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. 2023 Aug 18;102(11):103043. doi: 10.1016/j.psj.2023.103043

Evaluation of the immuno-stimulatory effect of aqueous neem (Azadirachta indica) leaf extract against highly pathogenic avian influenza (H5N8) in experimental chickens

Ahmed M Hegazy ⁎, Ola Hassanin *, Mai AM Hemele *, Maha Abdullah Momenah †, Fatimah A Al-Saeed ‡, Amani Osman Shakak §,#, Khaled A El-Tarabily ǁ,1, Mohamed T El-Saadony , Hala MN Tolba *
PMCID: PMC10520533  PMID: 37741118

Abstract

The recently detected clade 2.3.4.4 of the highly pathogenic avian influenza (HPAI) H5N8 virus in poultry encouraged us to study the efficacy of the 6 most extensively used saleable H5 poultry vaccinations (bivalent [AI + ND], Re-5 H5N1, H5N1, H5N3, monovalent AI, monovalent ND) with or without aqueous 8% neem (Azadirachta indica) leaf extract as an immunostimulant. One hundred thirty birds were randomly divided into 7 groups. Groups 1, 2, 3, 4, 5, and 6 were divided into 2 subgroups (G1a, G2a, G3a, G4a, G5a, G6a) and (G1b, G2b, G3b, G4b, G5b, G6b) with 10 birds each. Subgroups (G1a, G2a, G3a, G4a, G5a, G6a) received the (bivalent [AI + ND], Re-H5N1, H5N1, H5N3, monovalent AI, monovalent ND) vaccines, while subgroups (G1b, G2b, G3b, G4b, G5b, G6b) received the same previous vaccination but treated with neem leaf extract administrated 2 d before and after vaccination, and G7 with 10 birds was kept unvaccinated as positive control group. Clinical signs of the challenged group showed conjunctivitis, closed eyes, cyanosis in comb and wattle, ocular discharge, and greenish diarrhea, while postmortem lesions showed congested trachea and lung, hemorrhage on the shank, proventriculus, and pancreas; gelatinous fluid submandibular, congestion of all organs (septicemia), mottled spleen. The clinical signs and lesions were mild in neem leaf extract treated with bivalent vaccine and Re-H5N1 while moderate in monovalent vaccine and H5N3 with or without neem leaf extract treated and reached severe in the group immunized with H5N1 with or without neem leaf extract treatment. The protection levels in the bivalent vaccine (AI + ND), Re-5 H5N1, and H5N3 treated with neem leaf extract, were 80%, 80%, and 60%, respectively, while bivalent vaccine (AI + ND), Re-5 H5N1 and H5N3 without treatment were 60%, 60%, and 40%, respectively. The virus shedding was prevented in groups vaccinated with bivalent vaccine and Re-H5N1 vaccine treated with neem leaf extract, while decreased in the group vaccinated with H5N3 with neem leaf extract and Re-H5N1 without neem leaf extract compared with H5N3, H5N1, and monovalent vaccine. The immunological response after vaccination was stronger in the bivalent vaccine group than in the other commercial vaccine groups treated with neem leaf extract, with geometric mean titer (GMTs) of 315.2 and 207.9 at the third and fourth weeks, respectively. The use of immunostimulant antiviral medicinal plants, such as neem, completely protected chicken flocks against HPAI (H5N8) and prevented AI virus shedding, leading us to the conclusion that the use of bivalent vaccines induces a higher immune response than other different commercial vaccines.

Key words: aqueous neem leaf extract, avian influenza virus H5N8, bivalent vaccine, broiler chicken, Clade 2.3.4.4

INTRODUCTION

Egypt's national economy is significantly impacted by the poultry business, which also ranks as one of the country's most important economic sectors (Attia et al., 2022). Investing in Egypt's poultry industry can yield substantial returns due to the country's sizable domestic market for the consumption of poultry meat, which also has great development potential on account of the country's growing population and increasing per capita consumption, which in turn is being driven by the country's improving economic situation (Hassouneh et al., 2012; ElMasry et al. 2017; Attia et al., 2022). In Egypt, the kinds of animal protein that are more easily accessible and have prices that are more affordable include chicken and egg products (Kandeil et al., 2018; Rohaim et al., 2021; Setta et al., 2023). Chickens are the most common type of commercially important poultry in Egypt (Kandeil et al., 2018; Swelum et al., 2021; Abd El-Hack et al., 2022; El-Saadony et al., 2022). Farming chickens and other fowl is common in Egypt, both commercial and backyard (Rohaim et al., 2021).

Avian influenza (AI) is a contagious viral disease that spreads around the world and is caused by viruses from the Orthomyxoviridae family known as influenza A virus (Yehia et al., 2018). Only influenza A viruses are known to infect birds (Adere and Mukaria, 2023). Infections in birds can result in a wide variety of clinical symptoms, depending on the host species, virus strain, host immunological condition, presence of any secondary aggravating pathogens, and environmental factors (Adere and Mukaria, 2023). AI is divided into 16 different hemagglutinin (HA) subtypes and 9 different neuraminidase (NA) subtypes according to the antigenic characteristics of the surface glycoproteins HA and neuraminidase NA (Yehia et al., 2018).

During avian flu pandemics, flocks of chickens had substantial financial losses as a result of increased mortality, decreased output, greater condemnations, and costs associated with quarantine and eradication efforts (Tarek et al., 2021).

The highly pathogenic avian influenza (HPAI) H5N8 subtype was first described in China in 2010 as a result of a unique reassortant of HA and NA from various AI subtypes (Zhao et al., 2013), and was later assigned to clade 2.3.4.4 (Smith and Donis 2015). The new reassortant of the HPAI H5N8 virus, clade 2.3.4.4a, was discovered in wild and domestic birds in South Korea in January 2014 (Lee et al., 2014). Since then, it has spread to various countries in Asia, and even as far as Europe and North America (Lee et al., 2015; Saito et al., 2015).

The most widespread HPAI virus epidemic in the last decade began in Russia in 2016/2017 with the discovery of a novel reassortant HPAI H5N8 virus, clade 2.3.4.4b, and quickly spread to many countries in Europe, Asia, and Africa via migrating birds (Fusaro et al., 2019; Lycett et al., 2020). Since 2016/2017, HPAI H5N8 viruses of clade 2.3.4.4b have been evolving through reassortment with other influenza A subtypes, resulting in a variety of genotypes (Lycett et al., 2020).

Sobolev et al. (2021) classified HPAI H5N8 viruses of clade 2.3.4.4 into 2 categories (A and B). From this year until the present, the HPAI H5N8 has undergone considerable changes in virulence, antiviral treatment resistance, and immune system tolerance. These developments have occurred at a quick rate (Yehia et al., 2020; Salaheldin et al., 2022; Kandeil et al., 2023; Setta et al., 2023).

Birds infected with HPAI H5N8 viruses had clinical indications of inappetence, weakness, and ruffled feathers. They clustered together with respiratory symptoms such as coughing, sneezing, rales, nasal and ocular secretions, facial edema, and greenish diarrhea. In addition to the symptoms of septicemia, which include comb cyanosis and wattle with hemorrhages on the subcutaneous tissues of the shank, as well as neurological signs such as tremors and head tilting, the infected bird may also exhibit signs of sepsis (Setta et al., 2023).

