Poultry Health Research Articles

Exploring the potential of artificial intelligence in feed formulation to advance poultry health and One-Health

Exploring the potential of artificial intelligence in feed formulation to advance poultry health and One-Health

Isolation and characterization of duck sewage source Salmonella phage P6 and antibacterial activity for recombinant endolysin LysP6

Salmonella is a globally prevalent foodborne pathogen, and adverse events caused by S. Enteritidis and S. Typhimurium are extremely common. With the emergence of drug resistance, there is an urgent need for efficient and specific lytic bacteriophages as alternative to antibiotics in clinical practice. In this study, phage P6 was isolated and screened from effluent and fecal samples from duck farm environments to specifically lyse the duck sources S. Typhimurium and S. Enteritidis. Phage P6 belongs to the genus Lederbergvirus, unclassified Lederbergvirus species. The phage P6 genome did not contained non-coding RNA, virulence genes and drug resistance genes, indicating that phage P6 was biologically safe for clinical applications. Phage P6 lysed 77.78% (28/36) of multidrug-resistant Salmonella and reduced biofilms formed by S. Enteritidis CVCC 3377, 4, and 24, and S. Typhimurium 44 by 44% to 75% within 3 h, and decreased Salmonella in duckling feces by up to 1.64 orders of magnitude. Prokaryotic expression of endolysin LysP6 lysed the chloroform-treated bacterial outer membrane from different serotypes of duck-derived Salmonella and E. coli standard strain ATCC 25922. The host range was expanded compared to phage P6, and the growth of Salmonella was effectively inhibited by LysP6 in conjunction with the membrane permeabilizer EDTA within 24 h. Therefore, phage P6 and phage-derived endolysins LysP6 are suitable for application as potent biocontrol agents to improve poultry health and food safety.

Survey of the husbandry and biosecurity practices of backyard chicken keepers in the UK.

This survey investigated the housing, feeding, health and welfare of backyard chickens kept in the UK. Information was collected via an online questionnaire active from May to July 2021. The survey asked about flock demographics, housing, diet, enrichment provision, if the flock was registered with the Animal Plant and Health Agency (APHA) and the reason, preventative health care and biosecurity, and the incidence and methods of euthanasia. The majority of flocks (48.8%) consisted of one to five birds, were located in rural areas (58%) and were kept as pets (77%) and/or for eggs (71.6%). Enrichment was provided by 78.4% of keepers. Most respondents (69%) were aware of the Animal Plant and Health Agency poultry registration scheme, with 32.8% being registered with the scheme. Kitchen scraps were fed by 29% of keepers. Veterinary services were used by 63.6% of keepers, although 4% said they struggled to find a veterinarian willing to treat backyard poultry. New additions to the flock were isolated by 70.2% of keepers. The survey was distributed through poultry-specific Facebook groups and via chicken rescue centres, so it is not a truly random sample of backyard chicken keepers. All survey-based studies have an inherent element of subjectivity. The survey identified biosecurity, humane euthanasia training, veterinary training in backyard poultry medicine, and enrichment provision as areas where improvements can be made to improve poultry health and welfare and reduce the risks of infectious disease transmission.

A broad-spectrum vaccine candidate against H5 viruses bearing different sub-clade 2.3.4.4 HA genes

The global spread of H5 clade 2.3.4.4 highly pathogenic avian influenza (HPAI) viruses threatens poultry and public health. The continuous circulation of these viruses has led to their considerable genetic and antigenic evolution, resulting in the formation of eight subclades (2.3.4.4a–h). Here, we examined the antigenic sites that determine the antigenic differences between two H5 vaccine strains, H5-Re8 (clade 2.3.4.4g) and H5-Re11 (clade 2.3.4.4h). Epitope mapping data revealed that all eight identified antigenic sites were located within two classical antigenic regions, with five sites in region A (positions 115, 120, 124, 126, and 140) and three in region B (positions 151, 156, and 185). Through antigenic cartography analysis of mutants with varying numbers of substitutions, we confirmed that a combination of mutations in these eight sites reverses the antigenicity of H5-Re11 to that of H5-Re8, and vice versa. More importantly, our analyses identified H5-Re11_Q115L/R120S/A156T (H5-Re11 + 3) as a promising candidate for a broad-spectrum vaccine, positioned centrally in the antigenic map, and offering potential universal protection against all variants within the clade 2.3.4.4. H5-Re11 + 3 serum has better cross-reactivity than sera generated with other 2.3.4.4 vaccines, and H5-Re11 + 3 vaccine provided 100% protection of chickens against antigenically drifted H5 viruses from various 2.3.4.4 antigenic groups. Our findings suggest that antigenic regions A and B are immunodominant in H5 viruses, and that antigenic cartography-guided vaccine design is a promising strategy for selecting a broad-spectrum vaccine.

