Comparative study of conventional and molecular epidemiology of Newcastle disease in wild and domestic birds in the Rift Valley region of Ethiopia

Comparative study of conventional and molecular epidemiology of Newcastle disease in wild and domestic birds in the Rift Valley region of Ethiopia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Comparative study of conventional and molecular epidemiology of Newcastle disease in wild and domestic birds in the Rift Valley region of Ethiopia Gelana Haile Terara, Belete Kuraz Abebe, Mulugeta Tesfaye Alemayehu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7221776/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Newcastle Disease (ND) remains a significant threat to the health and productivity of poultry. This study aimed to evaluate the prevalence of ND in domestic and wild chickens. A total of 150 both domestic and wild birds were sampled from Bishoftu, Hawassa, and Batu. Serum samples were analyzed for ND antibodies using serological assays, while 60 pooled swab samples were subjected to RT-PCR for NDV detection. The overall seroprevalence of ND was found to be 12.7%, with Bishoftu exhibiting the highest prevalence at 20%, followed by Hawassa at 12% and Batu at 6%. No statistically significant differences were noted based on sex (p > 0.05) or age (p > 0.05). RT-PCR detected NDV in 16.7% of the swab samples, with a higher prevalence observed in cloacal swabs (20%) compared to tracheal swabs (13.3%). Risk analysis indicated a significantly increased odds ratio (OR) of 4.2 for ND infection in Bishoftu relative to Batu. Additionally, males were found to be 1.8 times more likely to be infected than females, although age did not significantly affect the risk of infection. These findings highlight the regional variability in ND prevalence, with Bishoftu showing the highest rates. Both serological and molecular analyses demonstrate a notable presence of NDV, particularly in cloacal swabs. Newcastle Disease poultry serology risk factors Figures Figure 1 Figure 2 1. Introduction Newcastle Disease (ND), caused by the Newcastle disease virus (NDV), remains a significant global threat to the poultry industry, affecting both developing and developed economies due to its highly contagious nature and substantial economic impacts (Absalón et al. 2019 ; Hu et al. 2022 ; Moustapha et al. 2023 ; Dharmayanti et al. 2024 ). Despite advancements in vaccination and biosecurity measures, ND continues to inflict considerable losses in regions where these interventions are inadequately implemented, thereby threatening productivity and international poultry trade (Dimitrov et al. 2017 ; Bello et al. 2018 ; Hu et al. 2022 ). Given ND's potential to disrupt food security and induce economic instability, there is an urgent need for comprehensive epidemiological research to inform targeted disease control strategies (Drewnowski, 2022 ). The Rift Valley region of Ethiopia, recognized as a crucial area for poultry production, exhibits significant vulnerability attributable to its heterogeneous environmental conditions, encompassing both highland and lowland ecosystems, as well as diverse poultry management practices. These environmental factors include variable temperatures, seasonal precipitation patterns, and the coexistence of commercial and subsistence poultry farming systems. Although large-scale poultry operations may benefit from contemporary management techniques, numerous smallholder farmers continue to employ traditional farming practices that often lack adequate biosecurity and disease control protocols. This deficiency exacerbates the region's susceptibility to outbreaks of NDV (Asfaw, Ameni, Medhin, Gumi, Hagos, et al. 2021; Hailegebreal et al. 2022 ). However, the insufficient data regarding the prevalence of ND across different districts has hindered the implementation of effective control measures, despite the region's importance for national food security and economic development (Asfaw, Ameni, Medhin, Gumi and Wieland, 2021 ). Previous studies have not sufficiently addressed the spatial and demographic factors that contribute to the spread of ND, nor have they employed advanced diagnostic technologies (Betela, Hailegebreal and Shiferaw, 2023 ). This study presents the first comprehensive epidemiological analysis of ND in the Rift Valley, focusing on three key districts: Bishoftu, Hawassa, and Batu. By utilizing both serological assays and real-time reverse transcription polymerase chain reaction (RT-PCR) techniques, we assessed the prevalence of ND and its associated risk factors, while examining demographic variables such as sex, age, and geographical location. Through the integration of these advanced diagnostic methodologies, our research aims to provide a detailed understanding of ND dynamics in a region that is crucial to Ethiopia's poultry industry. ND is a highly contagious disease that severely impacts poultry industries worldwide. Risk factors for ND include housing conditions (free-range vs. confined), contact with wild birds, and demographic factors like gender, age, and region. Free-range poultry are more exposed to wild birds, increasing the risk of NDV infection (Chaka et al. 2013 ). This study aimed to evaluate ND prevalence in domestic and wild chickens in Ethiopia, with a focus on identifying the impact of these risk factors on ND occurrence. The outcomes of this study are anticipated to significantly enhance ND surveillance efforts in the Rift Valley, refine vaccination strategies, and strengthen biosecurity measures. Our data-driven methodology establishes a framework for evidence-based interventions aimed at enhancing the health and productivity of Ethiopia's poultry sector, in accordance with the objectives set forth in Ethiopia’s Livestock Development Policy and Strategy (2015)(Shapiro et al., 2015 ) and the National Poultry Development Strategy 2022–2031 (Fekadu et al. 2023 ). By tackling the incidence of ND and improving diagnostic capabilities, the outcomes of this study contribute to the overarching goals of healthy disease control measures and increasing poultry productivity. These findings are essential for facilitating policy implementation, advancing biosecurity practices, and bolstering public health initiatives, with significant implications for disease control strategies not only within Ethiopia but also across the African continent and beyond. 2. MATERIALS AND METHODS 2.1. Ethical standards This research was conducted in accordance with the ethical standards for animal research established by Addis Ababa University and relevant Ethiopian laws. The experimental protocol was reviewed and approved by the Animal Research Ethical Review Committee at Addis Ababa University (Certificate Reference No. VM/ERC/06/05/017). 2.2. Study areas The research was conducted in the Rift Valley regions of the Eastern Shewa Zone, specifically within the Bishoftu and Batu districts of the Oromia Region, as well as in the Hawassa district of the Southern Nations, Nationalities, and Peoples (SNNP) Region. These districts are notable for their frequent visitation by migratory birds, which are attracted to the nearby water reservoirs that provide sustenance for wildlife. The investigation focused on urban markets within each of the selected districts. Geographically, the study areas are located between latitudes 7° 09 'N and 8°45'N and longitudes 38°32'E and 39°17'E, covering an approximate width of 40–60 km and extending over 100 km in length. The climate of this semi-arid region is characterized by erratic and unreliable precipitation, with annual rainfall averaging between 500 and 900 mm. The rainfall pattern is bimodal, featuring long rains from June to September and short rains from February to April (Figure 1). 2.3. Study population The study subjects were sourced from the Rift Valley regions of Oromia and the Southern Nations, Nationalities, and Peoples' (SNNP) region in Ethiopia. Specimens were collected from the fecal droppings of wild birds, swabs from pigeons, and domestic backyard chickens. For the domestic chickens, tracheal and cloacal swabs, as well as serum samples, were obtained from individuals across various districts, categorized by sex and age. Age classification followed the criteria established by [15], which designates chickens aged 3 to 6 months as young and those older than 6 months as adults. The backyard chickens selected for this study had no prior vaccination history and were deemed healthy, a crucial factor for the serological analysis. For wild birds, fresh fecal droppings and swabs were collected using a purposive sampling approach. Pigeons were humanely captured for swab collection. The study locations, which included natural water bodies, facilitated interactions between domestic poultry and wild birds. Additionally, a significant number of poultry were transported to local markets to meet high consumer demand, particularly during festive periods. 2.3.1. Sample size determination The sample size was determined using the previously documented prevalence rate of 11.6% [16], with a 95% confidence interval and a 5% margin of error. The calculation followed the formula proposed by [17] to ascertain the required sample size, as illustrated below: In order to assess the seroprevalence and perform molecular characterization of NDV, an initial sample size of 157 birds per population was calculated. However, due to logistical limitations in the field, a total of 300 domestic chicken samples and 70 wild bird samples were ultimately collected (Figure 2). For the domestic chickens, 150 serum samples were subjected to the Hemagglutination Inhibition (HI) test, resulting in the identification of 19 positive samples. Furthermore, 150 tracheal and cloacal swabs were obtained and subsequently pooled in groups of five for RNA extraction and reverse transcription polymerase chain reaction (RT-PCR) analysis, with seven pooled samples (representing 35 individual birds) testing positive for NDV. In the case of wild birds, 60 pooled fecal droplet samples and 10 swab samples were collected. The RNA extraction and RT-PCR analysis indicated that all pooled fecal samples were negative, while five out of the ten swab samples yielded positive results. All samples were preserved at a temperature of 4°C and subsequently transported to the National Animal Health Diagnostic and Investigation Center (NAHDIC) in Sebeta for further analysis. Although the sample size for wild birds did not meet the initially estimated threshold of 157, the findings still offer significant insights into the circulation of NDV within both domestic and wild bird populations. 