The comparative pathogenesis study revealed that in comparison with the other HPAI H5N8 viruses, the genotype F446 was more pathogenic, and showed more efficient viral replication and transmissibility in chickens in Egypt. The genotype F446 also showed a high viral titer than HPAI H5N1 and short mean death time at the third day after inoculation with 106 and 105 EID50, which revealed a conservation of certain H5N8 genotypes and a decrease in the incidence of H5N1.

ABSTRACT: 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 1β (IL1β) 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-β and TNFα than apparently healthy ones.

The highly pathogenic avian influenza (HPAI) H5N8 virus was first detected in Egypt in late 2016. Since then, the virus has spread rapidly among different poultry sectors, becoming the dominant HPAI H5 subtype reported in Egypt. Different genotypes of the HPAI H5N8 virus were reported in Egypt; however, the geographic patterns and molecular evolution of the Egyptian HPAI H5N8 viruses are still unclear. Here, extensive epidemiological surveillance was conducted, including more than half a million samples collected from different poultry sectors (farms/backyards/live bird markets) from all governorates in Egypt during 2019–2021. In addition, genetic characterization and evolutionary analyses were performed using 47 selected positive H5N8 isolates obtained during the same period. The result of the conducted surveillance showed that HPAI H5N8 viruses of clade 2.3.4.4b continue to circulate in different locations in Egypt, with an obvious seasonal pattern, and no further detection of the HPAI H5N1 virus of clade 2.2.1.2 was observed in the poultry population during 2019–2021. In addition, phylogenetic and Bayesian analyses revealed that two major genotypes (G5 and G6) of HPAI H5N8 viruses were continually expanding among the poultry sectors in Egypt. Notably, molecular dating analysis suggested that the Egyptian HPAI H5N8 virus is the potential ancestral viruses of the European H5N8 viruses of 2020–2021. In summary, the data of this study highlight the current epidemiology, diversity, and evolution of HPAI H5N8 viruses in Egypt and call for continuous monitoring of the genetic features of the avian influenza viruses in Egypt.

Highly pathogenic avian influenza (HPAI) viruses continue to circulate worldwide, causing numerous outbreaks among bird species and severe public health concerns. H5N1 and H5N8 are the two most fundamental HPAI subtypes detected in birds in the last two decades. The two viruses may compete with each other while sharing the same host population and, thus, suppress the spread of one of the viruses. In this study, we performed a statistical analysis to investigate the temporal correlation of the HPAI H5N1 and HPAI H5N8 subtypes using globally reported data in 2015–2020. This was joined with an in-depth analysis using data generated via our national surveillance program in Egypt. A total of 6412 outbreaks were reported worldwide during this period, with 39% (2529) as H5N1 and 61% (3883) as H5N8. In Egypt, 65% of positive cases were found in backyards, while only 12% were found in farms and 23% in live bird markets. Overall, our findings depict a trade-off between the number of positive H5N1 and H5N8 samples around early 2017, which is suggestive of the potential replacement between the two subtypes. Further research is still required to elucidate the underpinning mechanisms of this competitive dynamic. This, in turn, will implicate the design of effective strategies for disease control.

The H9N2 subtype of avian influenza A virus (aIAV) is circulating among birds worldwide, leading to severe economic losses. H9N2 cocirculation with other highly pathogenic aIAVs has the potential to contribute to the rise of new strains with pandemic potential. Therefore, rapid detection of H9 aIAVs infection is crucial to control virus spread. A qualitative reverse transcription recombinase polymerase amplification (RT-RPA) assay for the detection of aIAV subtype H9N2 was developed. All results were compared to the gold standard (real-time reverse transcription polymerase chain reaction (RT-PCR)). The RT-RPA assay was designed to detect the hemagglutinin (HA) gene of H9N2 by testing three pairs of primers and a probe. A serial concentration between 106 and 100 EID50 (50% embryo infective dose)/mL was applied to calculate the analytical sensitivity. The H9 RT-RPA assay was highly sensitive as the lowest concentration point of a standard range at one EID50/mL was detected after 5 to 8 min. The H9N2 RT-RPA assay was highly specific as nucleic acid extracted from H9 negative samples and from other avian pathogens were not cross detected. The diagnostic sensitivity when testing clinical samples was 100% for RT-RPA and RT-PCR. In conclusion, H9N2 RT-RPA is a rapid sensitive and specific assay that easily operable in a portable device for field diagnosis of aIAV H9N2.