Clinicopathological and Laboratory Diagnosis of Newcastle Disease

Clinicopathological and Laboratory Diagnosis of Newcastle Disease | 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 Clinicopathological and Laboratory Diagnosis of Newcastle Disease Dessalew Habte, Dehinet Terefe This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6391479/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 New castle disease is highly contagious, commonly occurring and overwhelming viral disease affecting poultry and significantly cause global socioeconomic impact. This study documents the diagnostic approaches of Newcastle diseases from poultry that were referred from Modgo veterinary clinic to VTH of AAU-CVMA, Bishoftu, with major complaints of occurrence of a disease and death of hens in his private poultry farm around Modjo town. Owner complaints also indicated that most of the hens showed symptoms of incoordination, unable to fed, depression, weakness, greenish watery diarrhea, followed by sudden death. Up-on clinical examination, lying down, swelling of face, twisting of neck, paralysis of legs, greenish diarrhea and arched position of the hens’ body were observed. During postmortem inspection, marked congestion of trachea with hemorrhages, pinpoint hemorrhages on proventriculus, enlarged and hemorrhagic caecal tonsils, hemorrhagic lesions in the intestinal wall and inflamed cloudy and congested air sacs were observed. EDDIE App based smart phone diagnosis was not used in this case, since it has no data for poultry diseases. Cloacal and tracheal swab samples showed a positive result for NCDV. Newcastle disease is one of the most economically important poultry disease characterized by high morbidity, mortality, and spreading nature. However, rapid clinical and consistent detection of the disease with isolation of suspected hens, proper hygiene, vaccination and strict biosecurity measures can reduce the losses associated with this disease. Infectious Diseases Diagnosis Hens. Newcastle disease Postmortem examination Figures Figure 1 Figure 2 Figure 3 1. INTRODUCTION Newcastle disease (NCD ) is one of the most commonly occurring, overwhelming, and highly contagious viral infections that affect many species of domestic and wild birds worldwide to varying degrees. It considerably hampers the global poultry industry of the world. Because of its huge socioeconomic importance and potential to rapidly spread to naive birds in the neighborhood, it is included among the list of avian diseases that must be notified to the OIE immediately upon recognition. In the poultry industry, it remains a constant threat and is a limiting disease for producers of poultry globally. The variety of clinical presentations and the emergence and spread of new genetic variants make recognition and diagnosis challenging (OIE, 2013; Bello et al., 2018 ; Abah et al., 2020 ). Newcastle disease (ND) is a viral disease of poultry caused by a single-strand, non-segmented, negative-sense RNA virus which is known as Newcastle disease virus (NCDV) in the genus Avianorthoavulavirus1 (AOAV-1) in the family Paramyxoviridae (Dimitrov et al., 2019 ). There are 9 serotypes of Avianrthovulavirus, but all isolates of Newcastle disease virus (NDV) belong to serotype 1, therefore NDV is synonymous with AOAV-1. Although all NDV isolates belong to a single serotype (AOAV-1), there is great genetic variability among different strains. Based upon phylogenetic reconstruction, NDV can be divided into 2 classes (I and II), each of those respectively subdivided into 9 and 10 genotypes (Sutton et al., 2019 ; Messaï et al., 2019 ; Quin et al., 2016 ). Newcastle disease is transmitted most often by direct contact with diseased or carrier birds that may shed the virus contaminating the environment. It can also transmitted by contaminated food, water, equipment, and human clothing with feces and respiratory discharges. ND viruses can survive for several weeks in the environment, especially in cool weather and shed during the incubation period and for a short time during recovery. Other wild birds have also been shown to have caused outbreaks in domestic poultry (Dimitrov et al., 2019 ; Abdisa and Tagesu, 2017 ; Absalón et al., 2019 ). Clinical signs of NDV vary widely, and are dependent on factors like the pathotype, host species, age and co-infection with other organisms, environmental stress and immune status. Different strains of NDV are distinguished into five pathotypes that cause clinical signs. Velogenic viruses are responsible for acute lethal infection showing frequently haemorrhagic lesions of the digestive tract of dead birds; Neurotropic velogenic viruses cause acute neurological signs and often-high mortality which follows respiratory distress; Lesogenic viruses produce moderate respiratory disease with high mortality only in young birds and Lentogenic viruses cause mild infections of respiratory tract and asymptomatic enteric signs (Quin et al., 2016 ). In addition, aggravating factors like immune depression, and concurrent infections may result in clinical signs induced by milder strains to resemble those of more virulent strains in the field (OIE, 2013; Sutton et al., 2019 ). Generally, NCD may involve signs of depression, greenish diarrhea, prostration, edema of the head and wattles. In the velogenic one, clinical signs often begin with listlessness, increased respiration ending with death. Diarrhoea is frequently seen. Surviving birds may develop nervous signs such as muscular tremors, paralysis and torticollis (Senne et al., 2005 ; Dimitrov et al., 2017 ; OIE, 2013). In neurotropic velogenic type, the clinical signs are marked by severe respiratory disease shortly followed by neurological signs. With all pathotypes of Newcastle Disease, egg production decreases dramatically and sometimes leading to complete egg laying cessation and death. Virulent ND strains may replicate in vaccinated birds, but the clinical signs will be greatly diminished depending on the antibody level (Hana et al., 2018 ; Mushtaq and Al-Zuhariy, 2023 ). Avianorthoavulavirus1 infections are usually diagnosed by clinical signs, virus isolation, inoculating from embryonated chicken egg, PCR, haemagglutination inhibition, serology in non-vaccinating countries (WOAH, 2012; Yune and Abdela, 2017 ). The prompt recognition of highly contagious OIE notifiable diseases like ND or AI and differentiation from other viral infections of poultry, causing similar clinical signs, such as gumboro disease is essential for the implementation of appropriate control measures and to limit economic losses (Murree et al., 2016 ; Mushtaq and Al-Zuhariy, 2023 ). There is no treatment for Newcastle disease, although treatment with antibiotics reduces secondary infections. Appropriate vaccination with live atenuated or inactivated vaccine is the only consistent control technique. However, under field conditions vaccination alone is not sufficient to control the disease. It must therefore be accompanied by good hygiene, management and biosecurity procedures to reduce the losses (Ban-Bo, 2013; Sharif et al., 2014 ; Ahmad et al. 2015 ). Therefore the current case study describes and documents the case of Newcastle disease and its diagnostic approaches through clinical, laboratory, postmortem and other diagnostic findings. 2. STUDY METHODS AND MATERIALS 2.1. Description of the Case Four hens were referred from Modgo Veterinary clinic and presented to the Veterinary teaching hospital (VTH) of Addis Ababa University at College of Veterinary Medicine and Agriculture (AAU-CVMA), Bishoftu, with major complaints of occurrence of a disease and death of some of the hens in one of privately owned poultry farm around Modgo town. The owner complaints also indicated that, the hens first showed symptoms of incoordination, unable to fed, greenish watery diarrhea, depression, weakness followed by sudden death of some of his hens and some of the hens are left in the farm showing similar clinical signs. Based on clinical examination, lying down, swelling of the face, twisting of the neck and greenish diarrhea, paralysis of legs and arched position of the hens’ body were observed (Fig. 1 ). The Ethiopian differential diagnosis and information environment (EDDIE) smart phone App-based diagnosis as a disease diagnosis and treatment aid tool was used in this case report but didn’t provide the required information because it didn’t work for the diagnosis of poultry diseases as appropriate data regarding poultry and pet animal disease are not uploaded or installed. Based on history, occurrence of an outbreak and typical clinical signs, the case was tentatively diagnosed as Newcastle disease which is differentially diagnosed from Mareks Disease, Avian Influenza and Infectious Bursal Disease. 2.2. Postmortem Examination The hens were sent to AAU-CVMA’s postmortem room for postmortem examination after the consensus of the owner that the result will be reported to him and he was advised to isolate or quarantine healthy hens from suspected one and should follow hygienic precautions and early vaccination of the rest of the hens with following strict biosecurity. During postmortem examination, pinpoint haemorrhages on the tips of glands in the proventriculus, enlarged and haemorrhagic caecal tonsils, haemorrhagic lesions in the intestinal wall (in the lymphoid aggregates) and marked congestion of trachea with haemorrhages were observed. The air sacs appeared inflamed cloudy and congested that contained cheesy (caseous) material. These postmortem findings indicate the pathological lessions of Newcastle disease (Fig. 2 ). 2.3. Laboratory Investigation and Findings Cloacal swab samples from two live hens and tracheal tissue (during postmortem) samples were collected with sterile swabs and directly placed into a screw caped cryovial tubes that contain viral transport media (VTM). Then, the samples were transported to National animal health institute (NAHI), Sebeta, Ethiopia for confirmation of the case. The laboratory finding by real time PCR showed a positive result for New castle disease for both samples from live hens and postmortem (Fig. 3 ). Therefore, based on history, clinical signs, postmortem and laboratory results, the case was finally diagnosed as New Castle disease. 