Enhanced phylogenetic resolution of Newcastle disease outbreaks using complete viral genome sequences from formalin-fixed paraffin-embedded tissue samples.

Highly virulent Newcastle disease virus (NDV) causes Newcastle disease (ND), which is a threat to poultry production worldwide. Effective disease management requires approaches to accurately determine sources of infection, which involves tracking of closely related viruses. Next-generation sequencing (NGS) has emerged as a research tool for thorough genetic characterization of infectious organisms. Previously formalin-fixed paraffin-embedded (FFPE) tissues have been used to conduct retrospective epidemiological studies of related but genetically distinct viruses. However, this study extends the applicability of NGS for complete genome analysis of viruses from FFPE tissues to track the evolution of closely related viruses. Total RNA was obtained from FFPE spleens, lungs, brains, and small intestines of chickens in 11 poultry flocks during disease outbreaks in Pakistan. The RNA was randomly sequenced on an Illumina MiSeq instrument and the raw data were analyzed using a custom data analysis pipeline that includes de novo assembly. Genomes of virulent NDV were detected in 10/11 birds: eight nearly complete (> 95% coverage of concatenated coding sequence) and two partial genomes. Phylogeny of the NDV complete genome coding sequences was compared to current methods of analysis based on the full and partial fusion genes and determined that the approach provided a better phylogenetic resolution. Two distinct lineages of sub-genotype VIIi NDV were identified to be simultaneously circulating in Pakistani poultry. Non-targeted NGS of total RNA from FFPE tissues coupled with de novo assembly provided a reliable, safe, and affordable method to conduct epidemiological and evolutionary studies to facilitate management of ND in Pakistan.

Comparison of protection and viral shedding following vaccination with Newcastle disease virus strains of different genotypes used in vaccine formulation.

Newcastle disease (ND) is a devastating disease and cause high t mortality and morbidity in poultry and nonpoultry avian species worldwide. An intensive vaccination program against ND is a routine practice in Pakistan like other developing countries, but still frequent outbreaks have been recorded in the field. In this study, vaccines prepared from ND viruses corresponding to four different genotypes were compared, to determine if the phylogenetic distance between vaccine and challenge strain influences the protection induced the amount of challenge virus shed. In the experiment, 1-day-old pathogen-free Hubbard chicks were divided into five groups and all groups except control were received live LaSota vaccine. The chicks were re-vaccinated at day 5 and were given oil-adjuvanted inactivated vaccines prepared from one of four different inactivated NDV strains including SFR-55 (genotype-VIIi), Chicken-12 (XIIIb), Mukteswar (III), and LaSota (II), and control group was treated with PBS only. Pre- and post-challenge serum was collected from all groups and tested for antibody against NDV using hemagglutination inhibition (HI) assay. After challenged with virulent SFR-55, the birds were examined daily for morbidity and mortality and were monitored at selected intervals for viral shedding. All the vaccines induced high immune response, and all the groups except the control induced > 84% protection against vNDV challenge. The vaccine genetically and antigeneically similar with challenge NDV strain reduced oral shedding significantly as compared to mismatched strains. From the present study, it was concluded that genotype-matched vaccine has potential to result in better protection by limiting the viral shedding.

Presence of virulent Newcastle disease virus in vaccinated chickens in farms in Pakistan.

One year after a virulent Newcastle disease virus (vNDV) outbreak in Pakistan, the causative strain was present in vaccinated chickens of multiple farms despite the existence of high-average NDV-specific antibody titers (>4.75 log2). The data suggest a possible role of vaccinated birds as reservoirs of vNDV.

Antibacterial activity of herbal extracts against multi-drug resistant Escherichia coli recovered from retail chicken meat.

Increasing incidence rate of multiple drug resistance in Escherichia coli (E. coli) due to extensive uses of antibiotics is a serious challenge to disease treatment. Contaminated retail chicken meat is one of the major sources of spread of multi drug resistant (MDR) E. coli. Current study has been conducted to study the prevalence of MDR E. coli in retail chicken meat samples from Lahore city of Pakistan and it was found that 73.86% of E. coli isolates have MDR pattern. In vitro evaluation of antibacterial activity of crude ethanolic extracts of six herbs against MDR E. coli phenotypes has revealed that clove and cinnamon have maximum zones of inhibition as compared to other herbal extracts. Mint and coriander gave the intermediate results while garlic and kalonji showed the least antibacterial activity against the MDR E. coli phenotypes using the agar well diffusion technique. Average Minimum Inhibitory Concentrations (MICs) for clove, mint, cinnamon, coriander, kalonji and garlic extracts were 1.15, 1.38, 0.5, 1.99, 2.41, 8.60 mg/mL respectively using the broth micro dilution method. The results obtained in present study were revealed that crude ethanol extracts of selected herbs have had significant antibacterial activity. Hence they can be used as promising alternatives of antimicrobials against MDR E. coli species and can be used for cooked food preservation.

