Phylogenetic analysis, genetic diversity, and epidemiology of pigeon paramyxovirus type 1 in China.

Pigeon paramyxovirus type 1 (PPMV-1) poses significant economic challenges to the pigeon industry in China. However, information about the prevalence, genetic diversity, and epidemiology of PPMV-1 in China is still lacking. In this study, we isolated six strains of PPMV-1 from Hubei and Zhejiang provinces in 2022. All six isolates were found to belong to subgenotype VI.2.1.1.2.2. Five of them were identified as mesogenic and one as lentogenic. Multiple mutations were observed in the F and HN proteins of these isolates. Comprehensive analysis of global PPMV-1 strains highlighted the dominance of genotype VI, showing that VI.2.1.1.2.2 has been the dominant subgenotype since 2011. We also identified 36 host-specific amino acid substitutions that are unique to PPMV-1 in comparison to chicken-origin NDVs. The data reported here contribute to our understanding of the epidemiology, genetic diversity, and prevalence of PPMV-1 and serve as a valuable reference for the prevention and control of PPMV-1.

Spillover of Newcastle disease virus to Himalayan Griffon vulture: a possible food-based transmission.

The Newcastle disease virus (NDV) affects wild and domesticated bird species, including commercial poultry. Although the diversity of NDV in domestic chickens is well documented, limited information is available about Newcastle disease (ND) outbreaks in other bird species. We report an annotated sequence of NDV/Vulture/Borjuri/01/22, an avirulent strain of NDV reported from Borjuri, Northeast India, in Himalayan Griffon vulture. The complete genome is 15,186 bases long with a fusion protein (F) cleavage site 112GRQGR↓L117. The phylogenetic analysis based on the F protein gene and the whole genome sequence revealed that the isolate from the vulture belongs to genotype II, sharing significant homology with vaccine strain LaSota. The study highlights the possible spillover of the virus from domestic to wild species through the food chain.

Genetic and biological characterization of Newcastle disease viruses circulating in Bangladesh during 2010-2017: further genetic diversification of class II genotype XIII in Southcentral Asia.

Newcastle disease virus (NDV) is endemic in Bangladesh and is a major threat to commercial poultry operations. While complete fusion (F) genes are recommended for molecular characterization and classification of NDV isolates, heretofore, only partial F gene data have been available for Bangladeshi NDVs. To this end, we obtained the full-length F gene coding sequences of 11 representative NDVs isolated in Bangladesh between 2010 and 2017. In addition, one of the viruses (MK934289/chicken/Bangladesh/C161/2010) was used in an experimental infection of chickens to establish the viral pathotype and study gross and microscopic lesions. Phylogenetic analysis provided evidence that all studied Bangladeshi isolates belong to genotype XIII.2 of class II NDVs. Six of the viruses were isolated between 2010 and 2017 and grouped together with isolates from neighbouring India during 2013-2016. Another four Bangladeshi isolates (2010-2016) formed a separate monophyletic branch within XIII.2 and showed high nucleotide distance from the isolates from India and the other six Bangladeshi viruses within the sub-genotype; however, none of these groups fulfils all classification criteria to be named as a separate sub-genotype. The eleventh Bangladeshi virus studied here (C162) was genetically more distant from the remaining isolates. It out-grouped the viruses from sub-genotypes XIII.2.1 and XIII.2.2 and showed more than 9.5 % nucleotide distance from all genotype XIII sub-genotypes. This isolate may represent an NDV variant that is evolving independently from the other viruses in the region. The experimental infection in chickens revealed that the tested isolate (C161) is a velogenic viscerotropic virus. Massive haemorrhages, congestion and necrosis in different visceral organs, and lymphoid depletion in lymphoid tissues, typical for infection with velogenic NDV, were observed. Our findings demonstrate the endemic circulation of sub-genotype XIII.2 in Southcentral Asia and further genetic diversification of these viruses in Bangladesh and neighbouring India. This constant evolution of the viruses may lead to the establishment of new genetic groups in the region. Additional historical and prospective virus and surveillance data from the region and neighbouring countries will allow a more detailed epidemiological inference.

Development of a TaqMan loop-mediated isothermal amplification assay for the rapid detection of pigeon paramyxovirus type 1.