3. DISCUSSION Newcastle disease is the most economically important contagious poultry disease, affecting many domestic and wild avian species characterized by rapid spreading and mass mortality and can be transmitted to humans which is caused by Avianorthoavulavirus1. This disease has been classified as a notifiable diseases list of terrestrial animals that causes huge socioeconomic impact (Dimitrov et al., 2019 ; OIE, 2013). This agrees with the current outbreak of Newcastle disease, in which the owner of the farm has lost many hens and economic gain from egg due to death and disease of these hens and he is frustrating on the future consequences regarding his farm and economy. In this case report, the clinical signs observed, occurrence of outbreak of the disease, typical postmortem examination and laboratory findings indicated that the case is Newcastle disease, but the smart phone application based diagnosis evaluation was not used since the application was not encoded with relevant data for diagnosis of poultry diseases. The clinical signs presented from the current case report were in agreement with the research conducted by (Hasan et al., 2010 ; Batawui et al., 2021 ; Messaï et al., 2019 ). Then, the hens were passed for postmortem examination for more detail diagnosis for better finding and confirmation of the case. During postmortem examination, the findings observed in the current case report were marked congestion of trachea with haemorrhages and inflamed cloudy/congested air sacs, pinpoint haemorrhages on the tips of the glands in the proventriculus, enlarged and haemorrhagic caecal tonsils, haemorrhagic lesions in the intestinal wall, and these findings were in agreement with (Ekou et al., 2014 ; WOAH, 2022) who conducted postmortem examination of Newcastle disease suspected birds and revealed the case as the Newcastle disease. The diagnosis of Newcastle disease can be made by using different approaches like complete diagnosis with clinical, laboratory and postmortem examinations for the confirmation of the virus. However, pathogenicity of the virus strain, host age, host species, stress, secondary infections, environmental conditions, viral dose, host immune status, and route of exposure may play a role in determining incubation time and disease severity in different circumstances (Esmaeelzadeh-Dizaji et al., 2022 ; Zhan et al., 2022 ; Moustapha et al., 2023 ). These findings are also in line with the findings of the present study that showed and conducted the diagnosis of Newcastle disease by clinical, laboratory and postmortem examination techniques. Newcastle disease control and prevention strategy must include strict biosecurity that will prevent virus strains from introduction with the poultry and adequate administration of effective vaccines. These findings are in agreement with the research conducted by Dimitrov et al., ( 2017 ) and Ahmad et al., 2015 ). In addition, serological monitoring of the immunological status of vaccinated flocks should be performed to assess the antibody response to the administered vaccines. Immediate isolation or quarantine of suspected hens from health one should be done with proper sanitary and husbandry practices (Nurzijah et al., 2022 ; Smith et al., 2023 ; Mushtaq and Al-Zuhariy, 2023 ). These control and preventive measures were also agreed with the present study. 4. CONCLUSION AND RECOMMENDATIONS This study showed and documents that Newcastle disease is one of the most economically important poultry disease characterized by high rate of morbidity, mortality and spreading nature. Newcastle disease is endemic in many developing countries and causes health threat to the global poultry industry. This disease is sometimes misdiagnosed due to serological and molecular diagnostic tests for NDV that have certain limitations as a result of cross-reactivity of NDV with other homologous avian paramyxoviruses and variability in the nucleotide sequence of the NDV F gene. This finding also indicated that Newcastle disease can be rapidly and properly diagnosed by clinical, laboratory and postmortem findings to avoid the losses. Based on the above conclusions, rapid and consistent detection of the disease with isolation of suspected hens, following hygienic and strict biosecurity measures and properly scheduled vaccination can reduce the losses associated with Newcastle disease in poultry farms. Declarations The animal owner consented to participate in any diagnostic tests on his animals to control and prevent the disease outbreak in and around his farm. Funding There is no funding organization for this manuscript. It is self-supported. Data Availability declaration No/Not applicable (this manuscript does not report data generation or analysis). Competing Interest Declaration We declared that the authors have no competing interests as defined by BMC, or other interests that might be perceived to influence the results and/or discussion reported in this paper Consent to Publish Declaration Not applicable Ethics Approval and Consent to Participate This work was done for the routine diagnosis of diseases and the affected animals were infected by a disease that kills them once affected. Regarding the consent, the owner was volunteer to do a postmortem examination on his affected hens for the confirmation of