Epigallocatechin gallate improves oleic acid-induced hepatic steatosis in laying hen hepatocytes via the MAPK pathway

Fatty liver disease in laying hens, characterized by excessive lipid accumulation in hepatocytes, poses significant challenges to poultry health and production efficiency. In this study, we investigated the therapeutic potential of epigallocatechin gallate (EGCG), a bioactive compound found in green tea, in mitigating oleic acid (OA)-induced hepatic steatosis in primary chicken hepatocytes. Treatment with EGCG effectively attenuated lipid deposition by downregulating lipid synthesis-related genes. Moreover, EGCG mitigated oxidative stress, inflammation, DNA damage, and apoptosis induced by OA, thereby preserving hepatocyte viability. Mechanistically, EGCG exerted its protective effects by modulating the p38 MAPK signaling pathway. Our findings suggest that EGCG holds promise as a therapeutic agent for managing fatty liver disease in poultry, offering insights into novel strategies for improving poultry health and production outcomes.

Transcriptome Analysis of Hypothalamic-Pituitary-Ovarian Axis Reveals circRNAs Related to Egg Production of Bian Chicken.

The hypothalamic-pituitary-ovarian (HPO) axis plays a pivotal role in the regulation of egg production in chickens. In addition to the traditional understanding of the HPO axis, emerging research highlights the significant role of circRNAs in modulating the functions of this axis. In the study, we collected hypothalamus, pituitary, and ovarian tissues from low-yielding and high-yielding Bian chickens for transcriptome sequencing. We identified 339, 339, and 287 differentially expressed (DE) circRNAs with p_value < 0.05 and |log2 (fold change)| ≥ 1 in hypothalamus, pituitary, and ovarian tissues. The Gene Ontology (GO) enrichment analysis for the source genes of DE circRNAs has yielded multiple biological process (BP) entries related to cell development, the nervous system, and proteins, including cellular component morphogenesis, cell morphogenesis, nervous system development, neurogenesis, protein modification process, and protein metabolic process. In the top 30 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, we observed the enrichment of the GnRH signaling pathway in both the hypothalamus and the pituitary, solely identified the GnRH secretion pathway in the pituitary, and discovered the pathway of oocyte meiosis in the ovary. Furthermore, given that circRNA primarily functions through the ceRNA mechanism, we constructed ceRNA regulatory networks with DE circRNAs originating from the GnRH signaling pathway, GnRH secretion, ovarian steroidogenesis, steroid hormone biosynthesis, and the estrogen signaling pathway. Finally, several important ceRNA regulatory networks related to reproduction were discovered, such as novel_circ_003662-gga-let-7b/miR-148a-3p/miR-146a-5p/miR-146b-5p and novel_circ_003538-gga-miR-7464-3p-SLC19A1. This study will contribute to advancements in understanding the involvement of circRNAs in the HPO axis, potentially leading to innovations in improving egg production and poultry health.

Molecular detection of avian pathogens in poultry red mite (Dermanyssus gallinae) in Algerian layer farms as a potential predictive tool

The poultry red mite Dermanyssus gallinae is a hematophagous ectoparasite of layer hens. Infestations with poultry red mites pose an increasing threat to the egg production industry, causing serious problems to animal health and welfare, directly or indirectly as a vector of several infectious agents. In this study, we aimed to investigate common avian pathogens in mites. The mite samples were collected from 58 poultry farms in 7 regions accounting for more than 70 % of the national egg production in Algeria. The presence of 13 avian pathogens was detected using DNA and RNA samples from mites collected. Results revealed significant associations between PRM and potential pathogens such as Escherichia coli, Salmonella enterica, fowlpox virus, and gallid herpesvirus 1. Pathogen detection in Dermanyssus gallinae could serve as an early diagnostic or a risk analysis tool for infectious diseases in poultry farms, facilitating effective disease management strategies. Despite further research being necessary to address uncertainties, such a strategy could be used to enhance the integrated management of poultry health.

