Repeated isolation of virulent Newcastle disease viruses of sub-genotype VIId from backyard chickens in Bulgaria and Ukraine between 2002 and 2013.

Here, we report the circulation of highly related virulent Newcastle disease viruses (NDV) in Bulgaria and Ukraine from 2002 until 2013. All of these NDV isolates have the same virulence-associated cleavage site ("113RQKR↓F117"), and selected ones have intracerebral pathogenicity index values ranging from 1.61 to 1.96. These isolates are most closely related to viruses circulating in Eastern Europe, followed by viruses isolated in Asia during the same period of time. Interestingly, the majority of the viruses were isolated from backyard poultry, suggesting the possibility of a "domestic" or "urban" cycle of maintenance. The molecular characterization of the nucleotide sequence of the complete fusion protein gene of the studied viruses suggests continued circulation of virulent NDV of sub-genotype VIId in Eastern Europe, with occasional introductions from Asia. Furthermore, the high level of genetic similarity among those isolates suggests that the NDV isolates of sub-genotype VIId from Bulgaria and Ukraine may have been part of a broader epizootic process in Eastern Europe rather than separate introductions from Asia or Africa. The continuous monitoring of backyard poultry flocks for the presence of circulating virulent NDV strains will allow early identification of Newcastle disease outbreaks.

Status of wild birds in Bulgarian zoos with regard to orthomyxovirus and paramyxovirus type 1 infections.

Newcastle disease virus (NDV) and avian influenza virus (AIV) are pathogens of major economic and social importance, and the diseases they cause are often devastating, particularly in domestic poultry. Both viruses are naturally found in a wide variety of wild birds, particularly aquatic species, where asymptomatic infection typically occurs. Wild birds are therefore considered to be a natural reservoir for both viruses. Wild birds kept in captivity are in an environment that promotes transmission of infection with both influenza and Newcastle disease viruses. This report describes a survey for the detection of antibodies against Newcastle disease and avian influenza A viruses using the hemagglutination inhibition test in samples from 88 wild birds from 38 species in four Bulgarian zoos. Samples with positive results against NDV were also tested against avian paramyxovirus type 3 (APMV-3). Real-time reverse-transcriptase PCR was also performed to detect viral RNA of NDV and AIV among 127 wild birds from 57 species from the same zoos. In 13 samples from seven avian species (ten birds from the family Phasianidae, two from the family Numidae, and one from the family Columbidae), antibodies against APMV-1 were detected. Seven birds, whose sera were APMV-1 positive, had been vaccinated. The other six birds (five Phasianidae representatives and one of the Columbidae family) had no immunization history. No antibodies against both H5 and H7 AIV and against APMV-3 were detected, and no RNA of NDV and AIV were detected.

Pathologic Evaluation of Influenza A H5N8 Infection Outbreaks in Mule Ducks in Bulgaria.

This article outlines pathomorphologic findings of a study involving commercial mule ducks with confirmed influenza A H5N8 infections after a series of outbreaks in Bulgaria. Examinations were carried out after performing necropsy on dead birds from three different age groups (up to 15, 20 to 30, and 40+ days of age) fattened on different farms. Among birds of all ages, gross lesions were present as lesions affecting the heart. Histologically, the myocardium exhibited severe intermyofibrillar edema, moderate to massive hemorrhages, and degenerative changes. All lesions resulted in single or multiple and small to massive myocardial infarctions. Other affected organs included the brain, lungs, liver, spleen, and pancreas. Nonpurulent lymphocytic encephalitis was found postmortem in ducks that had shown prior clinical nervous signs. Among ducks of all ages, a viral antigen in the cardiomyocytes and the epithelium of air capillaries was found through immunohistochemical detection methods. The results of the present study allowed us to conclude that the highly pathogenic avian influenza A H5N8 viral infection may manifest itself as a systemic illness in commercial mule ducks with septicemic lesions, resulting in high morbidity and mortality rates of up to 100%. Pathomorphologic lesions were somewhat different from those previously reported in wild waterfowl.

Evaluación patológica de brotes de infección por influenza A H5N8 en patos mula en Bulgaria. Este artículo describe los hallazgos patomorfológicos de un estudio que involucró patos mula comerciales con infecciones confirmadas de influenza A H5N8 después de una serie de brotes en Bulgaria. Los exámenes se llevaron a cabo después de realizar la necropsia en aves muertas de tres grupos de edad diferentes (hasta 15 días, de 20 a 30 días y más de 40 días de edad) engordadas en diferentes granjas. Entre las aves de todas las edades, las lesiones macroscópicas estaban presentes como lesiones afectando el corazón. Histológicamente, el miocardio exhibió edema intermyofibrillar severo, hemorragias moderadas a masivas y cambios degenerativos. Todas las lesiones resultaron en infartos de miocardio simples o múltiples y de pequeños a masivos. Otros órganos afectados incluyeron el cerebro, los pulmones, el hígado, el bazo y el páncreas. Se encontró encefalitis linfocítica no supurativa se encontró post mortem en patos que habían mostrado signos nerviosos clínicos previos. Entre los patos de todas las edades, se encontró un antígeno viral en los cardiomiocitos y el epitelio de los capilares aéreos a través de métodos de detección inmunohistoquímica. Los resultados del presente estudio nos permitieron concluir que la infección viral altamente patógena de la influenza aviar H5N8 puede manifestarse como una enfermedad sistémica en patos mulos comerciales con lesiones septicémicas, lo que resulta en altas tasas de morbilidad y mortalidad de hasta el 100%. Las lesiones patomorfológicas fueron algo diferentes de las reportadas previamente en aves acuáticas silvestres.

Pathological Changes in Natural Infection of Pheasants with Highly Pathogenic Avian Influenza A (H5N8) in Bulgaria.