the case that could help him to control and prevent the rest of his poultry from such a rapidly spreading and mass killing disease. Therefore, ethical approval was not necessary for this type of routine disease diagnosis. Acknowledgment The authors would like to forward heart felt acknowledgments to the College of Veterinary Medicine and Agriculture laboratory workers of Addis Ababa University, National Animal Health Institute and the owners of the hens who provided adequate information about the outbreak of the case in the surrounding area and his hens for postmortem examination. Authors' Contributions 1. Dessalew Habte: have made substantial contributions to the conception, design of the work, the acquisition, analysis and interpretation of data. 2. Dehninet Terefe: have made substantial contributions to the design of the work, the acquisition and analysis of the data. References Abah H, Shittu I, Abdu P, Aronu C (2020) Molecular characterization and phylogenetic analysis of Newcastle disease virus isolated from poultry in North Central States of Nigeria, Vol. 12 No. June, pp. 20–26 Abdisa T, Tagesu T (2017) Review on Newcastle Disease of Poultry and its Public Health Importance. J Veterinary Sci Technol. 08 03, available at https://doi.org/10.4172/2157-7579.1000441 Absalón A, Cortés-Espinosa D, Lucio E, Miller P, Afonso C (2019) Epidemiology, control, and prevention of Newcastle disease in endemic regions: Latin America, Tropical Animal Health and Production, pp. 1033–1048. 10.36103/m1rgrx05 Ahmad T, Umer M, Rehman A (2015) Prevention and Control of Newcastle Disease, No. September 2014. J Veterinary Med Sci, pp: 23–37 Ban-Bo A, Kebkiba B, Nadjilem D (2013) Factors favoring the emergence of the Newcastle disease in Chad. J Appl Bioscinces Vol 70(10):5591–5598. https://dx.doi.org/10.54203/scil.2023.wvj26 Bello A, Samuel A, Nayak B, Paldurai A, Xiao S, Aplogan G, Awoume K, Webby R, Ducatez M, Collins P, Samal S (2018) Phylogenetic and pathotypic characterization of Newcastle disease viruses circulating in West Africa and efficacy of a current vaccine. 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J Veterinary Med Anim Health 6(8):217–221 Esmaeelzadeh-Dizaji R, Molouki A, Hosseini H, Fallah-Mehrabadi M, Ziafati-Kafi Z, Takalou A, Eram N, Kumar N, Ashuri A, Sadri N et al (2022) Molecular characterization of a pigeon paramyxovirus type 1 virus isolated from Eurasian collared doves in Iran, 2017. Journal of Veterinary Science. 2022, 23, e29 Hana T, Senait B, Benti D (2018) Evaluation of the safety and efficacy of combined Newcastle disease, fowl pox and fowl typhoid vaccine under laboratory condition. J Veterinary Med Anim Health 10(2):60–66 Hasan RAK, Ali MH, Siddique MP, Rahman M, Islam MA (2010) Clinical and laboratory diagnoses of the Newcastle disease and infectious bursal diseases of chickens. Bangladesh J Veterinary Med 8(2):131–140 Mushtaq T, Al-Zuhariy F (2023) Evolution of the best vaccinal routes against Newcastle in the production stage of laying hens. Iraqi J Agri Sci 54(3):748–754 Messaï CR, Salhi O, Khelef D, Lounas A, Mohamed-Cherif A, Kaidi R, Aït-Oudhia K (2019) Serological, clinical, and risk factors of the Newcastle disease on broilers flocks in Algeria, Veterinary World, Vol. 12 No. 7, pp. 938–944 Moustapha A, Talaki E, Akourki A, Ousseini M (2023) Newcastle Disease Virus in Poultry: Current Status and Control Prospects. World Veterinary J 13(2):240–249. https://dx.doi.org/10.54203/scil.2023.wvj26 Murree B, Nizamani Z, Leghari I, Soomro N, Samo T, Samo F (2016) Pathology and Transmission of Expermental Velogenic Viscerotropic Newcastle Disease in Wild Pigeons, Broiler and Aseel Ccickens July-August. 28(4):3965–3971 Nurzijah I, Elbohy O, Kanyuka K, Daly J, Dunham S (2022) Development of plant-based vaccines for prevention of avian influenza and Newcastle disease in poultry. Vaccines 10(3):478. https://www.doi.org/10.3390/vaccines10030478 Office of International des Epizootics (OIE) (2013) Aetiology, Epidemiology, Diagnosis Prevetion and Control Refernces of Newcastle Disease. Vol. Apill, Las, pp 1–6 Quin PJ, Quin BK, Markey ME, Carte WJ, Donnelly, LeonaldSmith FC, I (2016) Veterinary Microbiology and Microbial Disease. Vet J 165(3):333 Senne DA, King DJ, Kapczynski DR (2005) Control of Newcastle disease by vaccination, Developments in Biologicals, Vol. 119 No. December, pp. 165–170 Sharif A, Ahmad T, Umer M, Rehman A, Hussain Z (2014) Prevention and control of Newcastle disease. Int J Agric Innovations Res 3(2):454–460. https://doi.org/10.3390/vetsci11100473 Smith T, O’Kennedy MM, Ross CS, Lewis NS, Abolnik C (2023) The production of Newcastle disease virus-like particles in Nicotiana benthamiana as potential vaccines. Front Plant Sci 14:1130910. https://www.doi.org/10.3389/fpls.2023.1130910 Sutton D, Allen D, Fuller C, Mayers J, Mollett B, Londt et al (2019) Development of an avian avulavirus 1 (AAvV-1) L-gene real-time RT-PCR assay using minor groove binding probes for application as a routine diagnostic tool. J Virol Methods 265:9–14. 