Genotypic and Pathotypic Characterization of Newcastle Disease Viruses from India

Newcastle disease virus (NDV) is an avian paramyxovirus that causes significant economic losses to the poultry industry in most parts of the world. The susceptibility of a wide variety of avian species coupled with synanthropic bird reservoirs has contributed to the vast genomic diversity of this virus as well as diagnostic failures. Since the first panzootic in 1926, Newcastle disease (ND) became enzootic in India with recurrent outbreaks in multiple avian species. The genetic characteristics of circulating strains in India, however, are largely unknown. To understand the nature of NDV genotypes in India, we characterized two representative strains isolated 13 years apart from a chicken and a pigeon by complete genome sequence analysis and pathotyping. The viruses were characterized as velogenic by pathogenicity indices devised to distinguish these strains. The genome length was 15,186 nucleotides (nt) and consisted of six non-overlapping genes, with conserved and complementary 3′ leader and 5′ trailer regions, conserved gene starts, gene stops, and intergenic sequences similar to those in avian paramyxovirus 1 (APMV-1) strains. Matrix gene sequence analysis grouped the pigeon isolate with APMV-1 strains. Phylogeny based on the fusion (F), and hemagglutinin (HN) genes and complete genome sequence grouped these viruses into genotype IV. Genotype IV strains are considered to have “died out” after the first panzootic (1926–1960) of ND. But, our results suggest that there is persistence of genotype IV strains in India

The immunogenicity and pathogenicity of Pasteurella multocida isolated from poultry in Namakkal

The growth of Indian poultry industry is phenomenal and the industry now ranks 5(th) in the world in egg production. Intensification of poultry farming was the way by which this growth was obtained. Such intensification has led to health problems associated with both existing and emerging diseases. Of the diseases affecting poultry, fowl cholera is a very important bacterial disease. Fowl cholera occurs worldwide and causes economic losses due to increased mortality, increased number of culls and a marked drop in egg production. The causative agent of fowl cholera namely Pasturella multocida was first isolated in 1878 by Bollinger (Carter and Chengappa, 1986). A recent survey on the prevalence of fowl cholera in Namakkal area reported an incidence of 4.17% in 0-10 wks, 8.47% in growers and 3.61% in layers (Daniel et al., 1998). There is little information regarding the serotype or pathogenicity of the Indian Pasturella multocida isolates associated with fowl cholera outbreaks. Though commercial vaccines are being used in chicken breeding farms, occasional outbreaks of fowl cholera still occur. Hence, this study was undertaken to characterize local isolates of Pasturella multocida associated with outbreaks in Namakkal as a first step in the development of a fowl cholera vaccine

TaqMan primer/Probe sequences for assessing basal expression levels of TLR3 and TLR7 mRNA.

<p>TaqMan primer/Probe sequences for assessing basal expression levels of TLR3 and TLR7 mRNA.</p

Virus replication in PBMCs stimulated with TLR3 and TLR7 agonists.

<p>a) Reduction in TCID₅₀ values of PPRV in goat PBMC on imiquimod treatment. Reduction in PPRV H gene expression levels in goat PBMC on treatment with b) poly I:C and c) imiquimod. Bars with the same superscript do not differ significantly. Significance is indicated when p<0.05. Values represent mean ± SD of 40-corrected CT in 5 individual animals per goat breed. Significantly higher PPRV viral loads observed in PBMC of Barbari and Tellicherry breeds as compared to Kanni and Salem Black. Significant reduction in PPRV levels on poly I:C and imiquimod treatment in all breeds.</p

Basal expression levels of TLR3 and 7 mRNA in Barbari, Tellicherry, Kanni and Salem Black goat breeds.

<p>Significantly higher basal TLR3 (p<0.001) and TLR7 (p<0.001) mRNA expression levels observed in Kanni and Salem Black breeds, compared to Barbari. Bars with the same superscript do not differ significantly. Values represent mean ± SD of 40-corrected CT of TLR3 and 7 in nine individual animals per breed (n = 9).</p

Cytokine levels in imiquimod, poly I:C or PPRV treated PBMCs.

<p>a) <b>TNFα,</b> b) IFNα, and c) IFNγ from supernatants of PPRV infected PBMC of different goat breeds. Bars with the same superscript do not differ significantly. Significance is indicated when p<0.05. PPRV infected PBMC from Kanni and Salem black breeds show higher production of TNFα, IFNα and IFNγ than Barbari. Poly I:C and imiquimod treated PBMC, similarly, show higher production of TNFα and IFNα in Kanni and Salem black breeds than in Barbari. TNFα and IFNγ levels are expressed as the corrected mean ± SD of optical density [OD] of treatment groups from which the OD of mock infected supernatants is subtracted. IFNα concentrations in the experimental samples are expressed as pg/ml.</p

Antiviral activity of human IFNα against PPRV.

<p>Reduction in PPRV viral load was observed in Vero cells pretreated with different concentrations of human IFNα. All the doses tested significantly reduced PPRV viral load (mean ± SD of 40-Ct). Statistical significance was defined as follows: ***<i>P</i><0.001.</p

Role of type I IFN in limiting PPRV replication.

<p>a) Imiquimod or b) PPRV induced IFN-alpha mRNA expression in buffalo and goat PBMCs in the presence and absence of the TLR7 antagonist (IRS 661). Cytokine mRNA expression was quantified at 3, 6, 12 and 24 h post stimulation by qRT-PCR assays using SYBR Green chemistry. Fold change in mRNA expression induced by Imiquimod or PPRV stimulation was calculated using mock induced cytokine mRNA expression levels as a calibrator. c) PPRV replication in goat and buffalo PBMC (at 24 h and 48 h) in the presence of conditioned medium (CM) from PPRV infected cells. The expression levels of viral hemagglutinin (H) mRNA levels expressed as a percentage inhibition in viral replication in the presence of CM when compared with the control (PBMC+PPRV). Statistical significance at <i>P</i><0.01. Values are mean ± SD of fold change/percent inhibition. B: water buffalo, G: goat, BPBMC: buffalo PBMC, GPBMC: goat PBMC.</p

Induction of cytokine genes of goat PBMCs with TLR3 and TLR7 agonists and PPRV.

<p>A) Fold changes in mRNA expression levels of IL1β, IL6, IL8, IL10, IL12p40, TNFα, IFNγ and IFNα in goat PBMC stimulated with a) poly I:C, b) imiquimod or c) infected with PPRV d) Goat and buffalo PBMCs infected with PPRV. Fold change was determined by the 2^−ΔΔCt formula. An upregulation in TNFα expression levels, consistent with a downregulation in IL10 levels, is observed in Kanni and Salem Black breeds of all treatment groups. In addition, upregulation of IFNα and IFNγ was observed in the PBMC of Kanni and Salem Black breeds after PPRV infection. PPRV stimulation resulted in an upregulation of IL1β and IFNα in buffalo PBMC and IL10, IL12p40 and IFNγ in goat PBMCs. Bars with the same superscript do not differ significantly. Significance is indicated when p<0.05. Values represent mean ± SD of 5 individual animals per goat breed.</p