2.4. Study design A cross-sectional study was conducted to evaluate the seroprevalence of Newcastle Disease (ND) in domestic chickens within the designated study areas (see Figure 2). Swab and fecal samples were collected from both domestic chickens and wild avian species for molecular characterization. Sampling was performed on two consecutive market days in each district to ensure an adequate sample size. In total, 67 volunteers and poultry traders participated in structured interviews using a questionnaire that had been translated into the local languages, Oromic and Amharic. The questionnaire addressed various topics, including methods of transporting chickens to market, domestic cleaning practices, historical occurrences of Newcastle Disease (ND) outbreaks, the seasonality of ND incidence, susceptibility based on age and breed, and the proximity of respondents' locations to lakes and markets (within a 10 km radius) where outbreaks had been documented. Furthermore, the questionnaire explored the potential for cross-contact between wild and domestic birds. Respondents were also asked about their understanding of ND transmission, prevention and control strategies, clinical symptoms (i.e., mass mortality, respiratory distress, diarrhea, ocular and nasal discharges, or neurological signs), and the vaccination history within their communities, as referenced in the study by [18]. 2.5. Sample collection and test procedure 2.5.1. Blood collection and serum preparation Blood samples were aseptically collected from the brachial vein of each chicken using 21-gauge sterile hypodermic needles and 3 mL syringes. Each syringe was properly labeled, and the blood samples were allowed to clot at a slight angle to facilitate serum separation. After the clot had formed, the serum was carefully harvested and transferred into sterile serum vials for long-term storage. The serum samples were preserved at -80°C to maintain their integrity prior to viral analysis. 2.5.2. Swab collection and sample processing A total of 150 chickens were sampled across three districts, with 50 birds collected from each district. For each bird, paired tracheal and cloacal swabs were obtained. These swabs were pooled in groups of five to enhance sample throughput while minimizing handling time and material costs. The pooled swabs were immediately stored in 2 mL cryovials containing freshly prepared viral transport medium (VTM), a standard procedure designed to preserve viral particles and prevent degradation. Each chicken contributed to both tracheal and cloacal swab pools, thereby providing a comprehensive assessment of viral shedding from both respiratory and intestinal sites. The pooled samples were subsequently stored at -80°C for future molecular analyses. During transportation to the National Animal Health Diagnostic and Investigation Center (NAHDIC) in Sebeta, Ethiopia, all samples were kept on ice to maintain their quality. Upon arrival, the samples were transferred to -80°C storage until processing began. 2.5.3. Collection of wild bird fecal samples Fecal samples were collected from wild birds in accordance with ethical standards and institutional guidelines, as approved by the Institutional Animal Care and Use Committee (IACUC). Fecal droplets were pooled from five distinct locations within the sampling area, ensuring that each droplet was likely sourced from a different individual bird. The pooled fecal material was homogenized and suspended at a 10% weight/volume (w/v) concentration in viral transport medium (VTM). This methodology preserved viral particles, which were subsequently analyzed for viral nucleic acids using molecular techniques. The collection and pooling strategy were designed to minimize stress on the animals while maximizing the efficiency of viral detection across a diverse population of wild birds. 2.5.4. Justification of sample size and statistical considerations The determination of sample size was based on an estimated viral prevalence and the necessary statistical power to identify significant results. Using standard statistical methodologies for prevalence studies, the target sample size for each district was calculated to be 157 chickens. In this investigation, a total of 150 birds were sampled across the three districts, which is slightly below the calculated target; however, this number was deemed sufficient for preliminary analysis, considering regional variability and the pooled sampling approach. In addressing potential concerns related to the pooled sampling methodology, it is important to emphasize that pooling was implemented to reduce costs and processing time, particularly for swabs and fecal samples. Pooling is a widely accepted strategy in epidemiological research that enables large-scale sampling when individual sampling is logistically impractical. However, the pooling of samples may affect detection sensitivity and statistical power. The impact of pooling on result accuracy was carefully evaluated during the study's design phase, and the sample size was chosen to balance practical limitations with the need for statistical rigor. 2.5.4.1. Haemagglutination (HA) Test: The haemagglutination (HA) test was performed using microplates in accordance with the guidelines established by the World Organisation for Animal Health (OIE). Inactivated antigens for the HA assay, along with positive and negative control sera for the haemagglutination inhibition (HI) test, were obtained from the Istituto Zooprofilattico Sperimentale delle Venezie, which serves as the OIE/FAO Reference Laboratory for Avian Influenza and Newcastle Disease located in Padua, Italy. The methodology involved preparing two-fold dilutions of virus suspensions (0.025 mL) in phosphate-buffered saline (PBS), which were subsequently transferred into V-bottomed microtiter plates. Following this, 0.025 mL of a 1% (v/v) suspension of chicken red blood cells (RBCs) was added to each well. The plates were then gently mixed and allowed to stand undisturbed at room temperature for approximately 30 minutes. The occurrence of haemagglutination was evaluated against a white background, in accordance with the OIE protocol (OIE, 2013). 2.5.4.2. Haemagglutination Inhibition (HI) Test The Hemagglutination Inhibition (HI) test was conducted to specifically identify hemagglutinating agents, following the guidelines established by the World Organisation for Animal Health (OIE) in 2013. The methodology utilized inactivated Newcastle Disease Virus (NDV) antigens derived from the LaSota strain, along with monospecific antiserum against NDVs and monoclonal antibodies targeting Pigeon paramyxoviruses, all obtained from the OIE/FAO Reference Laboratory. A series of two-fold dilutions of the virus suspension (0.025 mL) were prepared in phosphate-buffered saline (PBS), to which 4 hemagglutination units (4 HAU) of antigen were added in each well. The plates were then incubated at room temperature for 30 minutes. After this incubation period, 0.025 mL of a 1% suspension of chicken red blood cells (RBCs) was added to each well. The HI titer was determined by identifying the highest dilution of serum that completely inhibited the hemagglutinating activity of 4 HAU of antigen. Results were assessed by tilting the plate, with inhibition evaluated by comparing the sedimentation rate of the RBCs to that of control wells containing only PBS and RBCs (OIE, 2013). 2.5.4.3. Polymerase Chain Reaction (PCR) RNA extraction from swab and fecal samples was performed using the Qiagen® RNeasy Mini Kit, following the manufacturer's guidelines. Real-time reverse transcription PCR (RT-PCR) was conducted with an Applied Biosystems 7500 Fast Real-Time PCR Thermocycler [19]. The primer-probe set used for the specific detection of pathogenic strains of Newcastle Disease Virus (NDV) included Forward Primer F +4829, sequence (5’-GGT GAG TCT ATC CGG ARG ATA CAA G-3’), Reverse Primer F-4939, sequence (5’-AGC TGT TGC AAC CCC AAG-3’), and Probe F +4894 (5’-FAM–AAG CGT TTC TGT CTC CTT CCT CCA-BHQ-3’). The reaction mixture, with a total volume of 25 μL, consisted of 5 μL of 5x PCR buffer, 1 μL of each primer (at a concentration of 20 pmol), 1 μL of the probe (6 pmol), 0.8 μL of dNTPs, 1.25 μL of 25 mM MgCl2, 1 μL of RNase inhibitor (13.3 u/μL), 1 μL of Qiagen enzyme mix, and 6.95 μL of RNase-free water. The reverse transcription step involved incubation at 50°C for 30 minutes, followed by 15-minute incubation at 95°C. The cycling conditions included 40 cycles of denaturation at 94°C for 10 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 10 seconds (Wise et al., 2004). Signals from the reporter dye (FAM) were recorded during the extension phase, and the threshold cycle (Ct) for each sample was determined. Samples with a Ct value of less than 35 were classified as positive for the NDV F gene (OIE, 2013) 3. Data analysis Data obtained from laboratory tests were systematically recorded in Microsoft Excel and subsequently coded for statistical analysis. The analysis was performed using STATA Version 18.0. Logistic regression was employed to assess the risk factors associated with Newcastle Disease Virus (NDV) infection. Chi-square (χ²) tests were conducted to evaluate the associations between outcomes and risk factors when cell counts exceeded five. In instances where cell counts were smaller, Fisher’s exact test was utilized. A p-value of less than 0.05, corresponding to a 95% confidence interval, was considered statistically significant. 4. Results 4.1. Serology results A total of 150 chickens from three districts such as Bishoftu, Hawassa, and Batu were analyzed serologically for the presence of antibodies against Newcastle Disease (ND). The overall seroprevalence was found to be 12.7%, as determined by hemagglutination inhibition (HI) titers exceeding 2^−4, indicating prior exposure to ND. Among the districts, Bishoftu exhibited the highest prevalence at 20%, which was significantly greater than the 12% observed in Hawassa and the 6% in Batu (p = 0.04) (Table 1). These results suggest that the epidemiology of ND may be influenced by local environmental conditions or management practices, consistent with findings from previous studies. Table 1 : Sero-prevalence of ND by HI test in different districts Districts No. Examined No. positive Prevalence (%) P value χ 2 Bishoftu 50 10 20 0.04 4.4596 Hawassa 50 6 12 Batu 50 3 6 Total 150 19 12.7 Note : Statistical significance was assessed at a 95% confidence level (p < 0.05) was considered significant. The chi square (χ²) test was used to evaluate differences in seroprevalence across districts. A statistically significant difference was observed in Bishoftu (p = 0.04, χ² = 4.4596), indicating a higher seroprevalence compared to other districts. A more comprehensive examination of risk factors identified district location as the most significant predictor of ND infection, with chickens from Bishoftu being 4.2 times more likely to test positive than those from Batu (odds ratio [OR] = 4.2, p = 0.04). This association may be attributed to the higher density of poultry farms and market activities in Bishoftu, which could facilitate the transmission of the virus (Table 2). Table 2 : ND result by HI and associated risk factors. PCR result Odds ratio Std. error P value 95% CI Sex Male 1.8 0.94 0.28 0.62 - 5.06 Districts Bishoftu 4.2 2.90 0.04 1.05 -16.37 Hawassa 2.2 1.66 0.27 0.52 -9.61 Age Old 0.9 0.50 0.89 0.32 – 2.69 Constant 0.0448 0.03 0.00 0.001 - 0.20 4.2. Real-Time RT-PCR Results Real-time reverse transcription polymerase chain reaction (RT-PCR) was performed on 60 pooled swab samples, yielding an overall detection rate of 16.7% for Newcastle disease virus (NDV). The highest prevalence was observed in Bishoftu at 20%, which is consistent with serological findings, while both Hawassa and Batu exhibited a prevalence of 15%. The cycle threshold (Ct) values for the positive samples ranged from 15.35 to 30.50, with a mean Ct of 24.73, indicating variability in viral loads among the infected individuals (Table 3). Table 3: gene expression results using RT-PCR analysis Districts Sex Age Site of swab collected Ct. value Bishoftu Female Young Cloaca 20.35 Bishoftu Male Old Trachea 15.35 Bishoftu Male Old Trachea 25.77 Bishoftu Female Old Cloaca 25 Batu Male Old Trachea 30.5 Batu Female Old Cloaca 20 Batu Male Old Cloaca 24.5 Hawassa Male Old Cloaca 26.2 Hawassa Male Old Trachea 31.5 Hawassa Female Young Cloaca 28.1 Mean 24.73 4.3. Wild Bird Surveillance The monitoring of wild avian species has yielded significant results: out of 60 fecal samples collected from various wild birds, including ducks, sparrows, and guinea fowls, none tested positive for Newcastle Disease Virus (NDV). In contrast, five pooled samples from pigeons in Batu were found to be positive for virulent strains of NDV. These pigeon samples exhibited a mean Ct value of 26, suggesting active viral shedding. Recent literature has emphasized the potential role of pigeons in the transmission of NDV, particularly in rural areas, indicating that they may serve as reservoirs or amplifiers of ND outbreaks in nearby poultry populations. 