Development of an ELISA for the detection of fowl adenovirus serotype -4 utilizing fiber protein.

Hydropericardium syndrome (HPS), caused by the Fowl adenovirus 4 (FAdV-4) has led to significant financial losses for the poultry industry globally, including Pakistan over the past few years. Conventional serological methods are time consuming, laborious and less sensitive therefore, a rapid and sensitive ELISA kit is required for the reliable detection of FAdV-4 infection. In the current research, fiber proteins (1 &2) of FAdV-4 were successfully expressed in Escherichia coli and purified using metal affinity chromatography. Using these proteins as antigens, an indirect ELISA for detecting FAdV-4 infection was developed. The developed ELISA showed superior performances upon comparison with Serum neutralization test (SNT). This ELISA also showed reliable detection of FAdV specific antibodies in experimentally infected and vaccinated chickens. This assay produced good correlation on the samples collected from the field with SNT and found essential for large scale serology of the FAdV. No cross reactivity was observed in the ELISA following the testing of the serum samples of different other avian pathogens which showed that this ELISA is specific in detecting the FAdV infection. In conclusion, the developed Fiber protein ELISA is highly sensitive and specific in the detecting the FAdV infection and can be utilized for large scale sero-epidemiology of the disease.

Molecular investigation of foot-and-mouth disease virus circulating in Pakistan during 2014-17.

This study reports the molecular characterization of foot-and-mouth disease virus (FMDV) in the provinces of Punjab and Sindh, Pakistan during 2014-17. FMDV genome was detected in 42 and 41 out of 46 samples (epithelial tissue and saliva) by reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) and reverse transcriptase polymerase chain reaction (RT-PCR), respectively. Sequences of the complete VP1 coding region of the samples (n = 33) was achieved showing that 10, 4 and 19 samples belonged to serotype O, A and Asia1 respectively. Phylogenetic analysis of serotype O revealed that at least one novel sublineage within the ME-SA topotype is circulating in the region, named here as PAK-14. This sublineage showed similarity with the viruses circulating in Turkey and Pakistan during 2010 indicating that viruses circulating in these countries have common origin. Analysis of serotype A viruses revealed a new lineage is circulating in the region, reported here as A-PAK14 showing close identity with the strain prevalent in Pakistan during 2007. Circulation of these new linages in the region shows continuous evolution of the viruses. Two of the undisclosed serotype A sublineages within the Iran-05 lineage were also found circulating in the region. In addition, molecular investigation of the VP1 coding region sequences of serotype Asia1 strains revealed that they belong to Group-VII (Sindh-08). Interestingly some of the serotype Asia1 isolates (n = 6) showed 99.9% similarity (among themselves) although they were collected from different districts more than 100 Km apart from one another. This unusual conservation among serotype Asia1 over long distances can be explored by studying the role of wild animals, slaughter houses and milk collection centres in the spread the disease.

Isolation, Identification and Molecular Characterization of Highly Pathogenic Newcastle Disease Virus From Field Outbreaks

ABSTRACT Newcastle disease (ND) is a major infectious disease of the poultry caused by a virulent strain of Avian Paramyxovirus - 1, that is a single strand non-segmented negative sense RNA virus. ND virus is major threat to the poultry industry in many countries of the world. The study was aimed to isolate and identify Newcastle disease virus (NDV) by using a haemagglutination inhibition (HI) test and reverse transcription-polymerase chain reaction (RT-PCR) assay. A total 100 samples of infected and dead birds were collected from different poultry farms. The weight of the birds was ranged 1000-1200g. The birds were divided into 3 groups. Haemagglutination assay (HA) was performed to detect the presence of NDV in suspension of infected homogenized tissues and it was found that HA is not the best method to detect the virus when it is in trace amounts. RT-PCR using NDV specific primers analyzed different clinical and postmortem samples. Reverse transcriptase polymerase chain reaction and specific primers was used for determining the presence of viruses. It was found that the virus was present in most of the infected samples except the serum of infected birds. During multiple sequence alignment (MSA) it was found that, our isolates have high homology (98%) with other reported NDV isolates. Phylogenetic analysis revealed that our isolate was closely related with viscerotropic velogenic types of NDV, which are highly pathogenic Newcastle disease virus.