Nano-emulsified black soldier fly oil concerning performance traits, health, and immunity of broilers

In the antibiotic-free era, traditional antibiotics have been suggested as alternatives to antibiotic-based growth promoters. Among the various methods, self-nano-emulsifying drug delivery systems (SNEDDS) are increasingly utilized to improve the bioavailability of oils containing essential substances. In this study, we evaluated the effects of black soldier fly oil (BSFO) SNEDDS in chicken drinking water on growth performance, small intestine histomorphology, and poultry health status. We divided 225 male Indian River strain broiler chickens into five treatment groups, each consisting of 5 replicates. The chickens were reared from to 0 to 35 d of age in a controlled environmental housing system. The BSFO SNEDDS was administered via drinking water. Treatments included ordinary drinking water (P1), bacitracin (P2), and 10 mL/L (P3), 20 mL/L (P4), and 30 mL/L (P5) BSFO SNEDDS. The observed parameters included growth performance, carcass yield, blood hematology, intestinal histomorphology, digestive microflora, and immunoglobulin (Ig) levels. Data were analyzed using analysis of variance (ANOVA) and Duncan's test. The results indicated that administering BSFO SNEDDS via drinking water improved feed conversion (P < 0.05), enhanced the performance index (P < 0.05), increased carcass percentage (P < 0.001), and increased the weight of the carcass parts. Additionally, it increased villus height (P < 0.01), lowered jejunal pH (P < 0.001), reduced pathogenic bacteria in the jejunum, and decreased the leukocyte count. BSFO SNEDDS in drinking water is expected to be used as a traditional antibiotic with the potential to replace synthetic antibiotic growth promoters in broiler chickens.

Antimicrobial Resistance and Phylogenetic Relatedness of Salmonella Serovars in Indigenous Poultry and Their Drinking Water Sources in North Central Nigeria.

There is scant information on Salmonella in indigenous poultry in Nigeria. We investigated the occurrence and characterized Salmonella serovars in indigenous poultry and their drinking water sources to enhance the monitoring of the infection in poultry and to promote public health. We collected 1208 samples, poultry droppings (n = 1108), and water (n = 100) across 15 markets in North Central Nigeria. Salmonella spp. were isolated following World Organisation for Animal Health guidelines. Salmonella spp., confirmed through invA gene detection by a polymerase chain reaction assay, were 6.8% (75/1108) droppings and 3% (3/100) water. Susceptibility testing against 13 antimicrobials showed 60.3% (47/78) susceptibility to all the antimicrobials tested while 14.1% (11/78) were multidrug resistant. Serotyping and whole-genome sequencing were carried out on 44 of the isolates, and 23 different serovars were identified. Genomes of serovars Luedinghausen, Laredo, Widemarsh, and Lansing are being documented in Africa for the first time. Twenty (20) antimicrobial resistance (AMR) gene markers encoding for resistance to aminoglycosides, tetracyclines, sulphonamides, quinolones, trimethoprim, penicillins and phenicols were found. Phylogenetic cluster analysis showed close relatedness among isolates from different sources. This study shows both low Salmonella prevalence and AMR, but since uncommon serovars are circulating, continuous monitoring is recommended so as to ensure food safety and poultry health.

The effect of sodium butyrate administered in ovo on the health status and intestinal response in broiler chicken

A healthy gut is one of the main factors influencing bird response. Over the years, efforts have been made to improve intestinal health. One of the supporting methods may be enriching the diet with bioactive ingredients, including sodium butyrate (SB). One of the possible ways of administering such supplementation is in ovo technology. Over the years, research has shown that administering bioactive substances this way has a positive effect on the health status of chickens. The current study aimed to modify the gut microbiota of broiler chickens by in ovo stimulation on d 12 of egg incubation with SB and to determine the changes occurring in intestines. One thousand eggs were incubated and injected with 0.1, 0.3, or 0.5% SB on d 12 of incubation. The control group was injected with physiological saline. Samples collected for analysis were obtained postmortem from 42-day-old ROSS 308 broiler chickens. Growth performance parameters were also monitored during broiler rearing. Gene expression analysis showed significant changes in the levels of IL4, IFNγ, AvBD1, TJAP and MUC6 genes in the ileum. However, the IL8, MUC2 and MUC6 genes were significantly expressed in the cecal mucosa. These changes depended on the administered dose of butyrate. There was no effect of in ovo administration of various doses of SB on digesta pH, SCFA level and histological parameters. However, a significant increase in Bifidobacterium bacteria was detected in the ileum after administration of a dose of 0.5% SB and in the cecum after administration of a dose of 0.3%. Administration of SB in ovo has the potential to support intestinal health in poultry. The effects depend on the administered dose, while the results indicate a dose of 0.3% as the most optimal.