4.4. Questionnaire Results A structured questionnaire was administered to 67 poultry owners across three districts, revealing that nearly all respondents (98.5%) had experienced outbreaks of Newcastle Disease (ND) within the past years. A significant proportion (46.26%) of these outbreaks occurred during the short rainy season, which aligns with previous research indicating a seasonal increase in ND cases due to heightened avian mobility and elevated humidity levels that facilitate viral transmission (Table 4). Furthermore, 70.14% of participants reported outbreaks in areas where domestic chickens had either direct or indirect interactions with wild birds, underscoring the role of wildlife in the epidemiology of ND. Despite the high incidence of ND, only 1.49% of respondents demonstrated a comprehensive understanding of ND transmission dynamics and control strategies, highlighting a substantial knowledge gap that could hinder effective disease management efforts. Table 4 : Summary of responses from the questionnaire survey Category of Variable N = 67 % Way of transportation Carry by hand 41 61.19 By cart 14 20.89 By car 12 17.91 Respondents’ clean chicken’s room daily (% Yes) 60 89.55 Encountered ND outbreaks (% Yes) 66 98.5 Season of ND outbreaks Short rainy season 31 46.26 Long rainy season 19 28.35 Dry cool season 5 7.46 Throughout the year 2 2.98 Treatment method used Traditional 24 35.82 Veterinary clinic 25 37.31 No medication 17 25.37 Knowledge about ND (low/medium/high) 36 / 14 / 1 53.73 / 20.89 / 1.49 Age group most affected by ND Young 20 29.85 Layers 35 52.23 Mixed 9 13.43 Breed most affected by ND Local 32 47.76 Exotic 22 32.83 Cross 3 4.47 Observed ND outbreak by distance from lake 20 km 3 4.54 Observed ND outbreak by distance from market (% Yes) 49 73.13 ND outbreak linked to contact with wild birds (% Yes) 47 70.14 5. Discussion This research investigated the seroprevalence and molecular identification of NDV in backyard chickens and wild birds within the Rift Valley regions of Ethiopia. The overall seroprevalence of NDV in backyard chickens was found to be 12.7%, which aligns with previous findings from Hawassa (11.9%) [20], the central Rift Valley (11.6–13.26%) [16,21], and Adami Tulu Jido Kombolcha (16.7%) [22]. However, these results are lower than those reported in the Rift Valley (22.5%) [23] and Addis Ababa (28.6%) [24], yet higher than the figures from Kersna Kondaltity (5.6%) [15] and seasonal studies conducted in the Rift Valley (5.9–6%) [22]. Such discrepancies may be attributed to variations in sampling periods, flock management practices, and ecological conditions. A statistically significant difference in seroprevalence was noted across different districts (p = 0.035), with the highest prevalence recorded in Bishoftu (20%), followed by Hawassa (12%) and Batu (6%). The elevated prevalence in Bishoftu may be associated with its ecological environment, which supports a high population of migratory wild birds, potentially enhancing the transmission of NDV. Notably, none of the fecal samples collected from wild birds tested positive for NDV, corroborating previous research that indicates an ambiguous role of wild birds in the persistence of NDV [25]. Molecular analysis utilizing RT-PCR identified NDV in 16.7% of pooled swab samples, with the highest detection rate occurring in Bishoftu (20%), followed by Hawassa and Batu (15%). These results are consistent with earlier studies that reported an overall positivity rate of 8.4% for swab samples, with Bishoftu again showing the highest prevalence (12%) and Batu the lowest (2%) [16]. While RT-PCR effectively confirmed the presence of NDV, further molecular characterization is necessary to ascertain the virulence of the strains involved. The interaction between domestic chickens and wild birds, combined with inadequate biosecurity measures, likely facilitates the transmission of NDV. The identification of NDV in pigeons from Batu highlights the potential reservoir role of wild birds [26,27]. However, the lack of NDV detection in fecal samples from wild birds suggests that the environmental persistence of the virus may be influenced by climatic factors and patterns of viral shedding [28]. Previous studies have indicated that NDV can still be transmitted through excretions such as feces and respiratory secretions, underscoring the need for ongoing surveillance [25,29]. A questionnaire survey indicated that 98.5% of respondents had observed outbreaks of ND, primarily within a 10 km radius of live bird markets and lakes. The absence of vaccination, reliance on traditional treatment methods (35.8%), and limited access to veterinary services contribute to the uncontrolled transmission of NDV. Similar challenges have been documented in the Rift Valley [28] and Northern Ethiopia [25]. Live bird markets represent a significant transmission nexus, where clinically healthy birds may serve as asymptomatic carriers of virulent NDV strains [29]. 6. Conclusion This research revealed a seroprevalence of 12.7% for ND antibodies among backyard chickens, indicating a significant level of exposure to the virus in the examined regions. The application of RT-PCR identified NDV strains in 16.7% of the swab samples, underscoring the critical role of molecular diagnostics in the detection and management of ND outbreaks. The heightened seroprevalence observed in Bishoftu suggests that factors such as migratory wild birds and local ecological conditions may contribute to the transmission of the virus. Furthermore, the study highlights the influence of live bird markets and inadequate biosecurity practices on the spread of ND. Future research and surveillance programs should incorporate molecular diagnostic techniques, such as RT-PCR, to facilitate ongoing monitoring of NDV circulation and mitigate its effects on both commercial and traditional poultry farming systems. Abbreviations Not applicable Declarations Ethics approval and consent to participate Ethical approval for the present study was secured from the appropriate institutional review board, thereby ensuring compliance with all ethical and legal standards. Informed consent was obtained from all poultry owners, who were thoroughly informed about the study's aims, methodologies, and any potential risks involved. Participants provided written consent voluntarily, with the assurance that they could withdraw from the study at any point without facing any repercussions. All animal handling procedures adhered to established veterinary care standards to mitigate any distress or harm, with an emphasis on prioritizing the welfare of the birds and maintaining confidentiality throughout the research process. Consent for publication Not applicable Availability of data and materials Data for this study can be made available upon reasonable request by the corresponding author Competing Interests We have no competing interests to declare Funding Not applicable Authors' contributions Author Contributions: G.H. Terara, B.K. Abebe, M.T Alemayehu, F. Abunna, and F. Dawo involved in the conception of the research idea, methodology design, supervision, performed data analysis, and interpretation. G.H. Terara, B.K. Abebe and M.T Alemayehu participated in data analysis, drafted, prepared the manuscript for publication, and revision. 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Int Sch Res Not 2014:513961 Leta S, Endalew B (2010) Survey on village-based chicken production and utilization system in Mid Rift Valley of Oromia, Ethiopia. Glob Vet 5:198–203 Moustapha A, Talaki E, Akourki A, Ousseini M (2023) Newcastle disease virus in poultry: current status and control prospects. Worlds Vet J 2:240–249 Mulisa DD, W/Kiros MK, Alemu RB, Keno MS, Furaso A, Heidari A, Chibsa TR, Chunde HC (2014) Characterization of Newcastle Disease Virus and poultry-handling practices in live poultry markets. Ethiopia SpringerPlus 3:459 Shapiro BI, Gebru G, Desta S, Negassa A, Negussie K, Aboset G, Mechal H (2015) Ethiopia livestock master plan: Roadmaps for growth and transformation. [ILRI report]. International Livestock Research Institute (ILRI), Nairobi, Kenya Osman AM, Oladele OA, Ibrahim AM, Mahamoud MA, Mohamed MA, Nwachukwu NO (2021) Seroprevalence of Newcastle disease in backyard chickens in selected districts of Banadir region, Somalia. Trop Anim Health Prod 53(2):192 Thrusfield M, Christley R (2007) Veterinary epidemiology, 3rd edn. Wiley-Blackwell, Hoboken, pp 230–240 Wise MG, Suarez DL, Seal BS, Pedersen JC, Senne DA, King DJ, Kapczynski DR, Spackman E (2004) Development of a real-time reverse-transcription PCR for detection of Newcastle disease virus RNA in clinical samples. J Clin Microbiol 42:329–338 Zeleke A, Sori T, Gelaye E, Ayelet G (2005) Newcastle disease in village chickens in the southern and Rift Valley districts in Ethiopia. Int J Poult Sci 4:507–510 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7221776","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":492491176,"identity":"87e03668-945b-4767-a2c0-285b3bca332b","order_by":0,"name":"Gelana Haile Terara","email":"","orcid":"","institution":"Alage ATVET College","correspondingAuthor":false,"prefix":"","firstName":"Gelana","middleName":"Haile","lastName":"Terara","suffix":""},{"id":492491177,"identity":"75c77e7e-c309-4169-9f61-3a59d52b0949","order_by":1,"name":"Belete Kuraz Abebe","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYBACAwbmBgbGBhs5EOfAA+K0MIK0pBmDtSSQoOVwYgOIR5QWc/bGxgc/dzCnzw87/BBoi52cbgMBLZY9B5sNe8+w5W68nWYA1JJsbHaAkMNuJLZJ8Lbx5G6cnQDSciBxG0Et9x+2//zbJpFuODv9A5FabjC2MfO2GSTIS+cQaYtlT2KztOyZBMMN0jkFBxIMiPCLOfvhgx/f7vgvLz87ffOHDxV2cgS1IFwIVmlArHIQkG8gRfUoGAWjYBSMKAAA7i5KyQB5vfkAAAAASUVORK5CYII=","orcid":"","institution":"Werabe University","correspondingAuthor":true,"prefix":"","firstName":"Belete","middleName":"Kuraz","lastName":"Abebe","suffix":""},{"id":492491179,"identity":"821c51e6-d7dd-4aa8-8f94-93a57e2b2d8f","order_by":2,"name":"Mulugeta Tesfaye Alemayehu","email":"","orcid":"","institution":"Werabe University","correspondingAuthor":false,"prefix":"","firstName":"Mulugeta","middleName":"Tesfaye","lastName":"Alemayehu","suffix":""},{"id":492491180,"identity":"f8862e28-703e-4ce1-9aea-a9e62501eae1","order_by":3,"name":"Fufa Abunna","email":"","orcid":"","institution":"Addis Ababa University","correspondingAuthor":false,"prefix":"","firstName":"Fufa","middleName":"","lastName":"Abunna","suffix":""},{"id":492491181,"identity":"ecdd91d1-94cb-4577-9f4c-52a5c1886d84","order_by":4,"name":"Fufa Dawo","email":"","orcid":"","institution":"Addis Ababa University","correspondingAuthor":false,"prefix":"","firstName":"Fufa","middleName":"","lastName":"Dawo","suffix":""}],"badges":[],"createdAt":"2025-07-26 14:23:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7221776/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7221776/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89825625,"identity":"d6dccd73-2627-44ae-83cd-76118b73b2d8","added_by":"auto","created_at":"2025-08-25 12:33:42","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":90429,"visible":true,"origin":"","legend":"\u003cp\u003eMap showing study areas in the Rift Valley lakes of Oromia and SNNP, Ethiopia.