For the purpose of preventing and controlling AI pandemics, stringent biosafety standards are required in the poultry industry (Ijoma et al., 2020). Vaccine research and development is undergoing significant change. Thus, immunization may be able to prevent the terrible morbidity and death associated with HPAI in hens (Dey et al., 2023).

In Egypt, the antiviral activity of 4% neem (Azadirachta indicia) was examined against highly pathogenic AI (HPAI) in ducks (Hegazy et al., 2013), while the antiviral activity of 8% A. indicia was evaluated against chicken infectious anemia (CIA) (Hegazy et al., 2023). Both treatments offered 100% protection, and neither clinical symptoms nor death were noted.

The chemicals in neem have the potential to inhibit the growth of microorganisms, function as antioxidants, and control the activity of genes and proteins. Mahmood et al. (2014) reported that neem can increase resistance to the virus that causes Newcastle disease.

An 8% aqueous neem leaf extract shows antiviral efficacy against CIAV and AI, according to Hegazy et al. (2013, 2023). They also claimed to have isolated the first pure polyphenolic flavonoids quercetin, and ß-sitosterol from fresh neem leaf (Hegazy et al., 2023). Hegazy et al. (2018) also discovered that neem leaf extract has an antibacterial agent against Chlamydia psittaci. Spices and the oils derived from such spices have been used to treat a wide variety of infectious microorganisms (Naveed et al., 2013).

In the current study, we compared the efficacy of a bivalent vaccine (AI + ND), a monovalent AI vaccine, and a monovalent ND vaccine against AI virus (H5N8) (A/chicken-Cobb/Egypt/55/2019), with and without the addition of an immuno-stimulant  made from the leaves of the medicinal plant neem, at preventing infection and shedding of AI virus following challenge.

MATERIALS AND METHODS

Experimental Birds

One hundred thirty broiler chicks (1-day-old) were purchased from the EL-Dakahlia Poultry Company in Egypt and maintained in a floor-based system at the Faculty of Veterinary Medicine, Zagazig University, for use in the evaluation of multiple commercial vaccinations against HPAI H5N8 isolates. The birds were fed a meal containing 21% protein and yielding 3100 Kcal kg−1 from El-Eman Feed Millers in Egypt. The birds were housed in isolation cabinets with negative pressure, high efficiency, and air filtration in a biosafety level 3 facility. The Animal Welfare and Research Ethics Committee at Zagazig University, Faculty of Veterinary Medicine, Zagazig, Egypt, approved the study with approval number ZU-IACUC2F432022.

Preparation of 8% Aqueous Neem Leaf Extract

The leaves were picked from mature green trees in the agricultural orchards of Faculty of Agriculture, Zagazig University, Egypt. The leaves were dried in an oven at 37°C for 24 h before being pulverized in a metallic grinder. To make an 8% neem leaf extract, 80 g of powder was steeped for 5 to 8 h at room temperature in a nonmetallic jar with 1 L of hot-boiled distilled water as described by Leila (1977), and Hegazy et al. (2018). The aqueous neem leaf extract was administrated 2 d before and after vaccination with a dose of 50 mL liter−1 (Durrani et al., 2008).

Vaccines

Avian Influenza Vaccines

In order to carry out an evaluation, the most popular vaccines currently offered for sale on the market were purchased from Egyptian poultry vaccine wholesalers. The vaccines were typically based on reverse genetically created recombinant AI/H5 viruses with commercially oil-adjuvant inactivated Clades1, 2.2.1.2, or 2.3.4.

The first vaccine was Merial, China, with batch number 02206001, with Reassortant AI virus, inactivated (H5N1 subtype) (strain Re-5). This vaccine contains Chinese strain A/ Duck / Anhui / 1/ 2006, clade 2.3.4. The Re-5 vaccination was given a subcutaneous injection in the third center of the back of the neck at a volume of 0.5 mL for each bird.

The second vaccine was ME-VAC, Egypt, with batch number 2108110101, with ME Flu VAC, inactivated (H5N1 subtype). This strain contains Egyptian strain A/duck/ Egypt /M2583D /2010, Clade 2.2.1.2. The ME Flu VAC vaccine was injected subcutaneously at 0.5 mL per bird in the third middle of the neck.

The third vaccine was Poulvac FluFend AI, inactivated (H5N3 subtype), made in the USA by Zoetis with batch number 40020574. This vaccine comprises H5-HA of clade 1 HPAI H5N3 virus (A/chicken/Vietnam/C58/2004). The Poulvac vaccine was injected at 0.5 mL per bird subcutaneously in the third middle of the back of the neck.

The fourth vaccine was ValleyVac H5 plus-NDG7 manufactured by VACCINE VALLEY, 6th of October City, Giza, Egypt, inactivated Reassortant Avian influenza virus, H5N1 strain A\chicken\Egypt\ D10552B\ 2015) (H5N1), and inactivated Reassortant Avian influenza virus, H5N8 strain A\green-winged teal\Egypt\877\2016 (H5N8) and inactivated Newcastle Disease virus, Genotype VII, strain NDV-B7-RLQP-CH-EG-12, emulsified in oil adjuvant.

The fifth vaccine was ValleyVac Avian Flu H5 plus, manufactured by VACCINE VALLEY, inactivated Reassortant Avian influenza virus, H5N1 strain A\chicken\Egypt\ D10552B\ 2015) (H5N1), and inactivated Reassortant Avian influenza virus, H5N8 strain A\green-winged teal\Egypt\877\2016 (H5N8), emulsified in oil adjuvant.

The sixth vaccine was ValleyVac NDG7, manufactured by VACCINE VALLEY inactivated Newcastle Disease virus, Genotype VII, strain NDV-B7-RLQP-CH-EG-12, emulsified in oil adjuvant.

Challenge Virus

The challenge HPAI H5N8 virus A/chicken-Cobb/Egypt/55/2019 (Clade 2.3.4.4) with accession number ON724339 was obtained from Avian and Rabbit Medicine Department, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt. The virus was titrated on specific pathogen-free (SPF) embryonated chicken eggs (ECE) and used in laboratory challenge experiments. The titer of the HPAI virus was 106 EID50 (Kandeil et al., 2023).

Experimental Design

One hundred thirty birds were randomly divided into 7 equal groups of 20 birds/group with 10 birds in the seventh group (Table 1). All groups were vaccinated subcutaneously in the dorsal anterior of the neck with 0.5 mL per bird at 5 d of age and challenged intraocular with 0.1 mL of 107 EID50 with HPAI (H5N8 subtype) A/chicken-Cobb/Egypt/55/2019 (Clade 2.3.4.4) with accession number ON724339 at 3 wk postvaccination. G7 remained nonvaccinated and challenged as a control group. Serum samples were collected weekly from all birds for serological assessment. Tracheal swabs were collected at 2-, 4-, and 7-d postchallenge to record titers of viral shedding from 3 birds/subgroups. The chickens were observed twice daily for clinical signs, postmortem lesions, mortality, and morbidity for 10 d following the challenge.