10.1016/j.jviromet.2018.12.001 World Organization for Animal Health (WOAH) World Organization for Animal Health (2012). - Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. OIE, Paris World organization of animal health (WOAH) (2022) Manual of diagnostic tests and vaccines for terrestrial animals. Manual of diagnostic tests and vaccines for terrestrial animals 2022. Available at: https://www.woah.org/en/what-we-do/standards/codes-and-manuals/terrestrial-manualonline-access/ Yune N, Abdela N (2017) Update on Epidemiology, Diagnosis and Control Technique of Newcastle Disease. J Veterinary Sci Technol. 08 02, available at https://doi.org/10.4172/2157-7579.1000429 Zhan T, Lu X, He D, Gao X, Chen Y, Hu Z, Wang X, Hu S, Liu X (2022) Phylogenetic analysis and pathogenicity assessment of pigeon paramyxovirus type 1 circulating in China during 2007–2019. Trans boundary and Emerging Diseases, 2022, 69, 2076–2088 Additional Declarations The authors declare no competing interests. Supplementary Files SupplementaryTitlepageforNCD.docx CLINICOPATHOLOGICAL AND LABORATORY DIAGNOSIS OF NEWCASTLE DISEASE 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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INTRODUCTION","content":"\u003cp\u003eNewcastle disease (NCD\u003cb\u003e)\u003c/b\u003e is one of the most commonly occurring, overwhelming, and highly contagious viral infections that affect many species of domestic and wild birds worldwide to varying degrees. It considerably hampers the global poultry industry of the world. Because of its huge socioeconomic importance and potential to rapidly spread to naive birds in the neighborhood, it is included among the list of avian diseases that must be notified to the OIE immediately upon recognition. In the poultry industry, it remains a constant threat and is a limiting disease for producers of poultry globally. The variety of clinical presentations and the emergence and spread of new genetic variants make recognition and diagnosis challenging (OIE, 2013; Bello et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Abah et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNewcastle disease (ND) is a viral disease of poultry caused by a single-strand, non-segmented, negative-sense RNA virus which is known as \u003cem\u003eNewcastle disease virus (NCDV)\u003c/em\u003e in the genus Avianorthoavulavirus1 (AOAV-1) in the family Paramyxoviridae (Dimitrov et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). There are 9 serotypes of Avianrthovulavirus, but all isolates of Newcastle disease virus (NDV) belong to serotype 1, therefore NDV is synonymous with AOAV-1. Although all NDV isolates belong to a single serotype (AOAV-1), there is great genetic variability among different strains. Based upon phylogenetic reconstruction, NDV can be divided into 2 classes (I and II), each of those respectively subdivided into 9 and 10 genotypes (Sutton et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Messa\u0026iuml; et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Quin et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNewcastle disease is transmitted most often by direct contact with diseased or carrier birds that may shed the virus contaminating the environment. It can also transmitted by contaminated food, water, equipment, and human clothing with feces and respiratory discharges. ND viruses can survive for several weeks in the environment, especially in cool weather and shed during the incubation period and for a short time during recovery. Other wild birds have also been shown to have caused outbreaks in domestic poultry (Dimitrov et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Abdisa and Tagesu, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Absal\u0026oacute;n et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eClinical signs of NDV vary widely, and are dependent on factors like the pathotype, host species, age and co-infection with other organisms, environmental stress and immune status. Different strains of NDV are distinguished into five pathotypes that cause clinical signs. Velogenic viruses are responsible for acute lethal infection showing frequently haemorrhagic lesions of the digestive tract of dead birds; Neurotropic velogenic viruses cause acute neurological signs and often-high mortality which follows respiratory distress; Lesogenic viruses produce moderate respiratory disease with high mortality only in young birds and Lentogenic viruses cause mild infections of respiratory tract and asymptomatic enteric signs (Quin et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In addition, aggravating factors like immune depression, and concurrent infections may result in clinical signs induced by milder strains to resemble those of more virulent strains in the field (OIE, 2013; Sutton et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGenerally, NCD may involve signs of depression, greenish diarrhea, prostration, edema of the head and wattles. In the velogenic one, clinical signs often begin with listlessness, increased respiration ending with death. Diarrhoea is frequently seen. Surviving birds may develop nervous signs such as muscular tremors, paralysis and torticollis (Senne et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Dimitrov et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; OIE, 2013). In neurotropic