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7221776/v1/364f8b4ee4ff45b896fad4fd.jpg"},{"id":89825624,"identity":"7d2ffbb3-b9af-4fc9-8b8b-028b922742b0","added_by":"auto","created_at":"2025-08-25 12:33:42","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":95865,"visible":true,"origin":"","legend":"\u003cp\u003eIllustration of the study design investigating Newcastle Disease (ND) in both wild and domestic birds.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7221776/v1/734951f48b77ad9cac45c44e.jpg"},{"id":90737927,"identity":"ebd6a5e5-ca48-4ff2-8c55-aac4568690bb","added_by":"auto","created_at":"2025-09-06 23:46:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":881964,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7221776/v1/e1d4cc6e-f881-4626-977a-f327611c0e5f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative study of conventional and molecular epidemiology of Newcastle disease in wild and domestic birds in the Rift Valley region of Ethiopia","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eNewcastle Disease (ND), caused by the Newcastle disease virus (NDV), remains a significant global threat to the poultry industry, affecting both developing and developed economies due to its highly contagious nature and substantial economic impacts (Absal\u0026oacute;n et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Hu et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Moustapha et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Dharmayanti et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Despite advancements in vaccination and biosecurity measures, ND continues to inflict considerable losses in regions where these interventions are inadequately implemented, thereby threatening productivity and international poultry trade (Dimitrov et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Bello et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Hu et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Given ND's potential to disrupt food security and induce economic instability, there is an urgent need for comprehensive epidemiological research to inform targeted disease control strategies (Drewnowski, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe Rift Valley region of Ethiopia, recognized as a crucial area for poultry production, exhibits significant vulnerability attributable to its heterogeneous environmental conditions, encompassing both highland and lowland ecosystems, as well as diverse poultry management practices. These environmental factors include variable temperatures, seasonal precipitation patterns, and the coexistence of commercial and subsistence poultry farming systems. Although large-scale poultry operations may benefit from contemporary management techniques, numerous smallholder farmers continue to employ traditional farming practices that often lack adequate biosecurity and disease control protocols. This deficiency exacerbates the region's susceptibility to outbreaks of NDV (Asfaw, Ameni, Medhin, Gumi, Hagos, et al. 2021; Hailegebreal et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, the insufficient data regarding the prevalence of ND across different districts has hindered the implementation of effective control measures, despite the region's importance for national food security and economic development (Asfaw, Ameni, Medhin, Gumi and Wieland, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Previous studies have not sufficiently addressed the spatial and demographic factors that contribute to the spread of ND, nor have they employed advanced diagnostic technologies (Betela, Hailegebreal and Shiferaw, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThis study presents the first comprehensive epidemiological analysis of ND in the Rift Valley, focusing on three key districts: Bishoftu, Hawassa, and Batu. By utilizing both serological assays and real-time reverse transcription polymerase chain reaction (RT-PCR) techniques, we assessed the prevalence of ND and its associated risk factors, while examining demographic variables such as sex, age, and geographical location. Through the integration of these advanced diagnostic methodologies, our research aims to provide a detailed understanding of ND dynamics in a region that is crucial to Ethiopia's poultry industry.\u003c/p\u003e\u003cp\u003eND is a highly contagious disease that severely impacts poultry industries worldwide. Risk factors for ND include housing conditions (free-range vs. confined), contact with wild birds, and demographic factors like gender, age, and region. Free-range poultry are more exposed to wild birds, increasing the risk of NDV infection (Chaka et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). This study aimed to evaluate ND prevalence in domestic and wild chickens in Ethiopia, with a focus on identifying the impact of these risk factors on ND occurrence.\u003c/p\u003e\u003cp\u003eThe outcomes of this study are anticipated to significantly enhance ND surveillance efforts in the Rift Valley, refine vaccination strategies, and strengthen biosecurity measures. Our data-driven methodology establishes a framework for evidence-based interventions aimed at enhancing the health and productivity of Ethiopia's poultry sector, in accordance with the objectives set forth in Ethiopia\u0026rsquo;s Livestock Development Policy and Strategy (2015)(Shapiro et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and the National Poultry Development Strategy 2022\u0026ndash;2031 (Fekadu et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). By tackling the incidence of ND and improving diagnostic capabilities, the outcomes of this study contribute to the overarching goals of healthy disease control measures and increasing poultry productivity. These findings are essential for facilitating policy implementation, advancing biosecurity practices, and bolstering public health initiatives, with significant implications for disease control strategies not only within Ethiopia but also across the African continent and beyond.\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cp\u003e2.1. \u0026nbsp; \u0026nbsp; \u0026nbsp; Ethical standards\u003c/p\u003e\n\u003cp\u003eThis research was conducted in accordance with the ethical standards for animal research established by Addis Ababa University and relevant Ethiopian laws. The experimental protocol was reviewed and approved by the Animal Research Ethical Review Committee at Addis Ababa University (Certificate Reference No. VM/ERC/06/05/017).\u003c/p\u003e\n\u003cp\u003e2.2. \u0026nbsp; \u0026nbsp; \u0026nbsp;Study areas\u003c/p\u003e\n\u003cp\u003eThe research was conducted in the Rift Valley regions of the Eastern Shewa Zone, specifically within the Bishoftu and Batu districts of the Oromia Region, as well as in the Hawassa district of the Southern Nations, Nationalities, and Peoples (SNNP) Region. These districts are notable for their frequent visitation by migratory birds, which are attracted to the nearby water reservoirs that provide sustenance for wildlife. The investigation focused on urban markets within each of the selected districts. Geographically, the study areas are located between latitudes 7\u0026deg; 09 \u0026apos;N and 8\u0026deg;45\u0026apos;N and longitudes 38\u0026deg;32\u0026apos;E and 39\u0026deg;17\u0026apos;E, covering an approximate width of 40\u0026ndash;60 km and extending over 100 km in length. The climate of this semi-arid region is characterized by erratic and unreliable precipitation, with annual rainfall averaging between 500 and 900 mm. The rainfall pattern is bimodal, featuring long rains from June to September and short rains from February to April (Figure 1).\u003c/p\u003e\n\u003cp id=\"_Toc484555794\"\u003e2.3. \u0026nbsp;Study population\u003c/p\u003e\n\u003cp\u003eThe study subjects were sourced from the Rift Valley regions of Oromia and the Southern Nations, Nationalities, and Peoples\u0026apos; (SNNP) region in Ethiopia. Specimens were collected from the fecal droppings of wild birds, swabs from pigeons, and domestic backyard chickens. For the domestic chickens, tracheal and cloacal swabs, as well as serum samples, were obtained from individuals across various districts, categorized by sex and age. Age classification followed the criteria established by \u0026nbsp;[15], which designates chickens aged 3 to 6 months as young and those older than 6 months as adults.\u003c/p\u003e\n\u003cp\u003eThe backyard chickens selected for this study had no prior vaccination history and were deemed healthy, a crucial factor for the serological analysis. For wild birds, fresh fecal droppings and swabs were collected using a purposive sampling approach. Pigeons were humanely captured for swab collection. The study locations, which included natural water bodies, facilitated interactions between domestic poultry and wild birds. Additionally, a significant number of poultry were transported to local markets to meet high consumer demand, particularly during festive periods.\u003c/p\u003e\n\u003cp\u003e2.3.1. \u0026nbsp; Sample size determination\u003c/p\u003e\n\u003cp\u003eThe sample size was determined using the previously documented prevalence rate of 11.6% [16], with a 95% confidence interval and a 5% margin of error. The calculation followed the formula proposed by [17] to ascertain the required sample size, as illustrated below:\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\" style=\"width: 472px;\"\u003e\u003c/p\u003e\n\u003cp\u003eIn order to assess the seroprevalence and perform molecular characterization of NDV, an initial sample size of 157 birds per population was calculated. However, due to logistical limitations in the field, a total of 300 domestic chicken samples and 70 wild bird samples were ultimately collected (Figure 2). For the domestic chickens, 150 serum samples were subjected to the Hemagglutination Inhibition (HI) test, resulting in the identification of 19 positive samples. Furthermore, 150 tracheal and cloacal swabs were obtained and subsequently pooled in groups of five for RNA extraction and reverse transcription polymerase chain reaction (RT-PCR) analysis, with seven pooled samples (representing 35 individual birds) testing positive for NDV. In the case of wild birds, 60 pooled fecal droplet samples and 10 swab samples were collected.