Table 1.

Experimental design for evaluation of bivalent and monovalent vaccines against avian influenza virus (H5N8) (A/chicken-Cobb/Egypt/55/2019).

Groups Subgroups No of birds Vaccination Challenge
G1 A 10 Re-H5N1 Avian influenza virus (H5N8) (A/chicken-Cobb/Egypt/55/2019)
B 10 Re-H5N1 + neem 8%
G2 A 10 H5N1
B 10 H5N1 + neem 8%
G3 A 10 H5N3
B 10 H5N3 + neem 8%
G4 A 10 Bivalent vaccine only
B 10 Bivalent vaccine + neem 8%
G5 A 10 Monovalent AI
B 10 Monovalent AI + neem 8%
G6 A 10 Monovalent ND
B 10 Monovalent ND + neem 8%
G7 Control positive 10 Non vaccinated
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(G1a, G2a, G3a, G4a, G5a, G6a) received Re-H5N1 vaccine, H5N1 vaccine, H5N3 vaccine, the bivalent vaccine (AI + ND), monovalent vaccine AI H5, monovalent vaccine NDG7, while subgroups (G1b, G2b, G3b, G4b, G5b, G6b) were received the same previous vaccination and treated with 8% neem leaf extract received orally from 3 to 7 d of age. G7 was kept unvaccinated as positive control group.

Serology

Hemagglutination inhibition (HI) assay was performed using 4 HAU (Hemagglutination unit) of HPAIV (H5N8) strain (A/chicken-Cobb/Egypt/55/2019) using 1% chicken RBCs (red blood cells) as previously described by the World Organization for Animal Health (OIE) (2018). The serum samples were regularly collected from each subgroup weekly and HI titer was calculated according to the formula geometric mean titer (GMT) = antilog (average endpoint the tube number-1) X (log of the factor) + (log of reciprocal of first dilution) (Williams et al., 2016).

Avian Influenza Shedding

At 2, 4, and 7 d after a challenge, tracheal specimens were taken in 1 mL sterile antibiotic containing phosphate buffer saline (PBS) (penicillin and streptomycin) and centrifuged at 2,000 rpm for 10 min at 4°C. Virus titration was performed using quantitative real time polymerase chain reaction (qRT-PCR) on the supernatants (Löndt et al., 2008). RNA was extracted with the QIAamp Viral RNA Mini Kit (QIAGEN, Benelux B.V., Hulsterweg 82, Leiden, Venlo, Netherlands). A Qiagen one-step qRT-PCR kit (Qiagen, Netherlands) with sequence-specific primers and detectors (Metabion, GmbH, Steinkirchen, Germany) targeting H5 influenza viruses was utilized (Table 2). The reactions were performed using the ABI 7500 Real-Time PCR system (Applied Biosystems, Carlsbad, CA). A standard curve was established for viral quantification with RNA extracted from the titrated challenge, HPAI types H5N8 virus. Results were reported as EID50 mL equivalents−1.

Table 2.

Primers and probes of the H5 virus.

Virus Gene Primer/ probe sequence 5′-3′ References
AI H5 H5LH1
ACATATGACTAC CCACARTATTCA G		 (Löndt et al., 2008)
H5RH1
AGACCAGCT AYC ATGATTGC
H5PRO
[FAM]TCWACA GTGGCGAGT TCCCTAGCA[TAMRA]
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Statistical Analysis

The data were analyzed using SPSS version 25 (Armonk, IBM Corp., NY) and Graph Pad Prism 8.0.2 (GraphPad Software, Inc., Boston, MA). Data were described as mean ± SE. Two-way analysis of variance (ANOVA) was applied to test the differences in HI titer among groups on the experiment's first, second, third, and fourth weeks. The level of statistical significance was set at P < 0.05.

RESULTS

Clinical Findings

Survival curves of nonimmunized and immunized fowl after challenge using tested vaccines are shown in Figure 1. Specific signs of HPAI infection in the control positive group started as early as third day postchallenge (Figure 2, Figure 3 and Table 3). Birds suffered from conjunctivitis, closed eyes, cyanosis in comb and wattle, ocular discharge, and nervous signs in the form of torticollis and opisthotonus position appeared lately at 7 d postchallenge with greenish diarrhea. Mortality was 100% which started 24 h postinfection and reached 90% up to 10 d postinfection with a survival rate of 10%.

Figure 1.

Figure 1

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Survival curves of nonimmunized and immunized poultry with tested vaccines after challenge.

Figure 2.

Figure 2

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Clinical findings of the eye in experimentally inoculated chickens with H5N8. (A) severe conjunctivitis and completely closed eye in the positive control group and monovalent ND vaccine only or with 8% neem leaf extract; (B) less severe conjunctivitis in H5N3 vaccinated only group; (C) moderate conjunctivitis in H5N3 group with with 8% neem leaf extract; (D) mild conjunctivitis in bivalent and Re-H5N1 vaccine only; (E) nearly normal eye in bivalent and Re-H5N1 vaccine with 8% neem leaf extract; (F) severe conjunctivitis and partially closed eye in monovalent AI vaccine only or with 8% neem leaf extract; (G) sever conjunctivitis but not completely closed eye H5N1 vaccinated with or without 8% neem leaf extract group. Lesion score range (mild-moderate-sever) according to Landmann et al. (2021).

Figure 3.

Figure 3

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Correlation heat-map between groups representing the type of vaccination (Re-H5N1, H5N1, H5N3, bivalent vaccine, monovalent AI, monovalent ND, and treated with 8% neem leaf extract) and the clinical score. The degree of relationship is assessed based on the color scale on the right. The closer the color is to +1, the higher the correlation is, and the closer to 0, the lower the correlation. The heat map indicated a weak relationship between clinical signs and Re-H5N1; bivalent vaccine and treated with 8% neem leaf extract; the relationship of all clinical signs and H5N3; monovalent AI vaccine treated with 8% neem leaf extract was moderate. While the challenged group, monovalent ND vaccine and vaccines without 8% neem leaf extract showed a higher correlation.

Table 3.

Clinical observation of differentially avian influenza virus–vaccinated groups 10 d postchallenge.

Groups Subgroups Conjunctivitis Closed eye Cyanosis in comb and wattle Ocular discharge Greenish diarrhea Nervous sign Mortality %
Group 1 a 2\10 3\10 1\10 1\10 3\10 0\10 40%
b 2\10 2\10 2\10 1\10 4\10 0\10 20%
Group 2 a 7\10 7\10 8\10 8\10 10\10 2\10 80%
b 6\10 7\10 8\10 7\10 10\10 3\10 60%
Group 3 a 5\10 5\10 8\10 8\10 10\10 2\10 60%
b 6\10 5\10 4\10 4\10 6\10 1\10 40%
Group 4 a 1\10 2\10 2\10 1\10 1\10 1\10 30%
b 0\10 0\10 1\10 0\10 0\10 0\10 10%
Group 5 a 1\10 2\10 2\10 1\10 1\10 0\10 60%
b 2\10 2\10 1\10 3\10 2\10 1\10 40%
Group 6 a 4\10 2\10 2\10 6\10 4\10 4\10 60%
b 2\10 3\10 2\10 4\10 3\10 5\10 40%
Group 7 Positive control 7\10 7\10 8\10 8\10 10\10 4\10 100%
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(G1a, G2a, G3a, G4a, G5a, G6a) received Re-H5N1 vaccine, H5N1 vaccine, H5N3 vaccine, the bivalent vaccine (AI + ND), monovalent vaccine AI H5, monovalent vaccine NDG, while subgroups (G1b, G2b, G3b, G4b, G5b, G6b) received the same previous vaccination and treated with 8% neem leaf extract received orally from 3 to 7 d of age. G7 was kept unvaccinated as positive control group.