Pigeon paramyxovirus-1 (PPMV-1) is a strain of Newcastle disease virus (NDV) that has adapted to infect pigeons and poses a constant threat to the commercial poultry industry. Early detection via rapid and sensitive methods, along with timely preventative and mitigating actions, is important for reducing the spread of PPMV-1. Here, we report the development of a TaqMan loop-mediated isothermal amplification assay (TaqMan-LAMP) for rapid and specific detection of PPMV-1 based on the F gene. This system makes use of six novel primers and a TaqMan probe that targets nine distinct regions of the F gene that are highly conserved among PPMV-1 isolates. The results showed that the limit of detection was 10 copies µL-1 for PPMV-1 cDNA and 0.1 ng for PPMV-1 RNA. The reaction was completed within 25 min and was thus faster than conventional RT-PCR. Moreover, no cross-reactions with similar viruses or with peste des petits ruminants virus (PPRV) or NDV LaSota vaccine strains were observed under the same conditions. To evaluate the applicability of the assay, the TaqMan-LAMP assay and a commercial RT-PCR assay were compared using 108 clinical samples, and the concordance rate between two methods was found to be 96.3%. The newly developed PPMV-1 TaqMan-LAMP assay can therefore be used for simple, efficient, rapid, specific, and sensitive diagnosis of PPMV-1 infections.

Development and application of a novel triplex protein microarray method for rapid detection of antibodies against avian influenza virus, Newcastle disease virus, and avian infectious bronchitis virus.

Avian influenza virus (AIV), Newcastle disease virus (NDV), and avian infectious bronchitis virus (IBV) inflict immense damage on the global poultry industry annually. Serological diagnostic methods are fundamental for the effective control and prevention of outbreaks caused by these viruses. In this study, a novel triplex protein microarray assay was developed and validated for the rapid and simultaneous visualized detection of antibodies against AIV, NDV, and IBV in chicken sera. The AIV nuclear protein (NP), NDV phosphoprotein (P), and IBV nonstructural protein 5 (nsp5) were produced in a prokaryotic expression system, purified, and immobilized onto an initiator integrated poly(dimethylsiloxane) (iPDMS) film as probes to detect antibodies against these viruses in chicken sera. After optimization of the reaction conditions, no cross-reactivity was detected with infectious bursal disease virus, avian leukosis virus subgroup J and chicken anemia virus antisera. The lowest detectable antibody titers in this assay corresponded to hemagglutination inhibition (HI) titers of 24 and 21 for AIV and NDV, respectively, and to an IDEXX antibody titer of 103 for IBV, using the HI assay and IDEXX commercial ELISA kit as the reference methods. When156 serum samples were tested using the new assay, the HI test and the IBV IDEXX ELISA kit, the assay showed 96.8% (151/156), 97.4% (152/156) and 99.4% (155/156) diagnostic accuracy for detection of AIV, NDV and IBV antibody, respectively. The current study suggests that the newly developed triplex microarray is rapid, sensitive, and specific, providing a viable alternative assay for AIV, NDV, and IBV antibody screening in epidemiological investigations and vaccination evaluations.

Co-circulation of genotypes XIV.2 and XVIII.2 of avian paramyxovirus-1 (Newcastle disease virus) in backyard poultry in Niger.

The causative agent of Newcastle disease (ND) of poultry is the avian paramyxovirus-1, also commonly known as ND virus (NDV). Like in many developing countries, ND is endemic in Niger and has significant economic impact on commercial and backyard poultry production. NDVs were characterized in Niger between 2006 and 2008 and shown to belong to genotypes XIV.1 and XVII. In order to determine the current situation regarding the virus in Niger, tracheas (n = 384) were collected for the detection of NDV from both healthy (n = 335) and sick (n = 49) backyard poultry in 2019. Of these samples, 24 from sick chickens were positive for NDV by conventional RT-PCR. Sequencing of the fusion protein gene and phylogenetic analysis revealed that the viruses belonged to either genotype XIV.2 or XVIII.2. No NDVs of genotype XIV.1 or XVII were identified in the current study highlighting the dynamic nature of NDV circulation in Niger and the region.

Characterization of Newcastle disease virus in broiler flocks with respiratory symptoms in some provinces of Iran.