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe RNA extraction and RT-PCR analysis indicated that all pooled fecal samples were negative, while five out of the ten swab samples yielded positive results. All samples were preserved at a temperature of 4\u0026deg;C and subsequently transported to the National Animal Health Diagnostic and Investigation Center (NAHDIC) in Sebeta for further analysis. \u0026nbsp;Although the sample size for wild birds did not meet the initially estimated threshold of 157, the findings still offer significant insights into the circulation of NDV within both domestic and wild bird populations.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e2.4. \u0026nbsp; Study design\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eA cross-sectional study was conducted to evaluate the seroprevalence of Newcastle Disease (ND) in domestic chickens within the designated study areas (see Figure 2). Swab and fecal samples were collected from both domestic chickens and wild avian species for molecular characterization. Sampling was performed on two consecutive market days in each district to ensure an adequate sample size.\u003c/p\u003e\n\u003cp\u003eIn total, 67 volunteers and poultry traders participated in structured interviews using a questionnaire that had been translated into the local languages, Oromic and Amharic. The questionnaire addressed various topics, including methods of transporting chickens to market, domestic cleaning practices, historical occurrences of Newcastle Disease (ND) outbreaks, the seasonality of ND incidence, susceptibility based on age and breed, and the proximity of respondents\u0026apos; locations to lakes and markets (within a 10 km radius) where outbreaks had been documented. Furthermore, the questionnaire explored the potential for cross-contact between wild and domestic birds. Respondents were also asked about their understanding of ND transmission, prevention and control strategies, clinical symptoms (i.e., mass mortality, respiratory distress, diarrhea, ocular and nasal discharges, or neurological signs), and the vaccination history within their communities, as referenced in the study by [18].\u003c/p\u003e\n\u003cp\u003e2.5. \u0026nbsp; \u0026nbsp;Sample collection and test procedure\u003c/p\u003e\n\u003cp\u003e2.5.1. Blood collection and serum preparation\u003c/p\u003e\n\u003cp\u003eBlood samples were aseptically collected from the brachial vein of each chicken using 21-gauge sterile hypodermic needles and 3 mL syringes. Each syringe was properly labeled, and the blood samples were allowed to clot at a slight angle to facilitate serum separation. After the clot had formed, the serum was carefully harvested and transferred into sterile serum vials for long-term storage. The serum samples were preserved at -80\u0026deg;C to maintain their integrity prior to viral analysis.\u003c/p\u003e\n\u003cp\u003e2.5.2. Swab collection and sample processing\u003c/p\u003e\n\u003cp\u003eA total of 150 chickens were sampled across three districts, with 50 birds collected from each district. For each bird, paired tracheal and cloacal swabs were obtained. These swabs were pooled in groups of five to enhance sample throughput while minimizing handling time and material costs. The pooled swabs were immediately stored in 2 mL cryovials containing freshly prepared viral transport medium (VTM), a standard procedure designed to preserve viral particles and prevent degradation. Each chicken contributed to both tracheal and cloacal swab pools, thereby providing a comprehensive assessment of viral shedding from both respiratory and intestinal sites. The pooled samples were subsequently stored at -80\u0026deg;C for future molecular analyses. During transportation to the National Animal Health Diagnostic and Investigation Center (NAHDIC) in Sebeta, Ethiopia, all samples were kept on ice to maintain their quality. Upon arrival, the samples were transferred to -80\u0026deg;C storage until processing began.\u003c/p\u003e\n\u003cp\u003e2.5.3. Collection of wild bird fecal samples\u003c/p\u003e\n\u003cp\u003eFecal samples were collected from wild birds in accordance with ethical standards and institutional guidelines, as approved by the Institutional Animal Care and Use Committee (IACUC). Fecal droplets were pooled from five distinct locations within the sampling area, ensuring that each droplet was likely sourced from a different individual bird. The pooled fecal material was homogenized and suspended at a 10% weight/volume (w/v) concentration in viral transport medium (VTM). This methodology preserved viral particles, which were subsequently analyzed for viral nucleic acids using molecular techniques. The collection and pooling strategy were designed to minimize stress on the animals while maximizing the efficiency of viral detection across a diverse population of wild birds.\u003c/p\u003e\n\u003cp\u003e2.5.4. Justification of sample size and statistical considerations\u003c/p\u003e\n\u003cp\u003eThe determination of sample size was based on an estimated viral prevalence and the necessary statistical power to identify significant results. Using standard statistical methodologies for prevalence studies, the target sample size for each district was calculated to be 157 chickens. In this investigation, a total of 150 birds were sampled across the three districts, which is slightly below the calculated target; however, this number was deemed sufficient for preliminary analysis, considering regional variability and the pooled sampling approach. In addressing potential concerns related to the pooled sampling methodology, it is important to emphasize that pooling was implemented to reduce costs and processing time, particularly for swabs and fecal samples. Pooling is a widely accepted strategy in epidemiological research that enables large-scale sampling when individual sampling is logistically impractical. However, the pooling of samples may affect detection sensitivity and statistical power. The impact of pooling on result accuracy was carefully evaluated during the study\u0026apos;s design phase, and the sample size was chosen to balance practical limitations with the need for statistical rigor.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5.4.1. Haemagglutination (HA) Test:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe haemagglutination (HA) test was performed using microplates in accordance with the guidelines established by the World Organisation for Animal Health (OIE). Inactivated antigens for the HA assay, along with positive and negative control sera for the haemagglutination inhibition (HI) test, were obtained from the Istituto Zooprofilattico Sperimentale delle Venezie, which serves as the OIE/FAO Reference Laboratory for Avian Influenza and Newcastle Disease located in Padua, Italy. The methodology involved preparing two-fold dilutions of virus suspensions (0.025 mL) in phosphate-buffered saline (PBS), which were subsequently transferred into V-bottomed microtiter plates. Following this, 0.025 mL of a 1% (v/v) suspension of chicken red blood cells (RBCs) was added to each well. The plates were then gently mixed and allowed to stand undisturbed at room temperature for approximately 30 minutes. The occurrence of haemagglutination was evaluated against a white background, in accordance with the OIE protocol (OIE, 2013).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5.4.2. Haemagglutination Inhibition (HI) Test\u003c/strong\u003e\u003cbr\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eThe Hemagglutination Inhibition (HI) test was conducted to specifically identify hemagglutinating agents, following the guidelines established by the World Organisation for Animal Health (OIE) in 2013. The methodology utilized inactivated Newcastle Disease Virus (NDV) antigens derived from the LaSota strain, along with monospecific antiserum against NDVs and monoclonal antibodies targeting Pigeon paramyxoviruses, all obtained from the OIE/FAO Reference Laboratory. A series of two-fold dilutions of the virus suspension (0.025 mL) were prepared in phosphate-buffered saline (PBS), to which 4 hemagglutination units (4 HAU) of antigen were added in each well. The plates were then incubated at room temperature for 30 minutes. After this incubation period, 0.025 mL of a 1% suspension of chicken red blood cells (RBCs) was added to each well. The HI titer was determined by identifying the highest dilution of serum that completely inhibited the hemagglutinating activity of 4 HAU of antigen. Results were assessed by tilting the plate, with inhibition evaluated by comparing the sedimentation rate of the RBCs to that of control wells containing only PBS and RBCs (OIE, 2013).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5.4.3. Polymerase Chain Reaction (PCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA extraction from swab and fecal samples was performed using the Qiagen\u0026reg; RNeasy Mini Kit, following the manufacturer\u0026apos;s guidelines. Real-time reverse transcription PCR (RT-PCR) was conducted with an Applied Biosystems 7500 Fast Real-Time PCR Thermocycler [19]. The primer-probe set used for the specific detection of pathogenic strains of Newcastle Disease Virus (NDV) included Forward Primer F +4829, sequence (5\u0026rsquo;-GGT GAG TCT ATC CGG ARG ATA CAA G-3\u0026rsquo;), Reverse Primer F-4939, sequence (5\u0026rsquo;-AGC TGT TGC AAC CCC AAG-3\u0026rsquo;), and Probe F +4894 (5\u0026rsquo;-FAM\u0026ndash;AAG CGT TTC TGT CTC CTT CCT CCA-BHQ-3\u0026rsquo;). The reaction mixture, with a total volume of 25 \u0026mu;L, consisted of 5 \u0026mu;L of 5x PCR buffer, 1 \u0026mu;L of each primer (at a concentration of 20 pmol), 1 \u0026mu;L of the probe (6 pmol), 0.8 \u0026mu;L of dNTPs, 1.25 \u0026mu;L of 25 mM MgCl2, 1 \u0026mu;L of RNase inhibitor (13.3 u/\u0026mu;L), 1 \u0026mu;L of Qiagen enzyme mix, and 6.95 \u0026mu;L of RNase-free water. The reverse transcription step involved incubation at 50\u0026deg;C for 30 minutes, followed by 15-minute incubation at 95\u0026deg;C. The cycling conditions included 40 cycles of denaturation at 94\u0026deg;C for 10 seconds, annealing at 58\u0026deg;C for 30 seconds, and extension at 72\u0026deg;C for 10 seconds (Wise et al., 2004). Signals from the reporter dye (FAM) were recorded during the extension phase, and the threshold cycle (Ct) for each sample was determined. Samples with a Ct value of less than 35 were classified as positive for the NDV F gene (OIE, 2013)\u003c/p\u003e"},{"header":"3. Data analysis","content":"\u003cp\u003eData obtained from laboratory tests were systematically recorded in Microsoft Excel and subsequently coded for statistical analysis. The analysis was performed using STATA Version 18.0. Logistic regression was employed to assess the risk factors associated with Newcastle Disease Virus (NDV) infection. Chi-square (\u0026chi;\u0026sup2;) tests were conducted to evaluate the associations between outcomes and risk factors when cell counts exceeded five. In instances where cell counts were smaller, Fisher\u0026rsquo;s exact test was utilized. A p-value of less than 0.05, corresponding to a 95% confidence interval, was considered statistically significant.