Birds immunized with Re-H5N1 and bivalent (AI + ND) vaccines treated with 8% neem leaf extract (G1b & G4b) showed clinical signs nearly normal with a protection rate of 80% compared with Re-H5N1 and bivalent (AI + ND) vaccine only (G1a & G4a) which suffered from mild clinical manifestation with a protection rate of 60%.

In comparison, clinical manifestation in G2b was less severe than in G2a, with a 40% and 20% protection rate, respectively. However, G3b showed moderate clinical signs than G3a with a protection rate of 60% and 40%, respectively (Figure 1).

Interestingly, oral inclusion of 8% neem leaf extract in birds' drinking water improves the protection from mortality and HPAI-specific clinical signs in case of vaccination with bivalent vaccine (G4b), Re-H5N1(G1b), and H5N3 (G3b). In contrast, the lowest protection was reported from clinical signs, and mortality existed in birds receiving H5N1 (G2a).

Postmortem Findings

The gross lesions of the experimentally challenged broiler chickens in the control group showed congested trachea and lung, pericarditis, perihepatitis, hemorrhage on the shank, edema and gelatinous fluid submandibular, congestion of all organs (septicemia), nephritis, hemorrhage on proventriculus, hemorrhage on the pancreas and mottled spleen. The lesions were severe in the control group challenged with HPAI H5N8 and H5N1 vaccinated and treated groups; moderate in H5N3 vaccinated and treated while low as in neem treated with bivalent vaccine and Re-H5N1 (Table 4).

Table 4.

Postmortem findings of differentially avian influenza virus–vaccinated groups 10 d postchallenge.

Groups Subgroups Hemorrhage on shank Edema and gelatinous fluid submandibular Congestion of all organs (septicemia) Nephritis Hemorrhage on proventriculus Hemorrhage on pancreas Mottled spleen
G1 a 1\10 0\10 1\10 0\10 0\10 1\10 1\10
b 1\10 0\10 0\10 0\10 1\10 1\10 1\10
G2 a 1\10 2\10 2\10 1\10 3\10 4\10 5\10
b 2\10 3\10 2\10 2\10 5\10 3\10 1\10
G3 a 1\10 2\10 2\10 1\10 1\10 1\10 1\10
b 2\10 2\10 4\10 3\10 2\10 6\10 4\10
G4 a 1\10 2\10 2\10 1\10 0\10 1\10 1\10
b 0\10 0\10 1\10 0\10 0\10 0\10 0\10
G5 a 1\10 2\10 2\10 1\10 1\10 1\10 1\10
b 2\10 2\10 4\10 3\10 2\10 6\10 4\10
G6 a 4\10 2\10 2\10 6\10 4\10 2\10 3\10
b 2\10 3\10 2\10 2\10 3\10 5\10 5\10
G7 Positive control 10\10 8\10 10\10 5\10 8\10 7\10 8\10
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(G1a, G2a, G3a, G4a, G5a, G6a) received Re-H5N1 vaccine, H5N1 vaccine, H5N3 vaccine, the bivalent vaccine (AI + ND), monovalent vaccine AI H5, monovalent vaccine NDG7, while subgroups (G1b, G2b, G3b, G4b, G5b, G6b) received the same previous vaccination and treated with 8% neem leaf extract received orally from 3 to 7 d of age. G7 was kept unvaccinated as positive control group.

Immune Responses Postvaccination

To evaluate the humoral immune responses elicited from different commercial AI vaccines with different clades of origin, different groups of broiler chickens were vaccinated once at 5-day-olds with AI vaccines. The AI-HI-Ab responses were measured weekly up to the fourth week of age. The HI- Abs were assessed against H5N8 (clade 2.3.4) as antigens (Table 5 and Figure 4).

Table 5.

Evaluation of specific AI-HI-Ab responses postvaccination with different commercial avian influenza vaccines against influenza virus (H5N8) (A/chicken-Cobb/Egypt/55/2019).

Groups Subgroups First week Second week Third week Fourth week
G1 a 9.2 ± 0.25hi 21.1 ± 0.26defgh 147 ± 0.20ab 111.4 ± 0.22abc
b 12.1 ± 0.16gh 42.2 ± 0.27cde 207.9 ± 0.15a 157.6 ± 0.21ab
G2 a 10.6 ± 0.22h 9.8 ± 0.15h 64 ± 0.36bcd 34.3 ± 0.43defg
b 12.1 ± 0.16gh 19.7 ± 0.22efgh 39.4 ± 0.26cdef 35.3 ± 0.26defgh
G3 a 11.3 ± 0.22gh 13 ± 0.26fgh 21.1 ± 0.16defgh 45.3 ± 0.22cde
b 9.8 ± 0.26h 19.7 ± 0.15efgh 12.1 ± 0.26gh 55.3 ± 0.45h
G4 a 12.1 ± 0.25d 24.3 ± 0.26cd 157.6 ± 0.20bc 45.3 ± 0.25ab
b 9.8 ± 0.16d 48.5 ± 0.27bc 315.2 ± 0.0.27ab 157.6 ± 0.13a
G5 A 12.1 ± 0.22d 24.3 ± 0.15d 39.4 ± 0.15d 19.7 ± 0.21d
B 16 ± 0.16d 39.4 ± 0.22cd 194 ± 0.16cd 24.3 ± 0.16d
G6 A 12.1 ± 0.22d 39.4 ± 0.26d 12.1 ± 0.16cd 16 ± 0.166bc
B 9.8 ± 0.26d 64 ± 0.15cd 39.4 ± 0.16bc 32 ± 0.20ab
G7 Positive control 12.1 ± 0.25gh 3 ± 0.25ij 0 ± 0.25j 0 ± 0j
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(G1a, G2a, G3a, G4a, G5a, G6a) received Re-H5N1 vaccine, H5N1 vaccine, H5N3 vaccine, the bivalent vaccine (AI + ND), monovalent vaccine AI H5, monovalent vaccine NDG7, while subgroups (G1b, G2b, G3b, G4b, G5b, G6b) received the same previous vaccination and treated with 8% neem leaf extract received orally from 3 to 7 d of age. G7 was kept unvaccinated as positive control group.

a,b,c,d,e,f,h,i

According to Tukey's test means with different superscripts are statistically different at P<0.05.

Figure 4.

Figure 4

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Arithmetic mean HI titer results prechallenge serological response of broiler chickens after vaccination with different types of vaccines. Serum samples were collected weekly and tested against H5N8 to measure the level of H5N8 antibodies. Results are expressed as geometric mean titer (GMT ± SE) in vaccinated and nonvaccinated birds. Single AI mean monovalent AI vaccine; single ND mean monovalent ND vaccine.