velogenic type, the clinical signs are marked by severe respiratory disease shortly followed by neurological signs. With all pathotypes of Newcastle Disease, egg production decreases dramatically and sometimes leading to complete egg laying cessation and death. Virulent ND strains may replicate in vaccinated birds, but the clinical signs will be greatly diminished depending on the antibody level (Hana et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Mushtaq and Al-Zuhariy, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAvianorthoavulavirus1 infections are usually diagnosed by clinical signs, virus isolation, inoculating from embryonated chicken egg, PCR, haemagglutination inhibition, serology in non-vaccinating countries (WOAH, 2012; Yune and Abdela, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The prompt recognition of highly contagious OIE notifiable diseases like ND or AI and differentiation from other viral infections of poultry, causing similar clinical signs, such as gumboro disease is essential for the implementation of appropriate control measures and to limit economic losses (Murree et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Mushtaq and Al-Zuhariy, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). There is no treatment for Newcastle disease, although treatment with antibiotics reduces secondary infections. Appropriate vaccination with live atenuated or inactivated vaccine is the only consistent control technique. However, under field conditions vaccination alone is not sufficient to control the disease. It must therefore be accompanied by good hygiene, management and biosecurity procedures to reduce the losses (Ban-Bo, 2013; Sharif et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Ahmad et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Therefore the current case study describes and documents the case of Newcastle disease and its diagnostic approaches through clinical, laboratory, postmortem and other diagnostic findings.\u003c/p\u003e"},{"header":"2. STUDY METHODS AND MATERIALS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Description of the Case\u003c/h2\u003e \u003cp\u003eFour hens were referred from Modgo Veterinary clinic and presented to the Veterinary teaching hospital (VTH) of Addis Ababa University at College of Veterinary Medicine and Agriculture (AAU-CVMA), Bishoftu, with major complaints of occurrence of a disease and death of some of the hens in one of privately owned poultry farm around Modgo town. The owner complaints also indicated that, the hens first showed symptoms of incoordination, unable to fed, greenish watery diarrhea, depression, weakness followed by sudden death of some of his hens and some of the hens are left in the farm showing similar clinical signs. Based on clinical examination, lying down, swelling of the face, twisting of the neck and greenish diarrhea, paralysis of legs and arched position of the hens\u0026rsquo; body were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The Ethiopian differential diagnosis and information environment (EDDIE) smart phone App-based diagnosis as a disease diagnosis and treatment aid tool was used in this case report but didn\u0026rsquo;t provide the required information because it didn\u0026rsquo;t work for the diagnosis of poultry diseases as appropriate data regarding poultry and pet animal disease are not uploaded or installed. Based on history, occurrence of an outbreak and typical clinical signs, the case was tentatively diagnosed as Newcastle disease which is differentially diagnosed from Mareks Disease, Avian Influenza and Infectious Bursal Disease.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Postmortem Examination\u003c/h2\u003e \u003cp\u003eThe hens were sent to AAU-CVMA\u0026rsquo;s postmortem room for postmortem examination after the consensus of the owner that the result will be reported to him and he was advised to isolate or quarantine healthy hens from suspected one and should follow hygienic precautions and early vaccination of the rest of the hens with following strict biosecurity. During postmortem examination, pinpoint haemorrhages on the tips of glands in the proventriculus, enlarged and haemorrhagic caecal tonsils, haemorrhagic lesions in the intestinal wall (in the lymphoid aggregates) and marked congestion of trachea with haemorrhages were observed. The air sacs appeared inflamed cloudy and congested that contained cheesy (caseous) material. These postmortem findings indicate the pathological lessions of Newcastle disease (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Laboratory Investigation and Findings\u003c/h2\u003e \u003cp\u003eCloacal swab samples from two live hens and tracheal tissue (during postmortem) samples were collected with sterile swabs and directly placed into a screw caped cryovial tubes that contain viral transport media (VTM). Then, the samples were transported to National animal health institute (NAHI), Sebeta, Ethiopia for confirmation of the case. The laboratory finding by real time PCR showed a positive result for New castle disease for both samples from live hens and postmortem (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Therefore, based on history, clinical signs, postmortem and laboratory results, the case was finally diagnosed as New Castle disease.