INTRODUCTION: The study of histopathological changes caused by influenza A (H5N8) viral infection in bird species is essential for the understanding of their role in the spread of this highly infectious virus. However, there are few such studies under natural conditions in minor gallinaceous species. This article describes the pathomorphological findings in Colchis pheasants infected naturally with H5N8 during an epizootic outbreak in Bulgaria. MATERIAL AND METHODS: Samples of internal organs of 10 carcasses were collected for histopathological and immunohistochemical evaluation, virus isolation and identification, and nucleic acid detection. RESULTS: Consistent macroscopic findings were lesions affecting the intestine, heart, lung, and pancreas. Congestion and mononuclear infiltrate were common findings in the small intestine, as were necrosis and lymphoid clusters in the lamina propria of the caeca. Congestion with small focal necrosis and gliosis with multifocal nonpurulent encephalitis were observed in the brain. Myocardial interstitial oedema and degenerative necrobiotic processes were also detected. Immunohistological analysis confirmed systemic infection and revealed influenza virus nucleoprotein in all analysed organs. CONCLUSION: Variable necrosis was observed in the brain, liver, trachea, heart, small intestine, and caeca. Viral antigen was commonly found in the brain, heart, lung and trachea. Contact with migrating waterfowls was suspected as a reason for the outbreak.

Updated unified phylogenetic classification system and revised nomenclature for Newcastle disease virus.

Several Avian paramyxoviruses 1 (synonymous with Newcastle disease virus or NDV, used hereafter) classification systems have been proposed for strain identification and differentiation. These systems pioneered classification efforts; however, they were based on different approaches and lacked objective criteria for the differentiation of isolates. These differences have created discrepancies among systems, rendering discussions and comparisons across studies difficult. Although a system that used objective classification criteria was proposed by Diel and co-workers in 2012, the ample worldwide circulation and constant evolution of NDV, and utilization of only some of the criteria, led to identical naming and/or incorrect assigning of new sub/genotypes. To address these issues, an international consortium of experts was convened to undertake in-depth analyses of NDV genetic diversity. This consortium generated curated, up-to-date, complete fusion gene class I and class II datasets of all known NDV for public use, performed comprehensive phylogenetic neighbor-Joining, maximum-likelihood, Bayesian and nucleotide distance analyses, and compared these inference methods. An updated NDV classification and nomenclature system that incorporates phylogenetic topology, genetic distances, branch support, and epidemiological independence was developed. This new consensus system maintains two NDV classes and existing genotypes, identifies three new class II genotypes, and reduces the number of sub-genotypes. In order to track the ancestry of viruses, a dichotomous naming system for designating sub-genotypes was introduced. In addition, a pilot dataset and sub-trees rooting guidelines for rapid preliminary genotype identification of new isolates are provided. Guidelines for sequence dataset curation and phylogenetic inference, and a detailed comparison between the updated and previous systems are included. To increase the speed of phylogenetic inference and ensure consistency between laboratories, detailed guidelines for the use of a supercomputer are also provided. The proposed unified classification system will facilitate future studies of NDV evolution and epidemiology, and comparison of results obtained across the world.

Histopathologic Characterization and Shedding Dynamics of Guineafowl (Numida meleagris) Intravenously Infected with a H6N2 Low Pathogenicity Avian Influenza Virus.

Guineafowl of different ages were inoculated intravenously with a H6N2 wild waterfowl-origin low pathogenicity avian influenza virus (LPAIV). No clinical disease was observed. The infected birds had atrophy of the spleen, thymus, and cloacal bursa when compared with the noninfected control groups. The central and peripheral lymphoid tissues presented either lymphoproliferative or degenerative lesions that increased in intensity from 14 to 21 days postinoculation (DPI). Lymphoid depletion was present in the bursa, thymic lobes, and spleen T-dependent zone. In contrast, lymphoid proliferation was observed in liver, pancreas, and spleen B-dependent zone. Bronchus associated lymphoid tissue hyperplasia was observed in the lungs of the birds at 14 and 21 DPI. The virus was detected by virus isolation and reverse transcription PCR from both oropharyngeal and cloacal swabs with higher isolation rates from the latter. Most birds from the LPAIV inoculated groups shed virus up to 7 DPI. The virus was infrequently isolated from lung, kidney, liver, bursa, or spleen of infected birds until 14 DPI and from two samples (kidney and spleen, 1-yr-old birds) at 21 DPI. These data indicate that the wild bird-origin LPAIV used in this study caused pantropic infection in guineafowl when inoculated intravenously.

Presence of Vaccine-Derived Newcastle Disease Viruses in Wild Birds.

Our study demonstrates the repeated isolation of vaccine-derived Newcastle disease viruses from different species of wild birds across four continents from 1997 through 2014. The data indicate that at least 17 species from ten avian orders occupying different habitats excrete vaccine-derived Newcastle disease viruses. The most frequently reported isolates were detected among individuals in the order Columbiformes (n = 23), followed in frequency by the order Anseriformes (n = 13). Samples were isolated from both free-ranging (n = 47) and wild birds kept in captivity (n = 7). The number of recovered vaccine-derived viruses corresponded with the most widely utilized vaccines, LaSota (n = 28) and Hitchner B1 (n = 19). Other detected vaccine-derived viruses resembled the PHY-LMV2 and V4 vaccines, with five and two cases, respectively. These results and the ubiquitous and synanthropic nature of wild pigeons highlight their potential role as indicator species for the presence of Newcastle disease virus of low virulence in the environment. The reverse spillover of live agents from domestic animals to wildlife as a result of the expansion of livestock industries employing massive amounts of live virus vaccines represent an underappreciated and poorly studied effect of human activity on wildlife.