The increases in the AI-HI-Ab GMT in G1a (111.4 ± 0.22) were statistically significant (P˂ 0.05) when compared with birds vaccinated with H5N1 (clade 2.2.1.2) (G2a) at fourth week of age only, which had GMT (34.3 ± 0.43). Furthermore, the increases in the AI-HI-Ab titers in (G1a) were statistically significant (P˂ 0.05) when compared with birds vaccinated with H5N3 (clade 1) at the third week of age only with more than 2 log differences.

In the case of neem 8% oral treatment, except in the case of vaccination with H5N1 (G2b), there were numerical increases in the AI-HI-Ab responses in all the treated and vaccinated groups at second, third and fourth week of age when compared with their respective vaccinated only groups (Table 5 and Figure 4).

In G4b, which was immunized with bivalent vaccines (AI + ND) with 8% neem leaf extract, showed high GMT, which reached 315.2 ± 0.27 and 157.6 ± 0.13, respectively, at the third and fourth week that was much more than in group G4a, which immunized with bivalent vaccinations (AI+ND) only was 157.6 ± 0.20 and 45.3 ± 0.25, respectively. Our results indicated that bivalent vaccines (AI+ND) treated with 8% aqueous neem leaf extract (G4b) showed GMT higher than the monovalent vaccine with aqueous neem leaf extract of 8% (G5b) (Table 5 and Figure 4).

Detection of AIV Shedding During HPAI H5N8 Virus Challenge

The virus shedding was prevented in groups vaccinated with the bivalent vaccine (G4b) and Re-H5N1 vaccine (G1b) treated with 8% neem leaf extract, while decreased in the group vaccinated with H5N3 with 8% neem leaf extract and Re-H5N1 without 8% neem leaf extract compared with H5N3 and H5N1. The virus shedding levels from the trachea of some different commercial AI vaccines with or without 8% neem leaf extract at 2-, 4- & 7-d postchallenge with AI virus (H5N8) (A/chicken-Cobb/Egypt/55/2019) were estimated by qRT-PCR (Figure 5, Figure 6, Figure 7).

Figure 5.

Figure 5

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Titration of viral shedding from collected samples after the challenge of nonimmunized and immunized chickens with the Egyptian H5N8 isolate at 2, 4, and 7 d postinfection (dpi). Three tracheal swabs from 3 chickens from each subgroup at 2-, 4-, and 7-d postinfection were estimated by quantitative real-time (qRT-PCR). Virus shedding was monitored by log10 EID50 mL−1 titration for each collected sample from live birds. Each dot represents the viral titer of each chicken. The detection limit was <1 log10 EID50 0.1 mL−1.

Figure 6.

Figure 6

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Titration of viral shedding from collected samples after the challenge of nonimmunized and immunized chickens (Re-H5N1, H5N1, and H5N3 with or without 8% neem leaf extract) against the Egyptian H5N8 isolate (A/chicken-Cobb/Egypt/55/2019) at 2, 4, and 7 d postinfection (dpi). Three tracheal swabs from 3 chickens from each subgroup were collected; Re-H5N1 (sample 1-3); H5N1 (sample 4–6); H5N3 (sample 7–9); Re-H5N1+ neem 8% (sample 10–12); H5N1+ 8% neem leaf extract (sample 13–15); H5N3+ 8% neem leaf extract (16–18) and positive control (sample 19). Virus shedding was monitored by log10 EID50 mL−1 titration for each collected sample from live birds. The standard curve represents the viral titer of each chicken. The detection limit was <1 log10 EID50 0.1 mL−1.

Figure 7.

Figure 7

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Titration of viral shedding from collected samples after the challenge of nonimmunized and immunized chickens (bivalent vaccine AI + ND and monovalent AI vaccine with or without 8% neem leaf extract) against the Egyptian H5N8 isolate (A/chicken-Cobb/Egypt/55/2019) at 2, 4, and 7 d postinfection (dpi). Three tracheal swabs from 3 chickens from each subgroup were collected; bivalent vaccine (TH1–TH3); monovalent AI vaccine (TH4–TH6); bivalent vaccine + 8% neem leaf extract (TH7–TH9); monovalent AI vaccine + 8% neem leaf extract (TH10–TH12) and positive control (TH13). Virus shedding was monitored by log10 EID50 mL−1 titration for each collected sample from live birds. The standard curve represents the viral titer of each chicken. The detection limit was <1 log10 EID50 0.1 mL−1.

DISCUSSION

After the global poultry industry was devastated by the HPAIV H5N8 clade 2.3.4.4 pandemic, the H5N8 vaccine was successfully introduced in many countries to help mitigate the industry's massive financial losses (Antigua et al., 2019; Zeng et al., 2020; Cui et al., 2022).

Poultry should be immunized against HPAIV in order to reduce the risk of illness, mortality, viral excretion, and transmission from one bird to another. Poor vaccination or mismatched vaccines, on the other hand, may speed the evolution of a virus and result in its evasion of vaccine-induced antibodies (Swayne et al., 2014). According to Salaheldin et al. (2022), Egypt's routine AIV vaccine updates proved to be highly effective in reducing the risk that the virus posed to the general population's health and in preventing losses to the chicken business.

In broiler chickens, comparative research was conducted comparing 6 different commercially inactivated AI vaccinations against H5N8 with or without 8% neem leaf extract. One hundred thirty broiler chicks were separated into 7 groups: positive control, 6 vaccination groups (Re-H5N1, H5N1, H5N3, bivalent AI+ND, monovalent AI, and monovalent ND) with or without 8% neem leaf extract. Clinical signs of the challenged group showed conjunctivitis, closed eyes, cyanosis in comb and wattle, ocular discharge, and greenish diarrhea while postmortem lesions showed congested trachea and lung, hemorrhage on the shank, proventriculus, and pancreas; gelatinous fluid submandibular, congestion of all organs (septicemia), and mottled spleen. Previously, the same clinicopathological finding were reported by Swayne et al. (2020) and Djurdjević et al. (2023).

The clinical signs and lesions were low in bivalent vaccine and Re-H5N1 treated with 8% neem leaf extract compared with other vaccines. Nervous signs in the form of torticollis and opisthotonus position were recorded in our study lately at 7-d postchallenge in all groups except G1 and G4. Our results were in accordance with Ramzy (2016) and Setta et al. (2023).

The protection levels in the bivalent vaccine, inactivated strain Re-5 H5N1 and H5N3 treated with 8% neem leaf extract were 80%, 80%, and 60%, respectively, because neem is high in several components, particularly triterpenoids and glycosides, which are thought to be responsible for the herb's antiviral potency, as suggested by Hegazy et al. (2023).

On the other hand, the bivalent vaccine that was inactivated strain Re-5 H5N1 and H5N3 had a 60%, 60%, and 40% success rate without treatment respectively. Similar observations were made by Kandeil et al. (2018), who conducted an investigation on the effectiveness of 8 marketed inactivated AI vaccines based on multiple strains of the HPAI H5N1 virus against the HPAIV H8N8 virus. In addition, Kapczynski et al. (2017) utilized inactivated vaccinations or rHVT-H5 in hens, however they were unable to generate an appropriate level of protection against H5N8 clade 2.3.4.4 HPAI virus challenges.

