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.

In Vitro Characterization of chIFITMs of Aseel and Kadaknath Chicken Breeds against Newcastle Disease Virus Infection.

Newcastle disease (ND) is highly contagious and usually causes severe illness that affects Aves all over the world, including domestic poultry. Depending on the virus's virulence, it can impact the nervous, respiratory, and digestive systems and cause up to 100% mortality. The chIFITM genes are activated in response to viral infection. The current study was conducted to quantify the mRNA of chIFITM genes in vitro in response to ND viral infection. It also examined its ability to inhibit ND virus replication in chicken embryo fibroblast (CEF) cells of the Aseel and Kadaknath breeds. Results from the study showed that the expression of all chIFITM genes was significantly upregulated throughout the period in the infected CEF cells of both breeds compared to uninfected CEF cells. In CEF cells of the Kadaknath breed, elevated levels of expression of the chIFITM3 gene dramatically reduced ND viral growth, and the viral load was 60% lower than in CEF cells of the Aseel breed. The expression level of the chIFITMs in Kadaknath ranged from 2.39 to 11.68 log2 folds higher than that of control CEFs and was consistently (p < 0.01) higher than Aseel CEFs. Similar to this, theIFN-γ gene expresses strongly quickly and peaks at 13.9 log2 fold at 48 hpi. Based on these cellular experiments, the Kadaknath breed exhibits the potential for greater disease tolerance than Aseel. However, to gain a comprehensive understanding of disease resistance mechanisms in chickens, further research involving in vivo investigations is crucial.

Isolation and molecular characterization of infectious bronchitis virus from recent outbreaks in broiler flocks reveals emergence of novel strain in India.

In this study, two isolates of infectious bronchitis virus (IBV) from field outbreaks in 2008 (India/LKW/56/IVRI/08) and 2010 (India/NMK/72/IVRI/10) from broiler chickens in India were isolated and characterized. Reverse transcription polymerase chain reaction-restriction fragment length polymorphism of the entire S1 gene revealed that these isolates belong to two different genotypes, India/LKW/56/IVRI/08 as Mass strain whereas India/NMK/72/IVRI/10 as of different genotype. Nucleotide sequencing analysis showed that India/LKW/56/IVRI/08 shared 99 % homology with THA280252 (Thailand) and India/NMK/72/IVRI/10 shared greater than 99 % homology with 4/91 pathogenic strain (UK), JP/Wakayama/2/2004 (Japan) and TA03 (China), while the two Indian IBV isolates shared 73 % identity between them. Phylogenetic data allowed classification of two Indian isolates, India/LKW/56/IVRI/08 as having unique lineage within Mass genotype and India/NMK/72/IVRI/10 as of 4/91 genotype. Our study confirmed the presence of 4/91 (793/B) IBV nephropathogenic strain for the first time in India by virus isolation and sequencing.

Infectious laryngotracheitis: Etiology, epidemiology, pathobiology, and advances in diagnosis and control - a comprehensive review.

Infectious laryngotracheitis (ILT) is a highly contagious upper respiratory tract disease of chicken caused by a Gallid herpesvirus 1 (GaHV-1) belonging to the genus Iltovirus, and subfamily Alphaherpesvirinae within Herpesviridae family. The disease is characterized by conjunctivitis, sinusitis, oculo-nasal discharge, respiratory distress, bloody mucus, swollen orbital sinuses, high morbidity, considerable mortality and decreased egg production. It is well established in highly dense poultry producing areas of the world due to characteristic latency and carrier status of the virus. Co-infections with other respiratory pathogens and environmental factors adversely affect the respiratory system and prolong the course of the disease. Latently infected chickens are the primary source of ILT virus (ILTV) outbreaks irrespective of vaccination. Apart from conventional diagnostic methods including isolation and identification of ILTV, serological detection, advanced biotechnological tools such as PCR, quantitative real-time PCR, next generation sequencing, and others are being used in accurate diagnosis and epidemiological studies of ILTV. Vaccination is followed with the use of conventional vaccines including modified live attenuated ILTV vaccines, and advanced recombinant vector vaccines expressing different ILTV glycoproteins, but still these candidates frequently fail to reduce challenge virus shedding. Some herbal components have proved to be beneficial in reducing the severity of the clinical disease. The present review discusses ILT with respect to its current status, virus characteristics, epidemiology, transmission, pathobiology, and advances in diagnosis, vaccination and control strategies to counter this important disease of poultry.

