Newcastle disease virus (NDV) causes a highly contagious disease which continuously haunts the global poultry industry. The nature and molecular epidemiology of NDVs prevalent in recent outbreaks in India is poorly understood. This study aimed to characterize NDVs prevalent in vaccinated flocks in India using whole-genome sequencing and biological pathotyping. Twelve field isolates were collected from outbreaks which occurred in different parts of India and characterized as velogenic based on their intracerebral pathogenicity index (ICPI) and amino acid sequence at the F protein cleavage site. All 12 of the field isolates and five commonly used vaccine strains were selected for whole-genome sequencing using Ion Torrent PGM technology, yielding complete genome sequences for ten field isolates and all vaccine strains. The genome of all isolates was found to be 15 192 nt long with a high level of conservation across multiple genomic features with APMV-I viruses. Phylogenetic analysis and evolutionary distance calculations placed the isolates in genotypes II, IV and XIII. Revisiting other recently reported strains provided preliminary evidence of genotypes VI, VII and XVIII circulating in India. Comparison between the field and vaccine virus sequences revealed unique genomic and amino acid differences in important antigenic regions of the F and hemagglutinin-neuraminidase (HN) genes which can be targeted for site directed mutagenesis to evaluate the impact of these substitutions on virus pathogenicity. This study highlights the requirement to evaluate current vaccines through systematic protection assays to determine protection efficacy against field isolates.
Infectious bronchitis (IB) is a common, highly contagious, acute, and economically important viral disease of chickens caused by Infectious bronchitis virus (IBV, sp. Avian coronavirus). Five pooled tissue suspensions of 50 layer birds and one reference Massachusetts vaccine strain were inoculated into specific pathogen free (SPF) chicken egg for isolation of IBV. Reverse-transcription polymerase chain reaction (RT-PCR) was carried out using post inoculated allontoic fluid to amplify the spike (S) glycoprotein of S1 subunit of IBV. All the eggs inoculated with five pooled tissue samples and vaccine sample showed dwarfing and curling of SPF embryos indicative of IBV. All the five samples and the vaccine sample produced the expected amplicons of 466 bp by RT-PCR. The sequencing of five isolates revealed that all the five sequences were 99.09-100 % similar among themselves and showed 99.10-100 % nucleotide identity with the vaccine strain. On multiple sequence alignment it was found that our isolates were more similar at S1 subunit nucleotide level with the reference Ma5 and H120 vaccine strains than the reference Mass41 strain. The sequences of Anand isolates revealed further genetic changes in the circulating IBV in comparison to previous isolate of Gujarat as well as higher differences with the strains isolated in other states showing substantial changes at genetic level in Indian IBV isolates, which may partially explain the increasing incidences of IB in the country in spite of the vaccination.
A total of 34 clinical samples and four Marek's disease virus (MDV) vaccines were tested using primer BamH1/BamH2 in layer birds of poultry. Out of 34 samples tested for detection of MDV, 32 samples produced approximately 434 bp product. All the three HVT vaccines as well as SB-1 (MDV-2) vaccine failed to produce the expected amplicons, there by proving negative for the targeted 132 bp repeats of MDV genome by the primers BamH1/BamH2. Resultant PCR products of the field samples were purified and sequenced and resulted in 378 bp long sequences. PCR was found very satisfactory in detecting the presence of MDV either in feather follicle or in tissue samples. Sequencing study has proved beyond doubt that the two representative samples contained two 132 bp repeats indicating the virulent nature of the field virus.
Mastitis is one of the most common and burdensome diseases afflicting dairy animals. Among other causes of mastitis, staphylococci are frequently associated with clinical and subclinical mastitis. Although Staphylococcus aureus is the predominant species involved, Staphylococcus epidermidis and other coagulase-negative staphylococci are increasingly being isolated from cases of bovine mastitis. Although Staph. aureus and Staph. epidermidis can be easily differentiated based on their biochemical properties, such phenotypic identification is time consuming and laborious. This study aimed to rapidly identify Staph. aureus and Staph. epidermidis. Accordingly, a multiplex PCR was developed and we found that a single gene encoding the adhesin fibrinogen binding protein could be used to identify and differentiate the two species. Consequently, a multiplex reaction combining a triplex PCR for Staph. aureus and a duplex PCR for Staph. epidermidis was standardized, first using bacterial cultures and then with pasteurized milk spiked with live organisms or DNA extracted from the organisms. The test could specifically detect Staph. aureus and Staph. epidermidis even in the presence of a dozen other organisms. The limit of detection for detecting Staph. aureus and Staph. epidermidis separately was 10 to 100 cfu/mL for simplex PCR and 10 4 cfu/mL for multiplex PCR. Conversely, the limit was 10 6 cfu/ mL by multiplex PCR for simultaneous detection of both the organisms when spiked into culture medium or pasteurized milk. Overnight enrichment enhanced the assay sensitivity 100-fold. The assay had a high diagnostic sensitivity and specificity. The application of the test was verified on 602 field isolates of staphylococci that had been characterized earlier by phenotypic methods. Importantly, 25 coagulase-negative isolates were identified as Staph. aureus by the multiplex PCR. The test could be adapted for use in clinical diagnostic laboratories.
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