\u003c/p\u003e"},{"header":"4. Results","content":"\u003cp\u003e\u003cstrong\u003e4.1. Serology results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 150 chickens from three districts such as Bishoftu, Hawassa, and Batu were analyzed serologically for the presence of antibodies against Newcastle Disease (ND). The overall seroprevalence was found to be 12.7%, as determined by hemagglutination inhibition (HI) titers exceeding 2^\u0026minus;4, indicating prior exposure to ND. Among the districts, Bishoftu exhibited the highest prevalence at 20%, which was significantly greater than the 12% observed in Hawassa and the 6% in Batu (p = 0.04) (Table 1). These results suggest that the epidemiology of ND may be influenced by local environmental conditions or management practices, consistent with findings from previous studies.\u003c/p\u003e\n\u003cp id=\"_Toc484518807\"\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e: Sero-prevalence of ND\u0026nbsp;by HI test in different districts\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"697\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12.1951%;\"\u003e\n \u003cp\u003eDistricts\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.66%;\"\u003e\n \u003cp\u003eNo. Examined\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 20.66%;\"\u003e\n \u003cp\u003eNo. positive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.9383%;\"\u003e\n \u003cp\u003ePrevalence (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.6341%;\"\u003e\n \u003cp\u003eP value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.9125%;\"\u003e\n \u003cp\u003e\u0026chi;\u003cem\u003e\u003csup\u003e2\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12.1951%;\"\u003e\n \u003cp\u003eBishoftu\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.66%;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.33%;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.33%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.9383%;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.6341%;\"\u003e\n \u003cp\u003e0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.9125%;\"\u003e\n \u003cp\u003e4.4596\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12.1951%;\"\u003e\n \u003cp\u003eHawassa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.66%;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.33%;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.33%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.9383%;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.6341%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.9125%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12.1951%;\"\u003e\n \u003cp\u003eBatu\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.66%;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.33%;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.33%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.9383%;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.6341%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.9125%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12.1951%;\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.66%;\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.33%;\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.33%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.9383%;\"\u003e\n \u003cp\u003e12.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.6341%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.9125%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eNote\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e: Statistical significance was assessed at a 95% confidence level (p \u0026lt; 0.05) was considered significant. The chi square (\u0026chi;\u0026sup2;) test was used to evaluate differences in seroprevalence across districts. A statistically significant difference was observed in Bishoftu (p = 0.04, \u0026chi;\u0026sup2; = 4.4596), indicating a higher seroprevalence compared to other districts.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA more comprehensive examination of risk factors identified district location as the most significant predictor of ND infection, with chickens from Bishoftu being 4.2 times more likely to test positive than those from Batu (odds ratio [OR] = 4.2, p = 0.04). This association may be attributed to the higher density of poultry farms and market activities in Bishoftu, which could facilitate the transmission of the virus (Table 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e: ND result by HI and associated risk factors.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 21.6478%;\"\u003e\n \u003cp\u003ePCR result\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.4782%;\"\u003e\n \u003cp\u003eOdds ratio\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.3166%;\"\u003e\n \u003cp\u003eStd. error\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.4782%;\"\u003e\n \u003cp\u003eP value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.0792%;\"\u003e\n \u003cp\u003e95% CI\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 21.6478%;\"\u003e\n \u003cp\u003eSex\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" style=\"width: 78.3522%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 21.6478%;\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.4782%;\"\u003e\n \u003cp\u003e1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.3166%;\"\u003e\n \u003cp\u003e0.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.4782%;\"\u003e\n \u003cp\u003e0.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.0792%;\"\u003e\n \u003cp\u003e0.62 - 5.06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 21.6478%;\"\u003e\n \u003cp\u003eDistricts\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.4782%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.3166%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.4782%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.0792%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 21.6478%;\"\u003e\n \u003cp\u003eBishoftu\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.4782%;\"\u003e\n \u003cp\u003e4.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.3166%;\"\u003e\n \u003cp\u003e2.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.4782%;\"\u003e\n \u003cp\u003e0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.0792%;\"\u003e\n \u003cp\u003e1.05 -16.37\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 21.6478%;\"\u003e\n \u003cp\u003eHawassa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.4782%;\"\u003e\n \u003cp\u003e2.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.3166%;\"\u003e\n \u003cp\u003e1.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.4782%;\"\u003e\n \u003cp\u003e0.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.0792%;\"\u003e\n \u003cp\u003e0.52 -9.61\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 21.6478%;\"\u003e\n \u003cp\u003eAge\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" style=\"width: 78.3522%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 21.6478%;\"\u003e\n \u003cp\u003eOld\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.4782%;\"\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.3166%;\"\u003e\n \u003cp\u003e0.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.4782%;\"\u003e\n \u003cp\u003e0.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.0792%;\"\u003e\n \u003cp\u003e0.32 \u0026ndash; 2.69\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 21.6478%;\"\u003e\n \u003cp\u003eConstant\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.4782%;\"\u003e\n \u003cp\u003e0.0448\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.3166%;\"\u003e\n \u003cp\u003e0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.4782%;\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.0792%;\"\u003e\n \u003cp\u003e0.001 - 0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e4.2. Real-Time RT-PCR Results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eReal-time reverse transcription polymerase chain reaction (RT-PCR) was performed on 60 pooled swab samples, yielding an overall detection rate of 16.7% for Newcastle disease virus (NDV). The highest prevalence was observed in Bishoftu at 20%, which is consistent with serological findings, while both Hawassa and Batu exhibited a prevalence of 15%. The cycle threshold (Ct) values for the positive samples ranged from 15.35 to 30.50, with a mean Ct of 24.73, indicating variability in viral loads among the infected individuals (Table 3).\u003c/p\u003e\n\u003cp id=\"_Toc484518811\"\u003e\u003cstrong\u003eTable 3:\u0026nbsp;\u003c/strong\u003egene expression results using RT-PCR analysis\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"655\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0153%;\"\u003e\n \u003cp\u003eDistricts\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0153%;\"\u003e\n \u003cp\u003eSex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0153%;\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0458%;\"\u003e\n \u003cp\u003eSite of swab collected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.9084%;\"\u003e\n \u003cp\u003eCt. value\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0153%;\"\u003e\n \u003cp\u003eBishoftu\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0153%;\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0153%;\"\u003e\n \u003cp\u003eYoung\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0458%;\"\u003e\n \u003cp\u003eCloaca\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.9084%;\"\u003e\n \u003cp\u003e20.35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0153%;\"\u003e\n \u003cp\u003eBishoftu\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0153%;\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0153%;\"\u003e\n \u003cp\u003eOld\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0458%;\"\u003e\n \u003cp\u003eTrachea\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.9084%;\"\u003e\n \u003cp\u003e15.35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0153%;\"\u003e\n \u003cp\u003eBishoftu\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0153%;\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0153%;\"\u003e\n \u003cp\u003eOld\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0458%;\"\u003e\n \u003cp\u003eTrachea\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.9084%;\"\u003e\n \u003cp\u003e25.77\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0153%;\"\u003e\n \u003cp\u003eBishoftu\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0153%;\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0153%;\"\u003e\n \u003cp\u003eOld\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0458%;\"\u003e\n \u003cp\u003eCloaca\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.9084%;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0153%;\"\u003e\n \u003cp\u003eBatu\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0153%;\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0153%;\"\u003e\n \u003cp\u003eOld\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0458%;\"\u003e\n \u003cp\u003eTrachea\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.9084%;\"\u003e\n \u003cp\u003e30.