BACKGROUND: Newcastle disease, is one of the most important diseases of the poultry industry, has many economic losses. The aim of this study was to isolate and determine the molecular identity of Newcastle disease virus in 40 broiler flocks with respiratory symptoms in four provinces of Iran. METHODS AND RESULTS: Samples of farms with respiratory symptoms were collected from different regions of Isfahan, East Azerbaijan, Golestan, and Khuzestan provinces and inoculated into 9-day-old embryonated chicken eggs. The Reverse-transcription polymerase chain reaction (RT-PCR) was performed to detect the Newcastle disease virus in allantoic fluid. Of the 40 flocks, the virus was isolated and identified in 16 flocks. The PCR products of 16 isolates were sequenced, and a phylogenetic tree was drawn. Accordingly, six isolates were in genotype II and ten isolates were in subgenotype VII.1.1 (VIId) of class II. CONCLUSION: Both genotypes were present in all four provinces. The isolates of Khuzestan province showed the greatest diversity compared to the other three provinces. The similarity of isolates belonging to genotype II in this study was observed with Pakistan, China, and Nigeria, and other isolates were similar to previous isolates in Iran. Also, the highest amino acid sequence in the F-protein cleavage site was 112RRQKR/F117 for VII.1.1 (VIId) genotype isolates and 112GRQGR/L117 for II genotype isolates.

Comparison of three filter paper-based devices for safety and stability of viral sample collection in poultry.

General diagnosis of poultry viruses primarily relies on detection of viruses in samples, but many farms are located in remote areas requiring logistic transportation. Filter paper cards are a useful technology that offer an alternative for collecting and preserving samples without hazardous exposure. The goal of this study was to compare three filter papers: the Flinders Technology Associates filter (FTA®) card, dried blood spot (DBS) card and qualitative filter paper (FP) grade 2 to collect poultry samples. In particular, we have used Newcastle disease virus (NDV) to evaluate safety and a Marek's disease virus (MDV) attenuated vaccine (CVI988) to evaluate stability of viral DNA. This experiment was divided into two parts. The first part was to determine the DNA stability and detection limit of CVI988 in samples collected in different paper supports after four storage times (3, 7, 14 and 30 days post spot). The second part was to determine the safety of papers by evaluating the viral inactivation efficacy using NDV as a representative virus. Results showed that all papers could preserve CVI988 DNA at all times, with a detection limit of 0.5 PFU/5 µl for FTA® and DBS cards, and 5 PFU/5 µl for FP. Our results showed that the NDV remained viable and infectious on the DBS card and FP, while no viable virus was detected on the FTA® card, suggesting that the FTA® card was safest to use. Therefore, the use of the DBS card and FP for infectious sample collection should be discouraged and reconsidered. RESEARCH HIGHLIGHTS The detection limits of the FTA® card, DBS card and FP for CVI988 detection were 0.5, 0.5 and 5 PFU/5 µl, respectively. All three filter papers could preserve viral DNA for at least 30 days of post spot. The DBS card and FP are not suitable for collecting NDV samples, which is one of the major economical threats for the poultry industry worldwide.

Seroprevalence of Newcastle disease in backyard chickens in selected districts of Banadir region, Somalia.

A cross-sectional study was carried out in the period between January and April 2019 with the aim of establishing prevalence of Newcastle disease (ND) in backyard chickens in Banadir region of Somalia using indirect enzyme-linked immunosorbent assay (iELISA). A total of 373 unvaccinated free scavenging backyard chickens were sampled from five districts in Banadir region, namely Dharkenley, Hodan, Wadajir, Hawlwadag, and Daynile. The overall prevalence was found to be 39.4% (95% confidence interval: 34.6-44.4%) with a mean antibody titre of 3844.10 ± 263.3 (standard error). The seroprevalence of ND virus (NDV) antibody in Wadajir district was the highest (66.6%) followed by Hawlwadag, Daynile, Dharkenley, and Hodan with prevalence of 56%, 42.1%, 42.35%, and 10.6%, respectively, with statistically significant differences (P < 0.05). Adult chickens had significantly higher prevalence (43.8%) than growers (19.4%) (P < 0.05). The present study, which is the first of its kind in Somalia to the best of our knowledge, concluded that the disease is highly prevalent in the study area; therefore, molecular studies on the characteristics of circulating strains are to be carried out in order to develop an evidence-based control programme and minimize the economic and social impacts of ND on smallholders.

Experimental pigeon paramyxovirus-1 infection in chicken: evaluation of infectivity, clinical and pathological manifestations and diagnostic methods.