The immunological response to immunization in challenged birds was measured using the mean HI titer, which was evaluated by serological testing. The immune response postvaccination was higher in the group immunized with bivalent vaccine than in the group immunized with monovalent commercial vaccine treated with aqueous 8% neem leaf extract, which was 315.2 and 207.9 at the third and fourth weeks, respectively, because neem is rich in several components, particularly triterpenoids and glycosides, which were thought to have immunostimulant properties (Asghar et al. 2022; Hegazy et al., 2023).

When compared to the other vaccinated groups, birds immunized with an inactivated vaccine based solely on the Re-H5N1 strain (G1a) showed the strongest AI-HI-Ab responses at the third and fourth week of age. The GMT (147 ± 0.20, and 111.4 ± 0.22) against H5N8 agrees with the findings of Khelfa et al. (2016) who demonstrated that inactivated reassorted H5N1 AI is immunogenic and more protective than H5N3 vaccines than H5N1 vaccine.

The virus shedding levels from the trachea of some different commercial AI vaccines with or without 8% neem leaf extract at 2-, 4- & 7-d postchallenge with AI virus (H5N8) (A/chicken-Cobb/Egypt/55/2019) were estimated by qRT-PCR. The virus shedding was prevented in groups vaccinated with bivalent vaccine and Re-H5N1 vaccine treated with 8% neem leaf extract while decreased in the group vaccinated with H5N3 with 8% neem leaf extract and Re-H5N1 without 8% neem leaf extract compared with H5N3 and H5N1. These results were in agreement with Kandeil et al. (2018) and Khelfa et al. (2016) who stated that the Re-H5N1 commercial vaccine was able to completely protect chickens and significantly reduce virus shedding in comparison with other vaccines.

It is also worth noting that, according to Wu et al. (2023), the use of inactivated vaccines and low antigenically vaccines have a short time of antibody production, whereas the use of neem, which contains active constituents that act as antiviral and immunostimulant, prevents virus shedding as explained by Hegazy et al. (2023).

CONCLUSIONS

Vaccination programs must include bivalent vaccines, which induce a greater immune response than monovalent vaccines, as well as immunostimulant and antiviral medicinal plants such as A. indica (8% neem leaf extract) to completely protect chicken flocks against HPAI (H5N8) and prevent AI virus shedding. The introduction of bivalent vaccinations against the field strain reduced mortality, but it did not diminish losses from AIV-caused clinical signs and transmission among poultry flocks. A strict control strategy based on the use of immunostimulant and antiviral medicinal plants (neem leaf extract 8%) might be beneficial in preventing the transmission and circulation of AI virus (H5N8) (A/chicken-Cobb/Egypt/55/2019) strain among chicken flocks.

Acknowledgments

ACKNOWLEDGMENTS

The authors extend their appreciation to the Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2023R224), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia. The authors extend their appreciation to the Deanship of Scientific Research at King Khalid University for supporting this work under the grant number (R.G.P2 161-44). This project was funded by the Abu Dhabi Award for Research Excellence, Department of Education, and knowledge (Grant number: 21S105) to K. A. El-Tarabily.

Author Contributions: Conceptualization, A.M.H., O.H., M.A.M. H., K.A.E.-T., and H.M.N.T., formal Analysis, A.M.H., O.H., M.A.M. H., K.A.E.-T., and H.M.N.T., investigation, A.M.H., O.H., M.A.M. H., M.A.M., F.A.A.-S., A.O.S., K.A.E.-T., M.T.E.-S., and H.M.N.T., data curation, A.M.H., O.H., M.A.M. H., and H.M.N.T., writing original draft preparation A.M.H., O.H., M.A.M. H., and H.M.N.T., writing final manuscript and editing, A.M.H., O.H., M.A.M. H., M.A.M., F.A.A.-S., A.O.S., K.A.E.-T., M.T.E.-S., and H.M.N.T., visualization and Methodology, A.M.H., O.H., M.A.M. H., M.A.M., F.A.A.-S., A.O.S., K.A.E.-T., M.T.E.-S., and H.M.N.T., All authors have read and agreed to the published version of the manuscript.

DISCLOSURES

The authors declare no conflict of interest in the current work.