Efficacy &amp; Significance of LYSO HERB Herbal Lysine in the nutrition, metabolism and health in Poultry &amp; Efficacy and significance of the phytogenic METHO ADD Herbal Methionine as replacer of Synthetic DL- Methionine in nutrition and health of Poultry

In poultry industry, amino acids are supplied along with vitamins and minerals since these play a pivotal role as protein building units which are the essentialities of growth and metabolism. Of the 9 essential amino acids Methionine is the first limiting one followed by Lysine, these cannot be synthesized by animals hence to be obtained from external sources majorly the feed. Amino acids act as intermediates in all the metabolic and physiological functions of the bird like calcium absorption, production of collagen, antibodies, hormones, enzymes. Their deficiency causes de-pigmentation, reduced hemoglobin, poor FCR, reduced production, retarded growth, poor feathering and egg production, immune suppression and increased disease incidence. Cells in the body are constantly being broken down and restored, so the body needs these amino acids to produce new hormones and restore the body. Of late the use of synthetic Lysine and Methionine as supplement has increased due to high demand of meat. The safety of these is questionable due to higher pricing, emergence of drug resistance, residual toxicity and other side effects. Therefore, a renewed interest in developing natural alternatives to maintain production, performance and health in poultry, hence Phytogenic herbals are considered the best option.. The results obtained here provide clear evidence of the potential of Phytogenic Herbal Lysine as Lyso Herb and Herbal Methionine as Metho Add as substitute of synthetic Lysine &amp; Methionine with unaltered activity throughout, without reducing growth performance, increasing fat accretion and changing serum biochemical characteristics, with better performance and economics.

Transmission dynamics of avian influenza viruses in Egyptian poultry markets

Live bird markets (LBMs) are considered hotspots for Avian Influenza Viruses (AIVs). In such markets, AIVs pose threats to both poultry and public health. Within LBMs, AIVs spread through various routes, including direct contact, environmental contamination, and aerosol transmission. Unique factors in Egyptian LBMs, such as the coexistence of wild and domestic birds, increase transmission risks between birds as well as spill-overs into exposed humans. Understanding the transmission dynamics of AIVs is vital for implementing effective control measures. We conducted a study in four Egyptian LBMs located in Mediterranean coast cities from November 2021 to March 2023. In this study we tested 3,971 samples from poultry, wild birds, and the environment, out of which 692 (17.4%) were positive for AIV. Poultry exhibited a higher prevalence (42.2%) than wild birds (34.4%). Environmental samples, including water (30.8%), surfaces (17.2%), and air (18.2%), also tested positive for AIV. Diverse AIV subtypes, including H5N1, H9N2, H5/H9 co-infection, and H5N8, were detected among avian species and the environment. Temporal analysis revealed fluctuating IAV positivity rates from November 2021 to March 2023. These results emphasize the importance of continuous surveillance, resource allocation, and multisectoral collaboration to protect poultry and human health, and prevent novel influenza strains’ emergence in Egyptian LBMs.

66. Optimizing the use of antibiotics: Towards multi-criteria systems for monitoring the health and well-being of pigs and poultry

66. Optimizing the use of antibiotics: Towards multi-criteria systems for monitoring the health and well-being of pigs and poultry

Grape Seed Oil: Health Benefits and Useful Impacts on Livestock Performance and Products Quality – A Review

Abstract Grape seed oil (GSO) is a rich source of unsaturated fatty acids, antioxidants, and phenolic compounds that have been shown to affect physiological disorders associated with chronic diseases positively. The article demonstrates GSO’s chemical structure and health benefits, including its ability to alleviate oxidative stress, modulate cytokines, exhibit antimicrobial activity, and potentially prevent cancer. The review highlights the positive impact of GSO on livestock growth and products quality, particularly when combined with grape pomace in broiler and rabbits diets. Including grape seed extract in animals’ diets improved carcass traits under oxidative stress conditions. It may also help reduce total lipid levels and lower LDL cholesterol levels, benefiting heart health. The article emphasizes the need for more studies to better understanding of the optimal bioactivity of GSO and to determine the recommended doses of GSO for human consumption. The review suggests that GSO could be a promising natural feed additive for improving poultry health and product quality.