Several pigeon paramyxovirus-1 (PPMV-1) outbreaks in feral pigeons were described recently in Switzerland. The potential of PPMV-1 to induce the notifiable Newcastle disease in chickens is discussed controversially. Therefore, in order to study epidemiologically relevant parameters such as the kinetics of PPMV-1 replication and shedding as well as seroconversion after infection, chickens were infected experimentally with a Swiss PPMV-1 isolate. This generated also defined sample material for the comparison of diagnostic tests. The infectivity of the Swiss PPMV-1 isolate for chickens was demonstrated successfully by virus shedding after experimental inoculation. Our data suggest that long-lasting shedding for up to 60 days can occur in chickens infected with PPMV-1. The isolate used here was of low pathogenicity for chickens. Different quantitative reverse transcription PCR assays were evaluated with a set of Swiss PPMV-1 isolates, and various samples from experimentally infected chickens were analysed with respect to their suitability for viral RNA detection. At 14 days post-infection, virus genome was detected mainly in spleen, caecal tonsils, heart, cloacal swabs, liver, proventriculus, duodenum and kidney tissue samples. Overall, the level of virus replication was low. Not all assays used routinely in diagnostics were capable of detecting viral genome from the isolates tested. Possible explanations are the genetic divergence of PPMV-1 and the low level of viral RNA in the samples. In contrast, two methods that are not used routinely proved more suitable for virus-genome detection. Importantly, the collection of material from various different organs is recommended, in addition to the kidney and brain analysed routinely. In conclusion, this study shows that there is a need to reconsider the type of samples and the protocols used for the detection of PPMV-1 RNA in chickens.

Evaluation of the safety and protection efficacy of an attenuated genotype vii newcastle disease virus strain as a candidate vaccine.

Newcastle disease (ND) is a serious avian infectious disease, causing severe economic loss worldwide. Its prevention depends on comprehensive vaccination scheme against Newcastle disease virus (NDV). However, current vaccine strains are of different genotypes with prevalent circulating strains (genotype VII), with significant genetic distance. Our team previously generated a genotype matched attenuated NDV strain (rmNA-1). In this study, its safety and immunization efficacy were evaluated. Its lentogenic characteristic was stable for 25 generations in embryonated chicken eggs and for six generations in SPF chickens. Overdosed administration did not cause any clinical signs or pathogenic changes in chickens. As to its immunization effect, rmNA-1 stimulated a comparable serum NDV specific antibody level to a LaSota (genotype II) strain based commercial vaccine, and provided full protection against virulent genotype VII strain challenge, with significantly reduced virus shedding period.

First molecular characterization of avian paramyxovirus-1 (Newcastle disease virus) in Botswana.

Avian paramyxovirus-1 (APMV-1), the causative agent of Newcastle disease (ND) in domestic and wild avian species, has recently been reported and characterized in five southern African countries (i.e. Mozambique, Namibia, South Africa, Zambia and Zimbabwe). Since APMV-1s have never been characterized in Botswana, this study was undertaken to determine the genotype circulating in the country. Fourteen samples were collected from ND outbreaks in poultry in 2014, 2018 and 2019 and the complete fusion protein gene was sequenced. Phylogenetic analysis revealed that all of the viruses from Botswana clustered in genotype VII.2 (previously subgenotype VIIh) and that they were more related to viruses from South Africa and Mozambique than the other southern African countries (i.e. Namibia, Zambia and Zimbabwe).

Immunosensor-based rapid quantitative detection of Newcastle disease virus antibodies using innovative gold immunochromatographic assay.

AIMS: A novel quantitative method for rapid Newcastle disease virus (NDV) antibody detection was developed based on an innovative gold immunochromatographic assay with a quantitative immunosensor. METHODS AND RESULTS: NDV antibody-detecting test strips containing a two-reaction system and double-test lines (T1, T2) were prepared. The test results were judged according to the signal ratio between the test and control lines as measured by the quantitative immunosensor. The minimum detection limit of the test strips for NDV antibodies was 22 titres. In addition, the assay was highly specific because only NDV antibodies produced visible test lines on the strip. The clinical application of the strips was tested by detecting NDV antibodies in 506 serum samples collected from chickens. The results showed a coincidence of 92·49% with those of the haemagglutination inhibition assay. CONCLUSIONS: The strips were successfully prepared and showed high specificity towards NDV, sensitivity and stability. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes a new method for detection of NDV antibody and provides a reference basis for rapid and quantitative monitoring of NDV antibodies. This new method overcomes the limitation of the existing colloidal gold immunochromatography, which only produces qualitative or semi-quantitative results.