REFERENCES

  1. Abd El-Hack M.E., El-Saadony M.T., Elbestawy A.R., Nahed A., Saad A.M., Salem H.M., El-Tarabily K.A. Necrotic enteritis in broiler chickens: disease characteristics and prevention using organic antibiotic alternatives–a comprehensive review. Poult. Sci. 2022;101 doi: 10.1016/j.psj.2021.101590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Adere T., Mukaria M. The epidemiology of avian influenza and its significance for public health. JHTD. 2023;3:48–58. [Google Scholar]
  3. Antigua K.J.C., Choi W.S., Baek Y.H., Song M.S. The emergence and decennary distribution of clade 2.3.4.4 HPAI H5Nx. Microorganisms. 2019;7:156. doi: 10.3390/microorganisms7060156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Asghar H.A., Abbas S.Q., Arshad M.K., Jabin A., Usman B., Aslam M., Asghar A. Therapeutic potential of Azadirachta indica (Neem)-a comprehensive review. Sch. Int. J. Tradit. Complement. Med. 2022;5:47–64. [Google Scholar]
  5. Attia Y.A., Rahman M.T., Hossain M.J., Basiouni S., Khafaga A.F., Shehata A.A., Hafez H.M. Poultry production and sustainability in developing countries under the COVID-19 crisis: lessons learned. Animals. 2022;12:644. doi: 10.3390/ani12050644. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cui P., Zeng X., Li X., Li Y., Shi J., Zhao C., Qu Z., Wang Y., Guo J., Gu W., Ma Q. Genetic and biological characteristics of the globally circulating H5N8 avian influenza viruses and the protective efficacy offered by the poultry vaccine currently used in China. Sci. China Life Sci. 2022;65:795–808. doi: 10.1007/s11427-021-2025-y. [DOI] [PubMed] [Google Scholar]
  7. Dey P., Ahuja A., Panwar J., Choudhary P., Rani S., Kaur M., Sharma A., Kaur J., Yadav A.K., Sood V., Suresh Babu A.R., Bhadada S.K., Singh G., Barnwal R.P. Immune control of avian influenza virus infection and its vaccine development. Vaccines. 2023;11:593. doi: 10.3390/vaccines11030593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Djurdjević B., Polaček V., Pajić M., Petrović T., Vučićević I., Vidanović D., Aleksić-Kovačević S. Highly pathogenic avian influenza H5N8 outbreak in backyard chickens in Serbia. Animals. 2023;13:700. doi: 10.3390/ani13040700. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Durrani F.R., Sultan A., Jan M., Chand N., Durrani Z. Immunomodulatory and growth promoting effects of Neem (Azadirachta indica) leaf infusion in broiler chicks. Sarhad J. Agric. 2008;24:655–659. [Google Scholar]
  10. ElMasry I., Elshiekh H., Abdlenabi A., Saad A., Arafa A., Fasina F.O., Lubroth J., Jobre Y.M. Avian influenza H5N1 surveillance and its dynamics in poultry in live bird markets, Egypt. Transbound. Emerg. Dis. 2017;64:805–814. doi: 10.1111/tbed.12440. [DOI] [PubMed] [Google Scholar]
  11. El-Saadony M.T., Salem H.M., El-Tahan A.M., Abd El-Mageed T.A., Soliman S.M., Khafaga A.F., Abd El-Hack M.E. The control of poultry salmonellosis using organic agents: an updated overview. Poult. Sci. 2022;101 doi: 10.1016/j.psj.2022.101716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fusaro A., Zecchin B., Vrancken B., Abolnik C., Ademun R., Alassane A., Arafa A., Awuni J.A., Couacy-Hymann E., Coulibaly M.B., Gaidet N., Go-Maro E., Joannis T., Jumbo S.D., Minoungou G., Meseko C., Souley M.M., Ndumu D.B., Shittu I., Twabela A., Wade A., Wiersma L., Akpeli Y.P., Zamperin G., Milani A., Lemey P., Monne I. Disentangling the role of Africa in the global spread of H5 highly pathogenic avian influenza. Nat. Commun. 2019;10:5310. doi: 10.1038/s41467-019-13287-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hassouneh I., Radwan A., Serra T., Gil J.M. Food scare crises and developing countries: The impact of avian influenza on vertical price transmission in the Egyptian poultry sector. Food Policy. 2012;37:264–274. [Google Scholar]
  14. Hegazy A., El-Sisi M., Nazim A. Therapeutic evaluation of the antiviral activity of Azadirachta indica in comparison to amantadine hydrochloride against avian influenza virus infection in ducks. Zagazig Vet. J. 2013;41:35–56. [Google Scholar]
  15. Hegazy A.M.E., Kamel A., Rehan M.I.F., Tolba H.M.N. Effect of Azadirachta indica (Neem) Leaf extract as immunostimulant against chicken infectious anemia virus in broiler chickens. Slov. Vet. Res. 2023;60:5–15. [Google Scholar]
  16. Hegazy A., Tolba H., Abdelsamie L., Abdelaziz A., Abdel Moneim A. Effect of the medicinal plant (Azadirachta indica) on Chlamydophila psittaci infection in broiler chickens. Slov. Vet. Res. 2018;55:85–93. [Google Scholar]
  17. Ijoma S.I., Agusi E.R., Ifende V.I., Osemeke O.H., Columba V.T., Meseko C.A. Biosecurity challenges in the control of avian influenza in Nigeria. Sokoto J. Vet. Sci. 2020;18:150–157. [Google Scholar]
  18. Kandeil A., Patton C., Jones J.C., Jeevan T., Harrington W.N., Trifkovic S., Seiler J.P., Fabrizio T., Woodard K., Turner J.C., Crumpton J.C., Miller L., Rubrum A., DeBeauchamp J., Russell C.J., Govorkova E.A., Vogel P., Kim-Torchetti M., Berhane Y., Stallknecht D., Poulson R., Kercher L., Webby R.J. Rapid evolution of A (H5N1) influenza viruses after intercontinental spread to North America. Nat. Commun. 2023;14:3082. doi: 10.1038/s41467-023-38415-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kandeil A., Sabir J.S.M., Abdelaal A. Efficacy of commercial vaccines against newly emerging avian influenza H5N8 virus in Egypt. Sci. Rep. 2018;8:9697. doi: 10.1038/s41598-018-28057-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kapczynski D.R., Pantin-Jackwood M.J., Spackman E., Chrzastek K., Suarez D.L., Swayne D.E. Homologous and heterologous antigenic matched vaccines containing different H5 hemagglutinins provide variable protection of chickens from the 2014 US H5N8 and H5N2 clade 2.3.4.4 highly pathogenic avian influenza viruses. Vaccine. 2017;35:6345–6353. doi: 10.1016/j.vaccine.2017.04.042. [DOI] [PubMed] [Google Scholar]
  21. Khelfa D.G., Mourad A.A., Madian K., Nassif S.A. Efficacy of five commercial available inactivated avian influenza vaccines in both specific pathogen free (SPF) and commercial broiler chicks against challenging with the current recently isolated HPAI H5N1 (A/duck/Egypt/CLEVB-24_N00238/2015) field strain. Curr. Sci. Int. 2016;5:379–385. [Google Scholar]
  22. Landmann M., Scheibner D., Graaf A., Gischke M., Koethe S., Fatola O.I., Raddatz B., Mettenleiter T.C., Beer M., Grund C., Harder T., Abdelwhab E.M., Ulrich R. A semiquantitative scoring system for histopathological and immunohistochemical assessment of lesions and tissue tropism in avian Influenza. Viruses. 2021;13:868. doi: 10.3390/v13050868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lee Y.J., Kang H.M., Lee E.K., Song B.M., Jeong J., Kwon Y.K., Kim H.R., Lee K.J., Hong M.S., Jang I., Choi K.S., Kim J.Y., Lee H.J., Kang M.S., Jeong O.M., Baek J.H., Joo Y.S., Park Y.H., Lee H.S. Novel reassortant influenza A(H5N8) viruses, South Korea. Emerg. Infect. Dis. 2014;20:1087–1089. doi: 10.3201/eid2006.140233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lee D.H., Torchetti M.K., Winker K., Ip H.S., Song C.S., Swayne D.E. Intercontinental spread of Asian-origin H5N8 to North America through Beringia by migratory birds. J. Virol. 2015;89:6521–6524. doi: 10.1128/JVI.00728-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Leila S.F.M. A manual on some Philippine medicinal plants (preparation of drug materials) Bot. Soc. U. P. 1977;20:78–82. [Google Scholar]