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0153%;\"\u003e\n \u003cp\u003eBatu\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0153%;\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0153%;\"\u003e\n \u003cp\u003eOld\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0458%;\"\u003e\n \u003cp\u003eCloaca\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.9084%;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0153%;\"\u003e\n \u003cp\u003eBatu\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0153%;\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0153%;\"\u003e\n \u003cp\u003eOld\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0458%;\"\u003e\n \u003cp\u003eCloaca\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.9084%;\"\u003e\n \u003cp\u003e24.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0153%;\"\u003e\n \u003cp\u003eHawassa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0153%;\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0153%;\"\u003e\n \u003cp\u003eOld\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0458%;\"\u003e\n \u003cp\u003eCloaca\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.9084%;\"\u003e\n \u003cp\u003e26.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0153%;\"\u003e\n \u003cp\u003eHawassa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0153%;\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0153%;\"\u003e\n \u003cp\u003eOld\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0458%;\"\u003e\n \u003cp\u003eTrachea\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.9084%;\"\u003e\n \u003cp\u003e31.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0153%;\"\u003e\n \u003cp\u003eHawassa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0153%;\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0153%;\"\u003e\n \u003cp\u003eYoung\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0458%;\"\u003e\n \u003cp\u003eCloaca\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.9084%;\"\u003e\n \u003cp\u003e28.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0153%;\"\u003e\n \u003cp\u003eMean\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0153%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0153%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0458%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.9084%;\"\u003e\n \u003cp\u003e24.73\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e4.3. Wild Bird Surveillance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe monitoring of wild avian species has yielded significant results: out of 60 fecal samples collected from various wild birds, including ducks, sparrows, and guinea fowls, none tested positive for Newcastle Disease Virus (NDV). In contrast, five pooled samples from pigeons in Batu were found to be positive for virulent strains of NDV. These pigeon samples exhibited a mean Ct value of 26, suggesting active viral shedding. Recent literature has emphasized the potential role of pigeons in the transmission of NDV, particularly in rural areas, indicating that they may serve as reservoirs or amplifiers of ND outbreaks in nearby poultry populations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.4. Questionnaire Results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA structured questionnaire was administered to 67 poultry owners across three districts, revealing that nearly all respondents (98.5%) had experienced outbreaks of Newcastle Disease (ND) within the past years. A significant proportion (46.26%) of these outbreaks occurred during the short rainy season, which aligns with previous research indicating a seasonal increase in ND cases due to heightened avian mobility and elevated humidity levels that facilitate viral transmission (Table 4). Furthermore, 70.14% of participants reported outbreaks in areas where domestic chickens had either direct or indirect interactions with wild birds, underscoring the role of wildlife in the epidemiology of ND. Despite the high incidence of ND, only 1.49% of respondents demonstrated a comprehensive understanding of ND transmission dynamics and control strategies, highlighting a substantial knowledge gap that could hinder effective disease management efforts.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4\u003c/strong\u003e: Summary of responses from the questionnaire survey\u003c/p\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"835\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 43.9521%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCategory of Variable\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23.8323%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eN = 67\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 32.2156%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 43.9521%;\"\u003e\n \u003cp\u003eWay of transportation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 23.8323%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 32.2156%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 43.9521%;\"\u003e\n \u003cp\u003e\u0026nbsp; Carry by hand\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 23.8323%;\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 32.2156%;\"\u003e\n \u003cp\u003e61.19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 43.9521%;\"\u003e\n \u003cp\u003e\u0026nbsp; By cart\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 23.8323%;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 32.2156%;\"\u003e\n \u003cp\u003e20.89\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 43.9521%;\"\u003e\n \u003cp\u003e\u0026nbsp; By car\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 23.8323%;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 32.2156%;\"\u003e\n \u003cp\u003e17.91\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 43.9521%;\"\u003e\n \u003cp\u003eRespondents\u0026rsquo; clean chicken\u0026rsquo;s room daily (% Yes)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 23.8323%;\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 32.2156%;\"\u003e\n \u003cp\u003e89.55\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 43.9521%;\"\u003e\n \u003cp\u003eEncountered ND outbreaks (% Yes)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 23.8323%;\"\u003e\n \u003cp\u003e66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 32.2156%;\"\u003e\n \u003cp\u003e98.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 43.9521%;\"\u003e\n \u003cp\u003eSeason of ND outbreaks\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 23.8323%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 32.2156%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 43.9521%;\"\u003e\n \u003cp\u003e\u0026nbsp; Short rainy season\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 23.8323%;\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 32.2156%;\"\u003e\n \u003cp\u003e46.26\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 43.9521%;\"\u003e\n \u003cp\u003e\u0026nbsp; Long rainy season\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 23.8323%;\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 32.2156%;\"\u003e\n \u003cp\u003e28.35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 43.9521%;\"\u003e\n \u003cp\u003e\u0026nbsp; Dry cool season\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 23.8323%;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 32.2156%;\"\u003e\n \u003cp\u003e7.46\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 43.9521%;\"\u003e\n \u003cp\u003e\u0026nbsp; Throughout the year\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 23.8323%;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 32.2156%;\"\u003e\n \u003cp\u003e2.98\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 43.9521%;\"\u003e\n \u003cp\u003eTreatment method used\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 23.8323%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 32.2156%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 43.9521%;\"\u003e\n \u003cp\u003e\u0026nbsp; Traditional\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 23.8323%;\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 32.2156%;\"\u003e\n \u003cp\u003e35.82\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 43.9521%;\"\u003e\n \u003cp\u003e\u0026nbsp; Veterinary clinic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 23.8323%;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 32.2156%;\"\u003e\n \u003cp\u003e37.31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 43.9521%;\"\u003e\n \u003cp\u003e\u0026nbsp; No medication\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 23.8323%;\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 32.2156%;\"\u003e\n \u003cp\u003e25.37\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 43.9521%;\"\u003e\n \u003cp\u003e\u0026nbsp;Knowledge about ND (low/medium/high)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 23.8323%;\"\u003e\n \u003cp\u003e36 / 14 / 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 32.2156%;\"\u003e\n \u003cp\u003e53.73 / 20.89 / 1.49\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 43.9521%;\"\u003e\n \u003cp\u003eAge group most affected by ND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 23.8323%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 32.2156%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 43.9521%;\"\u003e\n \u003cp\u003e\u0026nbsp; Young\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 23.8323%;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 32.2156%;\"\u003e\n \u003cp\u003e29.85\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 43.9521%;\"\u003e\n \u003cp\u003e\u0026nbsp; Layers\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 23.8323%;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 32.2156%;\"\u003e\n \u003cp\u003e52.23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 43.9521%;\"\u003e\n \u003cp\u003e\u0026nbsp; Mixed\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 23.8323%;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 32.2156%;\"\u003e\n \u003cp\u003e13.43\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 43.9521%;\"\u003e\n \u003cp\u003eBreed most affected by ND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 23.8323%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 32.2156%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 43.9521%;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;Local\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 23.8323%;\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 32.2156%;\"\u003e\n \u003cp\u003e47.76\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 43.9521%;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;Exotic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 23.8323%;\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 32.2156%;\"\u003e\n \u003cp\u003e32.83\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 43.9521%;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;Cross\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 23.8323%;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 32.2156%;\"\u003e\n \u003cp\u003e4.47\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 67.7844%;\"\u003e\n \u003cp\u003eObserved ND outbreak by distance from lake\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 32.2156%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 43.9521%;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026lt;10 km\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 23.8323%;\"\u003e\n \u003cp\u003e55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 32.2156%;\"\u003e\n \u003cp\u003e83.33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 43.9521%;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;10\u0026ndash;20 km\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 23.8323%;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 32.2156%;\"\u003e\n \u003cp\u003e12.12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 43.9521%;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026gt;20 km\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 23.8323%;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 32.2156%;\"\u003e\n \u003cp\u003e4.54\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 43.9521%;\"\u003e\n \u003cp\u003eObserved ND outbreak by distance from market (% Yes)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 23.8323%;\"\u003e\n \u003cp\u003e49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 32.2156%;\"\u003e\n \u003cp\u003e73.13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 43.9521%;\"\u003e\n \u003cp\u003eND outbreak linked to contact with wild birds (% Yes)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 23.8323%;\"\u003e\n \u003cp\u003e47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 32.2156%;\"\u003e\n \u003cp\u003e70.14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n "},{"header":"5. Discussion ","content":"\u003cp\u003eThis research investigated the seroprevalence and molecular identification of NDV in backyard chickens and wild birds within the Rift Valley regions of Ethiopia. The overall seroprevalence of NDV in backyard chickens was found to be 12.7%, which aligns with previous findings from Hawassa (11.9%) [20], the central Rift Valley (11.6–13.26%) [16,21], and Adami Tulu Jido Kombolcha (16.7%) [22]. However, these results are lower than those reported in the Rift Valley (22.5%) [23] and Addis Ababa (28.6%) [24], yet higher than the figures from Kersna Kondaltity (5.6%) [15] and seasonal studies conducted in the Rift Valley (5.9–6%) [22]. Such discrepancies may be attributed to variations in sampling periods, flock management practices, and ecological conditions.\u003c/p\u003e\n\u003cp\u003eA statistically significant difference in seroprevalence was noted across different districts (p = 0.035), with the highest prevalence recorded in Bishoftu (20%), followed by Hawassa (12%) and Batu (6%). The elevated prevalence in Bishoftu may be associated with its ecological environment, which supports a high population of migratory wild birds, potentially enhancing the transmission of NDV. Notably, none of the fecal samples collected from wild birds tested positive for NDV, corroborating previous research that indicates an ambiguous role of wild birds in the persistence of NDV [25].\u003c/p\u003e\n\u003cp\u003eMolecular analysis utilizing RT-PCR identified NDV in 16.7% of pooled swab samples, with the highest detection rate occurring in Bishoftu (20%), followed by Hawassa and Batu (15%). These results are consistent with earlier studies that reported an overall positivity rate of 8.4% for swab samples, with Bishoftu again showing the highest prevalence (12%) and Batu the lowest (2%) [16]. While RT-PCR effectively confirmed the presence of NDV, further molecular characterization is necessary to ascertain the virulence of the strains involved.\u003c/p\u003e\n\u003cp\u003eThe interaction between domestic chickens and wild birds, combined with inadequate biosecurity measures, likely facilitates the transmission of NDV. The identification of NDV in pigeons from Batu highlights the potential reservoir role of wild birds [26,27]. However, the lack of NDV detection in fecal samples from wild birds suggests that the environmental persistence of the virus may be influenced by climatic factors and patterns of viral shedding [28]. Previous studies have indicated that NDV can still be transmitted through excretions such as feces and respiratory secretions, underscoring the need for ongoing surveillance [25,29].\u003c/p\u003e\n\u003cp\u003eA questionnaire survey indicated that 98.5% of respondents had observed outbreaks of ND, primarily within a 10 km radius of live bird markets and lakes. The absence of vaccination, reliance on traditional treatment methods (35.8%), and limited access to veterinary services contribute to the uncontrolled transmission of NDV. Similar challenges have been documented in the Rift Valley [28]\u0026nbsp; and Northern Ethiopia [25]. Live bird markets represent a significant transmission nexus, where clinically healthy birds may serve as asymptomatic carriers of virulent NDV strains [29].\u003c/p\u003e"},{"header":"6. Conclusion","content":"\u003cp\u003eThis research revealed a seroprevalence of 12.7% for ND antibodies among backyard chickens, indicating a significant level of exposure to the virus in the examined regions. The application of RT-PCR identified NDV strains in 16.7% of the swab samples, underscoring the critical role of molecular diagnostics in the detection and management of ND outbreaks. The heightened seroprevalence observed in Bishoftu suggests that factors such as migratory wild birds and local ecological conditions may contribute to the transmission of the virus. Furthermore, the study highlights the influence of live bird markets and inadequate biosecurity practices on the spread of ND. Future research and surveillance programs should incorporate molecular diagnostic techniques, such as RT-PCR, to facilitate ongoing monitoring of NDV circulation and mitigate its effects on both commercial and traditional poultry farming systems.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval for the present study was secured from the appropriate institutional review board, thereby ensuring compliance with all ethical and legal standards. Informed consent was obtained from all poultry owners, who were thoroughly informed about the study\u0026apos;s aims, methodologies, and any potential risks involved. Participants provided written consent voluntarily, with the assurance that they could withdraw from the study at any point without facing any repercussions. All animal handling procedures adhered to established veterinary care standards to mitigate any distress or harm, with an emphasis on prioritizing the welfare of the birds and maintaining confidentiality throughout the research process.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData for this study can be made \u0026nbsp; available upon reasonable request by the corresponding author\u0026nbsp;\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe have no competing interests to declare\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthor Contributions: G.H. Terara, B.K. Abebe, M.T Alemayehu, F. Abunna, and F. Dawo involved in the conception of the research idea, methodology design, supervision, performed data analysis, and interpretation. G.H. Terara, B.K. Abebe and M.T Alemayehu participated in data analysis, drafted, prepared the manuscript for publication, and revision. All authors read and approved of the final manuscript\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbsal\u0026oacute;n AE, Cort\u0026eacute;s-Espinosa DV, Lucio E, Miller PJ, Afonso CL (2019) Epidemiology, control, and prevention of Newcastle disease in endemic regions: Latin America. 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Worlds Vet J 2:240\u0026ndash;249\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMulisa DD, W/Kiros MK, Alemu RB, Keno MS, Furaso A, Heidari A, Chibsa TR, Chunde HC (2014) Characterization of Newcastle Disease Virus and poultry-handling practices in live poultry markets. Ethiopia SpringerPlus 3:459\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShapiro BI, Gebru G, Desta S, Negassa A, Negussie K, Aboset G, Mechal H (2015) Ethiopia livestock master plan: Roadmaps for growth and transformation. [ILRI report]. International Livestock Research Institute (ILRI), Nairobi, Kenya\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOsman AM, Oladele OA, Ibrahim AM, Mahamoud MA, Mohamed MA, Nwachukwu NO (2021) Seroprevalence of Newcastle disease in backyard chickens in selected districts of Banadir region, Somalia. Trop Anim Health Prod 53(2):192\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eThrusfield M, Christley R (2007) Veterinary epidemiology, 3rd edn. Wiley-Blackwell, Hoboken, pp 230\u0026ndash;240\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWise MG, Suarez DL, Seal BS, Pedersen JC, Senne DA, King DJ, Kapczynski DR, Spackman E (2004) Development of a real-time reverse-transcription PCR for detection of Newcastle disease virus RNA in clinical samples. J Clin Microbiol 42:329\u0026ndash;338\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZeleke A, Sori T, Gelaye E, Ayelet G (2005) Newcastle disease in village chickens in the southern and Rift Valley districts in Ethiopia. Int J Poult Sci 4:507\u0026ndash;510\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Newcastle Disease, poultry, serology, risk factors","lastPublishedDoi":"10.21203/rs.3.rs-7221776/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7221776/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNewcastle Disease (ND) remains a significant threat to the health and productivity of poultry. This study aimed to evaluate the prevalence of ND in domestic and wild chickens. A total of 150 both domestic and wild birds were sampled from Bishoftu, Hawassa, and Batu. Serum samples were analyzed for ND antibodies using serological assays, while 60 pooled swab samples were subjected to RT-PCR for NDV detection. The overall seroprevalence of ND was found to be 12.7%, with Bishoftu exhibiting the highest prevalence at 20%, followed by Hawassa at 12% and Batu at 6%. No statistically significant differences were noted based on sex (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) or age (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). RT-PCR detected NDV in 16.7% of the swab samples, with a higher prevalence observed in cloacal swabs (20%) compared to tracheal swabs (13.3%). Risk analysis indicated a significantly increased odds ratio (OR) of 4.2 for ND infection in Bishoftu relative to Batu. Additionally, males were found to be 1.8 times more likely to be infected than females, although age did not significantly affect the risk of infection. These findings highlight the regional variability in ND prevalence, with Bishoftu showing the highest rates. Both serological and molecular analyses demonstrate a notable presence of NDV, particularly in cloacal swabs.\u003c/p\u003e","manuscriptTitle":"Comparative study of conventional and molecular epidemiology of Newcastle disease in wild and domestic birds in the Rift Valley region of Ethiopia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-25 12:33:37","doi":"10.21203/rs.3.rs-7221776/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b1aefc2a-88f5-4002-80ee-5adbac7d5530","owner":[],"postedDate":"August 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-06T23:38:08+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-25 12:33:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7221776","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7221776","identity":"rs-7221776","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}