Identification of Newcastle disease virus subgenotype VII.2 in wild birds in Turkey.

BACKGROUND: Newcastle disease viruses (NDVs) can spread across continents via migratory birds. Hence, we investigated the frequency of NDV in both non-migratory and birds migrating on the Black Sea-Mediterranean flyway, in Istanbul, Turkey. Birds were trapped using nets placed around the Kucukcekmece lake Avcilar, Istanbul, in spring seasons of 2016 and 2018. In total, 297 birds belonging to 42 different species were trapped, categorized according to species and sex, and flocked oropharyngeal swabs were collected. In addition, flocked swabs were also collected from 115 mallards caught by hunters around Edirne and from 207 birds which had been treated in the Veterinary Faculty of Istanbul university-Cerrahpasa. Tissue samples were taken from dead wild birds brought by public to Veterinary Faculty. A total of 619 flocked oropharyngeal swabs were pooled into 206 samples. RNA was extracted from swabs and tissue samples. Real-time RT-PCR prob. assay was used to detect NDV-RNA in samples. RESULTS: There was no amplification in real time RT-PCR in samples taken from wild birds caught by traps. However, amplification of NDV-F gene was observed in oropharyngeal swabs taken from 2 waterfowls (Common Moorhen and Mallard), and in tissue samples taken from 2 little owls and 1 common kestrel. Sequencing and phylogenetic analyses of these 5 samples for NDV-F gene showed great similarity with NDV subgenotype VII.2 viruses. Analysis also showed that there is a high similarity with the F gene sequences previously reported from Turkey in 2012 and as well as the sequences from neighbouring countries Bulgaria and Georgia and geographically close country such as Pakistan. Although the strains found in this study are closely related, there is a relatively small degree of molecular divergence within 543 bp of F gene of the Turkish NDV isolate and strains detected in Israel, Pakistan, Iran, United Arab Emirates and Belgium. CONCLUSIONS: Our findings revealed the presence of subgenotype VII.2 of NDVs in wild birds in north west of Turkey and demonstrated some degree of molecular evolution when compared to the earlier NDV-VII.2 isolate in Turkey.

Sequence analysis and pathogenicity of Avian Orthoavulavirus 1 strains isolated from poultry flocks during 2015-2019.

BACKGROUND: Newcastle disease (ND) causes severe economic losses in poultry industry worldwide. Egyptian poultry industry suffered from severe economic losses since the isolation of Velogenic Newcastle disease virus (vNDV) genotype VIId in 2011 and up till now despite the use of different vaccination programs. So, this study aimed to isolate and characterize the vNDV from a total of 120 poultry flocks from ten provinces in the Egyptian Delta region with a history of respiratory manifestation, high mortalities or a decrease in egg production between 2015 and 2019. Seventy-three samples' allantoic fluid (73/120, 60.8%) were positive for hemagglutination with chicken RBCs. These samples were submitted to molecular examination using qRT-PCR specific primers for AOAV-1, highly pathogenic avian influenza (HPAI-H5), low pathogenic avian influenza (LPAI-H9) and infectious bronchitis virus (IBV). RESULTS: Fifty samples (50/120: 41.6%) were confirmed positive for AOAV-1, based on genetic analysis of matrix and fusion protein. The co-infection rate of other respiratory viral diseases examined was 1.6, 14.1, and 4.1%, for HPAI-H5, LPAI-H9, and IBV, respectively. Biologically, the intracerebral pathogenicity index of ten selected AOAV-1 isolates ranged from 1.70 to 1.98, which indicated the velogenic nature of these isolates. All the sixteen sequenced isolates were AOAV-1 genotype VII.1.1. The full F gene sequence of six examined AOAV-1 VII.1.1 isolates contained the seven neutralizing epitopes, and the glycosylation motif of six-potential sites for N linked glycosylation at residues 85, 191, 366, 447, 471, and 541. CONCLUSION: It could be concluded that the high prevalence of AOAV-1 genotype VII.1.1 in the Egyptian chicken flocks despite the intensive vaccination with live and killed ND vaccines, as all the 16 isolates tested were belonged to this genotype. Homologous vaccination is badly needed to control and reduce the spread of AOAV-1 genotype VII.1.1infection in Egyptian poultry flocks.