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. DISCUSSION","content":"\u003cp\u003eNewcastle disease is the most economically important contagious poultry disease, affecting many domestic and wild avian species characterized by rapid spreading and mass mortality and can be transmitted to humans which is caused by Avianorthoavulavirus1. This disease has been classified as a notifiable diseases list of terrestrial animals that causes huge socioeconomic impact (Dimitrov et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; OIE, 2013). This agrees with the current outbreak of Newcastle disease, in which the owner of the farm has lost many hens and economic gain from egg due to death and disease of these hens and he is frustrating on the future consequences regarding his farm and economy.\u003c/p\u003e \u003cp\u003eIn this case report, the clinical signs observed, occurrence of outbreak of the disease, typical postmortem examination and laboratory findings indicated that the case is Newcastle disease, but the smart phone application based diagnosis evaluation was not used since the application was not encoded with relevant data for diagnosis of poultry diseases. The clinical signs presented from the current case report were in agreement with the research conducted by (Hasan et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Batawui et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Messa\u0026iuml; et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Then, the hens were passed for postmortem examination for more detail diagnosis for better finding and confirmation of the case.\u003c/p\u003e \u003cp\u003eDuring postmortem examination, the findings observed in the current case report were marked congestion of trachea with haemorrhages and inflamed cloudy/congested air sacs, pinpoint haemorrhages on the tips of the glands in the proventriculus, enlarged and haemorrhagic caecal tonsils, haemorrhagic lesions in the intestinal wall, and these findings were in agreement with (Ekou et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; WOAH, 2022) who conducted postmortem examination of Newcastle disease suspected birds and revealed the case as the Newcastle disease.\u003c/p\u003e \u003cp\u003eThe diagnosis of Newcastle disease can be made by using different approaches like complete diagnosis with clinical, laboratory and postmortem examinations for the confirmation of the virus. However, pathogenicity of the virus strain, host age, host species, stress, secondary infections, environmental conditions, viral dose, host immune status, and route of exposure may play a role in determining incubation time and disease severity in different circumstances (Esmaeelzadeh-Dizaji et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhan et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Moustapha et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These findings are also in line with the findings of the present study that showed and conducted the diagnosis of Newcastle disease by clinical, laboratory and postmortem examination techniques.\u003c/p\u003e \u003cp\u003eNewcastle disease control and prevention strategy must include strict biosecurity that will prevent virus strains from introduction with the poultry and adequate administration of effective vaccines. These findings are in agreement with the research conducted by Dimitrov et al., (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and Ahmad et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In addition, serological monitoring of the immunological status of vaccinated flocks should be performed to assess the antibody response to the administered vaccines. Immediate isolation or quarantine of suspected hens from health one should be done with proper sanitary and husbandry practices (Nurzijah et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Smith et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Mushtaq and Al-Zuhariy, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These control and preventive measures were also agreed with the present study.\u003c/p\u003e"},{"header":"4. CONCLUSION AND RECOMMENDATIONS","content":"\u003cp\u003eThis study showed and documents that Newcastle disease is one of the most economically important poultry disease characterized by high rate of morbidity, mortality and spreading nature. Newcastle disease is endemic in many developing countries and causes health threat to the global poultry industry. This disease is sometimes misdiagnosed due to serological and molecular diagnostic tests for NDV that have certain limitations as a result of cross-reactivity of NDV with other homologous avian paramyxoviruses and variability in the nucleotide sequence of the NDV F gene. This finding also indicated that Newcastle disease can be rapidly and properly diagnosed by clinical, laboratory and postmortem findings to avoid the losses. Based on the above conclusions, rapid and consistent detection of the disease with isolation of suspected hens, following hygienic and strict biosecurity measures and properly scheduled vaccination can reduce the losses associated with Newcastle disease in poultry farms.