Repeated isolation of virulent Newcastle disease viruses in poultry and captive non-poultry avian species in Pakistan from 2011 to 2016.

Virulent viruses of the panzootic Avian avulavirus 1 (AAvV-1) of sub-genotype VIIi were repeatedly isolated (2011-2016) from commercial chickens and from multiple non-poultry avian species in Pakistan. These findings provide evidence for the existence of epidemiological links between Newcastle disease outbreaks in commercial poultry and infections with virulent AAvV-1 strains in other avian species kept in proximity to poultry. Our results suggest that the endemicity of Newcastle disease in Pakistan involves multiple hosts and environments.

Complete Genome Sequence of a Virulent Newcastle Disease Virus Strain Isolated from a Clinically Healthy Duck (Anas platyrhynchos domesticus) in Pakistan.

Here, we report the complete genome sequence of a virulent Newcastle disease virus (vNDV) strain, duck/Pakistan/Lahore/AW-123/2015, isolated from apparently healthy laying ducks (Anas platyrhynchos domesticus) from the province of Punjab, Pakistan. The virus has a genome length of 15,192 nucleotides and is classified as member of subgenotype VIIi, class II.

Complete Genome Sequence of a Recent Panzootic Virulent Newcastle Disease Virus from Pakistan.

The genome sequence of a new strain of Newcastle disease virus (NDV) (chicken/Pak/Quality Operations Lab/SFR-611/13) is reported here. The strain was isolated from a vaccinated chicken flock in Pakistan in 2013 and has panzootic features. The genome is 15,192 nucleotides in length and is classified in subgenotype VIIi of genotype VII, class II.

Identification of new sub-genotypes of virulent Newcastle disease virus with potential panzootic features.

Virulent Newcastle disease virus (NDV) isolates from new sub-genotypes within genotype VII are rapidly spreading through Asia and the Middle East causing outbreaks of Newcastle disease (ND) characterized by significant illness and mortality in poultry, suggesting the existence of a fifth panzootic. These viruses, which belong to the new sub-genotypes VIIh and VIIi, have epizootic characteristics and do not appear to have originated directly from other genotype VII NDV isolates that are currently circulating elsewhere, but are related to the present and past Indonesian NDV viruses isolated from wild birds since the 80s. Viruses from sub-genotype VIIh were isolated in Indonesia (2009-2010), Malaysia (2011), China (2011), and Cambodia (2011-2012) and are closely related to the Indonesian NDV isolated in 2007, APMV1/Chicken/Karangasem, Indonesia (Bali-01)/2007. Since 2011 and during 2012 highly related NDV isolates from sub-genotype VIIi have been isolated from poultry production facilities and occasionally from pet birds, throughout Indonesia, Pakistan and Israel. In Pakistan, the viruses of sub-genotype VIIi have replaced NDV isolates of genotype XIII, which were commonly isolated in 2009-2011, and they have become the predominant sub-genotype causing ND outbreaks since 2012. In a similar fashion, the numbers of viruses of sub-genotype VIIi isolated in Israel increased in 2012, and isolates from this sub-genotype are now found more frequently than viruses from the previously predominant sub-genotypes VIId and VIIb, from 2009 to 2012. All NDV isolates of sub-genotype VIIi are approximately 99% identical to each other and are more closely related to Indonesian viruses isolated from 1983 through 1990 than to those of genotype VII, still circulating in the region. Similarly, in addition to the Pakistani NDV isolates of the original genotype XIII (now called sub-genotype XIIIa), there is an additional sub-genotype (XIIIb) that was initially detected in India and Iran. This sub-genotype also appears to have as an ancestor a NDV strain from an Indian cockatoo isolated in 1982. These data suggest the existence of a new panzootic composed of viruses of subgenotype VIIi and support our previous findings of co-evolution of multiple virulent NDV genotypes in unknown reservoirs, e.g. as recorded with the virulent NDV identified in Dominican Republic in 2008. The co-evolution of at least three different sub-genotypes reported here and the apparent close relationship of some of those genotypes from ND viruses isolated from wild birds, suggests that identifying wild life reservoirs may help predict new panzootics.