Development and application of a triplex real-time PCR assay for simultaneous detection of avian influenza virus, Newcastle disease virus, and duck Tembusu virus.

BACKGROUND: Pathogens including duck-origin avian influenza virus (AIV), duck-origin Newcastle disease virus (NDV) and duck Tembusu virus (DTMUV) posed great harm to ducks and caused great economic losses to the duck industry. In this study, we aim to develop a triplex real-time polymerase chain reaction (PCR) assay to detect these three viruses as early as possible in the suspicious duck flocks. RESULTS: The detection limit of the triplex real-time PCR for AIV, NDV, and DTMUV was 1 × 101 copies/µL, which was at least 10 times higher than the conventional PCR. In addition, the triplex assay was highly specific, and won't cross-react with other duck pathogens. Besides, the intra-day relative standard deviation and inter-day relative standard deviation were lower than 4.44% for these viruses at three different concentrations. Finally, a total of 120 clinical samples were evaluated by the triplex real-time PCR, the conventional PCR and virus isolation, and the positive rates for these three methods were 20.83, 21.67, 19.17%, respectively. Taking virus isolation as the gold standard, the diagnostic specificity and positive predictive value of the three viruses were all above 85%, while the diagnostic sensitivity and negative predictive value of the three viruses were all 100%. CONCLUSION: The developed triplex real-time PCR is fast, specific and sensitive, and is feasible and effective for the simultaneous detection of AIV, NDV, and DTMUV in ducks.

Fenobody and RANbody-based sandwich enzyme-linked immunosorbent assay to detect Newcastle disease virus.

BACKGROUND: Traditional sandwich enzyme-linked immunosorbent assay (ELISA) using polyclonal and monoclonal antibodies as reagents presents several drawbacks, including limited amounts, difficulty in permanent storage, and required use of a secondary antibody. Nanobodies can be easily expressed with different systems and fused with several tags in their tertiary structure by recombinant technology, thus offering an effective detection method for diagnostic purposes. Recently, the fenobody (ferritin-fused nanobody) and RANbody (nanobody-fused reporter) have been designed and derived from the nanobody for developing the diagnostic immunoassays. However, there was no report about developing the sandwich ELISA using the fenobody and RANbody as pairing reagents. RESULTS: A platform for developing a sandwich ELISA utilizing fenobody as the capture antibody and RANbody as the detection antibody was firstly designed in the study. Newcastle disease virus (NDV) was selected as the antigen, from which 13 NDV-specific nanobodies were screened from an immunized Bactrian camel. Then, 5 nanobodies were selected to produce fenobodies and RANbodies. The best pairing of fenobodies (NDV-fenobody-4, 800 ng/well) and RANbodies (NDV-RANbody-49, 1:10) was determined to develop the sandwich ELISA for detecting NDV. The detection limits of the assay were determined to be 22 of hemagglutination (HA) titers and 10 ng of purified NDV particles. Compared with two commercial assays, the developed assay shows higher sensitivity and specificity. Meanwhile, it exhibits 98.7% agreement with the HA test and can detect the reference NDV strains belonging to Class II but not Class I. CONCLUSIONS: In the presented study, the 13 anti-NDV nanobodies binding the NDV particles were first produced. Then, for the first time, the sandwich ELISA to detect the NDV in the different samples has been developed using the fenobody and RANbody as reagents derived from the nanobodies. Considering the rapidly increasing generation of nanobodies, the platform can reduce the cost of production for the sandwich ELISA and be universally used to develop assays for detecting other antigens.

A retrospective study of Newcastle disease in Kenya.