Exploring the immunomodulatory and antioxidant capacities of Berberis aristata in avian lymphocytes

India is one of the leading producers for eggs and broilers globally and thus has witnessed significant growth in the poultry sector. Different poultry stressors can negatively impact the growth, gut health, immune function and production reproductive performances of poultry. Antibiotic growth promoters (AGPs) have been used in the poultry industry as they also combat stress in poultry along with growth promotion. But the use of antimicrobials as growth promoter raises the concern of antibiotic resistance and its potential hazardous effect on human health as well as the environment. Several other alternatives are available for AGPs such as acidifiers, probiotics, prebiotics, antibacterial peptides and phytobiotics. Phytobiotics are known to possess several benefits for poultry as well as livestock such as immunomodulation, anti-inflammatory effect, antioxidant effect, maintain intestinal health etc. One such important plant is Berberis aristata belonging to the family Berberidaceae which is used in traditional system of medicine for the management of various inflammatory disorders. In the present study aqueous root extract of Berberis aristata (BAE) was evaluated for its immunomodulatory and antioxidant activity. The extraction yield of the roots of Berberis aristata in water was about 9.78%. The BAE showed presence of phenolics, flavonoids, tannins, terpenoids and contained about 104.97 mg/gm of BAE and 83.14 mg/gm of BAE of phenolics and flavonoids, respectively. BAE showed significant antioxidant activity in DPPH free radical scavenging assay. Maximum non-cytotoxic dose (MNCD) of BAE for lymphocyte culture was determined to be 600 µg/ ml. Lymphocytes proliferation assay (LPA) showed that BAE enhanced lymphocyte proliferation of B-cells stimulated by lipopolysaccharide (LPS) and T-cells stimulated by Concanavalin A and Phytohaemagglutinin M (PHA). Treatment with BAE significantly reduced lipid peroxidation (LPO) and increased the level of reduced glutathione, superoxide dismutase, and catalase in chicken lymphocytes, thus indicating antioxidant potential of BAE. The study reveals significant immunomodulatory and antioxidant properties of BAE, thus suggesting that it could be a potential alternative to AGPs in poultry production. But further in vivo studies are required to explore its efficacy and safety for its application in commercial poultry farming.

Impact of Chlorella vulgaris Intake Levels on Performance Parameters and Blood Health Markers in Broiler Chickens.

This systematic review examines the effects of cumulative Chlorella vulgaris intake levels on broiler chickens, focusing on growth performance and systemic health markers. The review establishes a clear relationship between cumulative C. vulgaris intake and significant outcomes in poultry nutrition and health through a detailed analysis of various studies. The correlation analysis revealed that cumulative C. vulgaris intake levels ranging from 0.8 to 718 g/bird influenced growth rates and feed efficiency, following sigmoid models. Specifically, intakes of approximately 20 g/bird maximized final body weight (R2 = 0.616, p < 0.001), cumulative body weight gain (R2 = 0.627, p < 0.001) and daily weight gain (R2 = 0.639, p < 0.001). The feed conversion ratio also improved with increasing C. vulgaris intakes up to this level, although this was non-significant (R2 = 0.289, p = 0.117). In addition, similar cumulative C. vulgaris intake levels impacted plasma health markers in broilers, leading to reductions in triacylglycerols and cholesterol and improvements in immunoglobulin levels. These findings underscore the importance of carefully calibrated C. vulgaris supplementation strategies to optimise poultry growth and health without adverse effects. Future research should focus on refining C. vulgaris dosing guidelines and further exploring its long-term effects and mechanisms of action to enhance poultry health and production sustainability.

CircRUNX2.2, highly expressed in Marek's disease tumor tissues, functions in cis to regulate parental gene RUNX2 expression

Marek's disease (MD), an immunosuppression disease induced by Marek's disease virus (MDV), is one of the significant diseases affecting the health and productive performance of poultry. The roles of circular RNAs (circRNAs) in MD development were poorly understood. In this study, we found a circRNA derived from exon 6 of RUNX family transcription factor 2 (RUNX2) gene, named circRUNX2.2, was highly expressed in chicken tumorous spleens (TS) induced by MDV. Through fluorescence in situ hybridization and nuclear-cytoplasmic separation assay, we determined circRUNX2.2 was mainly located in the nucleus. Knockout experiments confirmed that the flanking complementary sequences (RCMs) mediated its circularization. Gain of function assay and dual luciferase reporter gene assay revealed that circRUNX2.2 could promote the expression of RUNX2 via binding with its promoter region. RNA antisense purification assay and mass spectrometry assay showed circRUNX2.2 could recruit proteins such as CHD9 protein. Knocking down CHD9 expression decreased the expression of RUNX2 gene, which confirmed the positive regulation that circRUNX2.2 on RUNX2 expression was probably facilitated via recruiting CHD9 protein. Functional experiments showed that circRUNX2.2 promoted the proliferation of the MD lymphoma-derived chicken cell line, MDCC-MSB1, which confirmed the potential oncogenic role of circRNX2.2 in tumor development. In conclusion, we found that the RUNX2-derived circRUNX2.2 can positively regulate the transcription of the parental gene RUNX2 in a cis-acting manner. The high expression of circRUNX2.2 in MD tumor tissues indicated that it might mediate MD lymphoma progression.