  26. Löndt B.Z., Nunez A., Banks J., Nili H., Johnson L.K., Alexander D.J. Pathogenesis of highly pathogenic avian influenza A/turkey/Turkey/1/2005 H5N1 in Pekin ducks (Anas platyrhynchos) infected experimentally. Avian Pathol. 2008;37:619–627. doi: 10.1080/03079450802499126. [DOI] [PubMed] [Google Scholar]
  27. Lycett S.J., Pohlmann A., Staubach C., Caliendo V., Woolhouse M., Beer M., Kuiken T., Global consortium for H5N8 and related influenza viruses Genesis and spread of multiple reassortants during the 2016/2017 H5 avian influenza epidemic in Eurasia. Proc. Natl. Acad. Sci. USA. 2020;117:20814–20825. doi: 10.1073/pnas.2001813117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mahmood M.S., Hussain I., Ahmad M.F., Khan A., Abbas R.Z., Rafiq A. Immunomodulatory effects of Pimpinella anisum L. (Aniseed) in broiler chicks against Newcastle disease and infectious bursal disease viruses. Bol. Latinoam. Caribe Plantas Med. Aromat. 2014;13:458–465. [Google Scholar]
  29. Naveed R., Hussain I., Mahmood M.S., Akhtar M. In vitro and in vivo evaluation of antimicrobial activities of essential oils extracted from some indigenous spices. Pak. Vet. J. 2013;33:413–417. [Google Scholar]
  30. Ramzy N. Viral respiratory diseases of chicken in Egypt (Review) Egypt. J. Chem. Environ. Health. 2016;2:38–44. [Google Scholar]
  31. Rohaim M.A., El Naggar R.F., Madbouly Y., AbdelSabour M.A., Ahmed K.A., Munir M. Comparative infectivity and transmissibility studies of wild-bird and chicken-origin highly pathogenic avian influenza viruses H5N8 in chickens. Comp. Immunol. Microbiol. Infect. Dis. 2021;74 doi: 10.1016/j.cimid.2020.101594. [DOI] [PubMed] [Google Scholar]
  32. Saito T., Tanikawa T., Uchida Y., Takemae N., Kanehira K., Tsunekuni R. Intracontinental and intercontinental dissemination of Asian H5 highly pathogenic avian influenza virus (clade 2.3.4.4) in the winter of 2014-2015. Rev. Med. Virol. 2015;25:388–405. doi: 10.1002/rmv.1857. [DOI] [PubMed] [Google Scholar]
  33. Salaheldin A.H., Elbestawy A.R., Abdelkader A.M., A.Sultan H., Ibrahim A.A., Abd El-Hamid H.S., Abdelwhab E.M. Isolation of genetically diverse H5N8 avian influenza viruses in poultry in Egypt, 2019–2021. Viruses. 2022;14:1431. doi: 10.3390/v14071431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Setta A., Yehia N., Shakak A.O., Al-Quwaie D.A., Al-Otaibi A.M., El-Saadony M.T., El-Tarabily K.A., Salem H. Molecular detection of highly pathogenic avian influenza H5N8 in commercial broiler chicken farms from 2019 to 2022. Poult. Sci. 2023;102 doi: 10.1016/j.psj.2023.102639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Smith G.J., Donis R.O. World Health Organization/World Organisation for Animal Health/Food and Agriculture Organization (WHO/OIE/FAO) H5 Evolution Working Group. Nomenclature updates resulting from the evolution of avian influenza A(H5) virus clades 2.1.3.2a, 2.2.1, and 2.3.4 during 2013-2014. Influenza. Other. Respir. Viruses. 2015;9:271–276. doi: 10.1111/irv.12324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sobolev I., Sharshov K., Dubovitskiy N., Kurskaya O., Alekseev A., Leonov S., Yushkov Y., Irza V., Komissarov A., Fadeev A., Danilenko D., Mine J., Tsunekuni R., Uchida Y., Saito T., Shestopalov A. Highly pathogenic avian influenza A (H5N8) virus clade 2.3. 4.4 b, Western Siberia, Russia, 2020. Emerg. Infect. Dis. 2021;27:2224–2227. doi: 10.3201/eid2708.204969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Swayne D.E., Spackman E., Pantin-Jackwood M. Success factors for avian influenza vaccine use in poultry and potential impact at the wild bird-agricultural interface. Ecohealth. 2014;11:94–108. doi: 10.1007/s10393-013-0861-3. [DOI] [PubMed] [Google Scholar]
  38. Swayne D.E., Suarez D.L., Sims L.D. In: Pages 210–256 in Diseases of Poultry. Swayne D.E., Boulianne M., Logue C.M., McDougald L.R., Nair V., Suarez D.L., de Wit S., Grimes T., Johnson D., Kromm M., Prajitno T.Y., Rubinoff I., Zavala G., editors. John Wiley & Sons; Hoboken, NJ: 2020. Influenza. eds. [Google Scholar]
  39. Swelum A.A., Elbestawy A.R., El-Saadony M.T., Hussein E.O., Alhotan R., Suliman G.M., Taha A.E., Ba-Awadh H., El-Tarabily K.A., Abd El-Hack M.E. Ways to minimize bacterial infections, with special reference to Escherichia coli, to cope with the first-week mortality in chicks: an updated overview. Poult. Sci. 2021;100:101039. doi: 10.1016/j.psj.2021.101039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tarek M., Naguib M.M., Arafa A.S., Tantawy L.A., Selim K.M., Talaat S., Sultan H.A. Epidemiology, genetic characterization, and pathogenesis of avian influenza H5N8 viruses circulating in Northern and Southern Parts of Egypt, 2017–2019. Animals. 2021;11:2208. doi: 10.3390/ani11082208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. World Organization for Animal Health (OIE). 2018. Avian influenza — infection with avian influenza viruses. Pages 821-84 in Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 2018. Accessed March 2019. https://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/3.03.04_AI.pdf.
  42. Williams S.M., Dufour-Zavala L., Jackwood M.W., Lee M.D., Lupiani B., Lupiani W.M., Spackman E., Woolcock P.R. 6th ed. American Association of Avian Pathologists, University of Pennsylvania; Kennett Square, PA: 2016. Page 400 in A Laboratory Manual for the Isolation, Identification, and Characterization of Avian Pathogens. [Google Scholar]
  43. Wu Q., Wei L., Du X., Sun W., Li S., Guo X., Jiang M., Liu J., Xue Z., Li H., Zhang T., Wang W., Ren G. Development and evaluation of Newcastle disease-avian influenza bivalent vector vaccines in commercial chickens. Int. Immunopharmacol. 2023;120 doi: 10.1016/j.intimp.2023.110363. [DOI] [PubMed] [Google Scholar]
  44. Yehia N., Hassan W.M.M., Sedeek A., Elhusseiny M.H. Genetic variability of avian influenza virus subtype H5N8 in Egypt in 2017 and 2018. Arch. Virol. 2020;165:1357–1366. doi: 10.1007/s00705-020-04621-7. [DOI] [PubMed] [Google Scholar]
  45. Yehia N., Naguib M.M., Li R., Hagag N., El-Husseiny M., Mosaad Z., Nour A., Rabea N., Hasan W.M., Hassan M.K., Harder T., Arafa A.A. Multiple introductions of reassorted highly pathogenic avian influenza viruses (H5N8) clade 2.3.4.4b causing outbreaks in wild birds and poultry in Egypt. Infect. Genet. Evol. 2018;58:56–65. doi: 10.1016/j.meegid.2017.12.011. [DOI] [PubMed] [Google Scholar]
  46. Zeng X., Chen X., Ma S., Wu J., Bao H., Pan S., Liu Y., Deng G., Shi J., Chen P., Jiang Y.P., Li Y.B. Protective efficacy of an H5/H7 trivalent inactivated vaccine produced from Re-11, Re-12, and H7-Re2 strains against challenge with different H5 and H7 viruses in chickens. J. Integr. Agric. 2020;19:2294–2300. [Google Scholar]
  47. Zhao K., Gu M., Zhong L., Duan Z., Zhang Y., Zhu Y., Zhao G., Zhao M., Chen Z., Hu S., Liu W., Liu X., Peng D., Liu X. Characterization of three H5N5 and one H5N8 highly pathogenic avian influenza viruses in China. Vet. Microbiol. 2013;163:351–357. doi: 10.1016/j.vetmic.2012.12.025. [DOI] [PubMed] [Google Scholar]

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