Newcastle disease (ND) is a major constraint to Kenya's poultry production, which is comprised of approximately 80% indigenous chickens (ICs; caged and free-range system) and 20% exotic chickens (intensive system). This study analyzed cases reported as suspected ND in Kenya between 2005 and 2015. Of the suspected 332 ND reported cases from the three production systems in 27 locations within six Kenyan Agro-Ecological Zones (AEZs), 140 diagnosed as infected with avian orthoavulavirus 1 (AOaV-1; formerly Newcastle disease virus) were present in every year in all AEZs. The numbers of AOaV-1-positive cases differed significantly (p < 0.05) between the production systems across the years depending on the season, climate, and location. In the free-range system, both ambient temperatures and season associated significantly (p = 0.001 and 0.02, respectively) with the number of cases, while in the intensive and caged systems, the positive cases correlated significantly with season and relative humidity, respectively (p = 0.05). Regardless of the production systems, the numbers of clinically sick birds positively correlated with the ambient temperatures (r = 0.6; p < 0.05). Failure to detect AOaV-1 in 58% of the ND cases reported, and mortalities exceeding the observed numbers of clinically sick birds suggest deficiencies in the current ND reporting and diagnostic system. Intensive farmers were the slowest in reporting the cases and diagnostic deficiencies were most evident by failure to test the exposure of ICs to natural infection with AOaV-1 and for the AOaV-1-negative cases lack of testing for other pathogens and/or AOaV-1 variants. This study indicates a need for improved surveillance and diagnostics in Kenyan domestic poultry.

Host immune responses of pigeons infected with Newcastle disease viruses isolated from pigeons.

Newcastle disease (ND), affecting over 250 bird species, is caused by the Newcastle disease virus (NDV). ND is one of the leading causes of morbidity and mortality in pigeons. Most studies investigating NDV in pigeons have focused on the epidemiology and pathogenicity of the virus. However, the host immune responses in pigeons infected with NDVs remains largely unclear. In this study, we investigated the host immune responses in pigeons infected with two NDV stains, a pigeon paramyxovirus type 1(PPMV-1) strain, GZH14, and a genotype II virus, KP08. Although no mortality was observed upon infection with either virus, obvious neurological effects were observed in the GZH14-infected pigeons but not in the KP08-infected pigeons. Both viruses could replicate in the examined tissues, namely brain, lung, spleen, trachea, kidney, and bursa of Fabricius. The expression level of RIG-I, IL-6, IL-1ß, CCL5, and IL-8 were up-regulated by both viruses in the brain, lung and spleen at 3 and 7 days post-infection. Notably, these proinflammatory cytokines and chemokines showed more intense expression in the brain, when induced by the GZH14 strain than with the KP08 strain. These results indicate that the intense inflammatory responses induced by PPMV-1 in the brain may be a critical determinant of neurological symptoms in pigeons infected with PPMV-1. Our study provides new insight into the pathogenicity of PPMV-1 in pigeons attributable to the host immune responses.

Evaluation of transmission potential and pathobiological characteristics of mallard originated Avian orthoavulavirus 1 (sub-genotype VII.2) in commercial broilers.

Newcastle disease (ND), caused by Avian orthoavulavirus 1 (AOAV-1), affects multiple avian species around the globe. Frequent disease outbreaks are not uncommon even in vaccinates despite routine vaccination and, in this regards, viruses of diverse genotypes originating from natural reservoirs (migratory waterfowls) play an important role in a disease endemic setting. Though genomic characterization of waterfowl originated viruses has been well-elucidated previously, there is a paucity of data on clinico-pathological assessment of mallard-originated sub-genotype VII.2 in commercial chickens. Hence, the current study was designed to evaluate its transmission potential, tissue tropism and micro- and macroscopic lesions in commercial broilers. Based on complete genome and complete F gene, phylogenetic analysis clustered the study isolate within genotype VII and sub-genotype VII.2 in close association with those reported previously from multiple avian species worldwide. The study strain was found to be velogenic on the basis of typical residue pattern in the F-protein cleavage site (112R-RQ-K-R↓F117), sever disease induction in chicken, tissue tropism and subsequent clinico-pathological characteristics. Giving a clear evidence of horizontal transmission, a 100% mortality was observed by 4th and 6th day post infection (dpi) in chickens challenged with the virus and those kept with the challenged birds (contact birds), respectively. The observed clinical signs, particularly the greenish diarrhea, and macroscopic lesions such as pinpoint hemorrhages in proventriculus and caecal tonsils were typical of the infection caused by an AOAV-1 in chickens. The virus exhibited a broad tissue tropism where genomic RNA corresponding to study virus was detected in all of the tissues collected from recently mortile and necropsied birds. The study concludes that mallard-originated Avian orthoavulavirus 1 is highly velogenic to commercial chicken and therefore ascertain continuous disease monitoring and surveillance of migratory/aquatic fowls to better elucidate infection epidemiology and subsequent potential impacts on commercial poultry.