Effects of gossypol acetate on growth, serum biochemical parameters, and intestinal health of goslings

Free gossypol (FG), the primary antinutritional component in cottonseed meal, can adversely affect the growth and health of poultry. Although younger geese are particularly sensitive to FG, the precise effects of FG on geese remain elusive. This study aimed to investigate the effects of gossypol acetate (GA), a form of FG, on the growth, serum biochemical parameters, and intestinal health of goslings. Seventy-two healthy male goslings, aged 7-day-old with similar body weight (BW), were randomly divided into 3 groups: a control group and 2 GA-treated groups (GA25 and GA50), which were orally administered GA (25 and 50 mg/kg BW) daily for 14 d. The results showed that oral administration of GA significantly suppressed BW, altered serum parameters, and impaired intestinal health in a dose- and time-dependent manner. Specifically, GA adversely affected intestinal morphology, induced oxidative stress, and inflammation, diminished immune function, and increased intestinal permeability and apoptosis of intestinal cells, consequently impairing nutrient absorption and utilization of goslings. Overall, these data indicate that GA adversely affects the growth, serum parameters, and intestinal health of goslings, providing valuable information further to understand the toxic effects of gossypol on goslings.

Melatonin prevents cadmium-induced osteoporosis by affecting the osteoblast and osteoclast differentiation and pyroptosis in duck

Cadmium (Cd), is a highly toxic environmental pollutant, which seriously threatens the health of poultry and humans. The occurrence of osteoporosis is the main manifestation of cadmium toxicity. Pyroptosis plays an important role in the development of osteoporosis. Melatonin has been shown to affect preserving bone health. However, the underlying mechanism has not been elucidated. In the present study, these functions of melatonin have been investigated in duck bone tissue and osteoblast during cadmium exposure. In vivo, the studies suggest that melatonin protects against cadmium-induced duck osteoporosis by improving the osteogenesis function, inhibiting bone resorption, and suppressing the occurrence of pyroptosis. In vitro, the findings demonstrated that melatonin alleviated the inhibition effect of cadmium on duck bone marrow-derived mesenchymal stem cells (BMSC) osteogenic differentiation, and suppressed the cadmium-induced osteoclast differentiation. In addition, we also found that melatonin prevents cytokines release of lactate dehydrogenase (LDH), interleukin-18 (IL-18), and interleukin-1β (IL-1β) by cadmium-induced, and reduces the expression of n-terminal Gasdermin D (N-GSDMD), alleviates the osteoblast death rate. In short, melatonin as a potential therapeutic agent has bright prospects in cadmium-induced bone toxicity.

Dual Gene Detection of H5N1 Avian Influenza Virus Based on Dual RT-RPA.

The H5N1 avian influenza virus seriously affects the health of poultry and humans. Once infected, the mortality rate is very high. Therefore, accurate and timely detection of the H5N1 avian influenza virus is beneficial for controlling its spread. This article establishes a dual gene detection method based on dual RPA for simultaneously detecting the HA and M2 genes of H5N1 avian influenza virus, for the detection of H5N1 avian influenza virus. Design specific primers for the conserved regions of the HA and M2 genes. The sensitivity of the dual RT-RPA detection method for HA and M2 genes is 1 × 10-7 ng/μL. The optimal primer ratio is 1:1, the optimal reaction temperature is 40 °C, and the optimal reaction time is 20 min. Dual RT-RPA was used to detect 72 samples, and compared with RT-qPCR detection, the Kappa value was 1 (p value < 0.05), and the clinical sample detection sensitivity and specificity were both 100%. The dual RT-RPA method is used for the first time to simultaneously detect two genes of the H5N1 avian influenza virus. As an accurate and convenient diagnostic tool, it can be used to diagnose the H5N1 avian influenza virus.