Molecular Survey of Respiratory and Immunosuppressive Pathogens Associated with Low Pathogenic Avian Influenza H9N2 Subtype and Virulent Newcastle Disease Viruses in Commercial Chicken Flocks.

The study was carried out in 48 poultry flocks to elucidate the roles of various complicating pathogens involved along with Newcastle disease (ND)/ low pathogenic avian influenza (LPAI) outbreaks. Necropsy was conducted and samples were collected for the isolation of Newcastle disease virus (NDV), Influenza A virus, infectious bronchitis virus (IBV), pathogenic bacteria; molecular detection of infectious laryngotracheitis virus (ILTV), fowl adeno virus (FAV), chicken anaemia virus (CAV), Mycoplasma synoviae (MS) and Mycoplasma gallisepticum (MG). The isolation results confirmed that 18/48 flocks (37%) were positive for the presence of hemagglutinating agents. Out of 18 hemagglutination (HA) positive flocks, 11 flocks (61%) were positive for both avian influenza virus (AIV) and NDV; 4 flocks (22%) were positive for NDV; and 3 flocks (17%) were positive for AIV. Sequence analysis of hemagglutinin and neuraminidase genes of AIV revealed that all were belonging to LPAI-H9N2 subtype. Sequence analysis of F gene of NDV revealed that they belong to virulent type. The PCR results confirmed the presence of three to seven etiological agents (CAV, FAV, ILTV, MG, MS and avian pathogenic E. coli along with LPAI/NDV from all the 18 HA-positive flocks. The detection rate of triple, quadruple, quintuple, sextuple and sevenfold infections was 17% (3 flocks), 28% (5 flocks), 11%, (2 flocks) 28% (5 flocks) and 17% (3 flocks), respectively. In conclusion, the disease complex involved more than one pathogen, primarily resulting from the interplay between LPAI-H9N2 and NDV; subsequently this could be exacerbated by co-infection with other agents which may cause exacerbated outbreaks that may otherwise go undetected in field.

Isolation and phylogenetic characterization of haemagglutinin and neuraminidase genes of H9N2 low pathogenicity avian influenza virus isolated from commercial layers in India.

Avian influenza is a highly infectious and dynamically evolving disease of birds causing high morbidity and mortality. It is caused by avian influenza virus (AIV) that belongs to the family Orthomyxoviridae. Two types of AIV have been described based on their pathogenicity viz. highly pathogenic avian influenza virus that causes severe disease with high mortality and low pathogenic avian influenza virus (LPAI) that generally causes asymptomatic infection or a mild disease. The H9N2 subtype is the widely circulated LPAI type in the world. The H9N2 subtype of was first reported from northern India in March 2003. However, systematical surveillance information for the evolution of H9N2 viruses in poultry flocks of Southern India is lacking. The present study reports the isolation and characterization of H9N2 isolates from the southern parts of the country during the period between May 2010 and September 2011. Out of the 30 poultry flocks investigated, six were found to be positive for HA activity. Further, all the six samples conformed as AIV. Partial nucleotide sequencing of the HA and NA genes revealed that all were belonging to the H9N2 subtype. Phylogenetically, the HA and NA genes of the H9N2 viruses from India clustered with those isolated from Bangladesh, Pakistan and the Middle East, although we were not able to conclude on their exact geographic origin.

Detection and partial genetic characterisation of a novel variant of Avian nephritis virus in Indian poultry flocks showing diverse clinical signs.

Avian nephritis virus (ANV) infects poultry flocks worldwide, but no confirmed cases have been reported from India so far. In the current study, disease investigation was carried out in 21 broiler flocks at different parts of India with clinical signs of nephritis, uneven and stunted growth, diarrhoea, reduced body weight, and mortality up to 9.72%. Out of the 21 flocks screened, two were found positive for ANV in RT-PCR assay. BLAST analysis revealed that the ANV of Indian origin was closely related to ANV-1 strains reported from Japan, Hungary and China. However, comparison of a small portion (~12% of nucleotides, i.e. ~60 nts, common site for ANV-1 and ANV-3, position 2200-2260 of ORF 1a gene) of the Indian ANV sequence with ANV-3 sequences revealed 89-93% identities with different ANV-3 isolates. Phylogenetically, ANV-1 forms three clades, and the Indian ANV clustered under clade II. This study confirms the existence of ANV in Indian poultry flocks and is the first report on the molecular detection and genetic characterisation of ANV from India.

Molecular Epidemiology of Nontyphoidal Salmonella in Poultry and Poultry Products in India: Implications for Human Health.

Human infections with non-typhoidal Salmonella (NTS) serovars are increasingly becoming a threat to human health globally. While all motile Salmonellae have zoonotic potential, Salmonella Enteritidis and Salmonella Typhimurium are most commonly associated with human disease, for which poultry are a major source. Despite the increasing number of human NTS infections, the epidemiology of NTS in poultry in India has not been fully understood. Hence, as a first step, we carried out epidemiological analysis to establish the incidence of NTS in poultry to evaluate the risk to human health. A total of 1215 samples (including poultry meat, tissues, egg and environmental samples) were collected from 154 commercial layer farms from southern India and screened for NTS. Following identification by cultural and biochemical methods, Salmonella isolates were further characterized by multiplex PCR, allele-specific PCR, enterobacterial repetitive intergenic consensus (ERIC) PCR and pulse field gel electrophoresis (PFGE). In the present study, 21/1215 (1.73 %) samples tested positive for NTS. We found 12/392 (3.06 %) of tissue samples, 7/460 (1.52 %) of poultry products, and 2/363 (0.55 %) of environmental samples tested positive for NTS. All the Salmonella isolates were resistant to oxytetracycline, which is routinely used as poultry feed additive. The multiplex PCR results allowed 16/21 isolates to be classified as S. Typhimurium, and five isolates as S. Enteritidis. Of the five S. Enteritidis isolates, four were identified as group D Salmonella by allele-specific PCR. All of the isolates produced different banding patterns in ERIC PCR. Of the thirteen macro restriction profiles (MRPs) obtained by PFGE, MRP 6 was predominant which included 6 (21 %) isolates. In conclusion, the findings of the study revealed higher incidence of contamination of NTS Salmonella in poultry tissue and animal protein sources used for poultry. The results of the study warrants further investigation on different type of animal feed sources, food market chains, processing plants, live bird markets etc., to evaluate the risk factors, transmission and effective control measures of human Salmonella infection from poultry products.

Molecular detection and characterization of infectious laryngotracheitis virus (Gallid herpesvirus-1) from clinical samples of commercial poultry flocks in India.

Although the existence of infectious laryngotracheitis virus (ILTV) in India was first reported in 1964, no reports are available regarding its molecular detection and characterization. The present study was aimed to detect and characterize ILTV from recent respiratory disease complex (RDC) outbreaks of commercial poultry flocks in different parts of the country by using envelope glycoprotein G gene (US4 gene) based PCR and sequencing. A total of thirty two flocks with a history of RDC were investigated. Overall, all the strains/breeds of birds and all ages of birds are equally susceptible and depending on the severity, the clinical signs and gross lesions were varied. Out of 32 flocks investigated 10 were found positive for ILTV infection by PCR. The phylogenetic analyses of eight representative sequences in the present study deciphered that Indian ILT viruses are closely related to chicken embryo origin vaccine strains of Italy, USA, China and Brazil.