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe animal owner consented to participate in any diagnostic tests on his animals to control and prevent the disease outbreak in and around his farm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere is no funding organization for this manuscript. It is self-supported.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo/Not applicable (this manuscript does not report data generation or analysis).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe declared that the authors have no competing interests as defined by BMC, or other interests that might be perceived to influence the results and/or discussion reported in this paper\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval and Consent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was done for the routine diagnosis of diseases and the affected animals were infected by a disease that kills them once affected. Regarding the consent, the owner was volunteer to do a postmortem examination on his affected hens for the confirmation of the case that could help him to control and prevent the rest of his poultry from such a rapidly spreading and mass killing disease. Therefore, ethical approval was not necessary for this type of routine disease diagnosis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to forward heart felt acknowledgments to the College of Veterinary Medicine and Agriculture laboratory workers of Addis Ababa University, National Animal Health Institute and the owners of the hens who provided adequate information about the outbreak of the case in the surrounding area and his hens for postmortem examination.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e1. Dessalew Habte: have made substantial contributions to the conception, design of the work, the acquisition, analysis and interpretation of data.\u003c/p\u003e\n\u003cp\u003e2. Dehninet Terefe: have made substantial contributions to the design of the work, the acquisition and analysis of the data.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbah H, Shittu I, Abdu P, Aronu C (2020) Molecular characterization and phylogenetic analysis of Newcastle disease virus isolated from poultry in North Central States of Nigeria, Vol. 12 No. June, pp. 20\u0026ndash;26\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbdisa T, Tagesu T (2017) Review on Newcastle Disease of Poultry and its Public Health Importance. 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Trans boundary and Emerging Diseases, 2022, 69, 2076\u0026ndash;2088\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"Diagnosis, Hens. Newcastle disease, Postmortem examination","lastPublishedDoi":"10.21203/rs.3.rs-6391479/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6391479/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNew castle disease is highly contagious, commonly occurring and overwhelming viral disease affecting poultry and significantly cause global socioeconomic impact. This study documents the diagnostic approaches of Newcastle diseases from poultry that were referred from Modgo veterinary clinic to VTH of AAU-CVMA, Bishoftu, with major complaints of occurrence of a disease and death of hens in his private poultry farm around Modjo town. Owner complaints also indicated that most of the hens showed symptoms of incoordination, unable to fed, depression, weakness, greenish watery diarrhea, followed by sudden death. Up-on clinical examination, lying down, swelling of face, twisting of neck, paralysis of legs, greenish diarrhea and arched position of the hens\u0026rsquo; body were observed. During postmortem inspection, marked congestion of trachea with hemorrhages, pinpoint hemorrhages on proventriculus, enlarged and hemorrhagic caecal tonsils, hemorrhagic lesions in the intestinal wall and inflamed cloudy and congested air sacs were observed. EDDIE App based smart phone diagnosis was not used in this case, since it has no data for poultry diseases. Cloacal and tracheal swab samples showed a positive result for NCDV. Newcastle disease is one of the most economically important poultry disease characterized by high morbidity, mortality, and spreading nature. However, rapid clinical and consistent detection of the disease with isolation of suspected hens, proper hygiene, vaccination and strict biosecurity measures can reduce the losses associated with this disease.\u003c/p\u003e","manuscriptTitle":"Clinicopathological and Laboratory Diagnosis of Newcastle Disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-10 08:06:47","doi":"10.21203/rs.3.rs-6391479/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":"a01512d8-ec77-4f94-a36d-873504c03df8","owner":[],"postedDate":"April 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":46941449,"name":"Infectious Diseases"}],"tags":[],"updatedAt":"2025-04-10T08:06:47+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-10 08:06:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6391479","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6391479","identity":"rs-6391479","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}