Molecular phylogenetics of Newcastle disease viruses isolated from vaccinated flocks during outbreaks in Southern India reveals circulation of a novel sub-genotype.

Newcastle disease (ND) is an economically important, contagious poultry viral disease reported across the globe. In India, ND is endemic and episodes of ND outbreaks despite strict vaccinations are not uncommon. We isolated and characterized seven ND viruses from vaccinated commercial poultry farms during severe disease outbreaks in Tamil Nadu, in Southern India, between April 2015 and June 2016. All the seven isolates were categorized as virulent by mean death time (48-54 hr) in embryonated chicken eggs. Also, their sequences carried the virulence signature of multi-basic amino acid residues in their fusion protein cleavage site (RRQ/RR/KRF). Phylogenetic and evolutionary distance analyses revealed circulation of a novel sub-genotype of genotype XIII, class II ND viruses, herein proposed as sub-genotype XIIIe. The genetic divergence between the circulating virulent strains and the vaccine strains could possibly explain the disease outbreak in the vaccinated flocks. Further, our study signifies the need to implement routine epidemiological surveillance and to revisit the current vaccination program.

Occurrence of Clostridium perfringens contamination in poultry feed ingredients: Isolation, identification and its antibiotic sensitivity pattern.

This work has been undertaken to study the occurrence of Clostridium perfringens contamination in the poultry feed ingredients and find out its in-vitro antibiotic sensitivity pattern to various antimicrobial drugs. Two hundred and ninety-eight poultry feed ingredient samples received at Poultry Disease Diagnosis and Surveillance Laboratory, Namakkal, Tamil Nadu in South India were screened for the presence of C. perfringens. The organisms were isolated in Perfringens agar under anaerobic condition and subjected to standard biochemical tests for confirmation. In vitro antibiogram assay has been carried out to determine the sensitivity pattern of the isolates to various antimicrobial drugs. One hundred and one isolates of C. perfringens were obtained from a total of 298 poultry feed ingredient samples. Overall positivity of 33.89% could be made from the poultry feed ingredients. Highest level of C. perfringens contamination was detected in fish meal followed by bone meal, meat and bone meal and dry fish. Antibiogram assay indicated that the organisms are highly sensitive to gentamicin (100%), chlortetracycline (96.67%), gatifloxacin (93.33%), ciprofloxacin (86.67%), ofloxacin (86.67%) and lincomycin (86.67%). All the isolates were resistant to penicillin-G. Feed ingredients rich in animal proteins are the major source of C. perfringens contamination.

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.

Haemorrhagic enteritis of turkeys - current knowledge.

Haemorrhagic enteritis virus (HEV), an adenovirus associated with acute haemorrhagic gastro-intestinal disease of 6-11-week old turkeys predominantly hampers both humoral and cellular immunity. Affected birds are more prone to secondary complications (e.g. colibacillosis and clostridiosis) and failure to mount an effective vaccine-induced immune response. HEV belongs to the new genus Siadenovirus. Feco-oral transmission is the main route of entry of the virus and it mainly colonizes bursa, intestine and spleen. Both naturally occurring virulent and avirulent strains of HEVs are serologically indistinguishable. Recent findings revealed that ORF1, E3 and fib genes are the key factors affecting virulence. The adoption of suitable diagnostic tools, proper vaccination and biosecurity measures have restrained the occurrence of disease epidemics. For diagnostic purposes, the best source of HEV is either intestinal contents or samples from spleen. For rapid detection highly sensitive and specific tests such as quantitative real-time PCR based on Taq man probe has been designed. Avirulent strains of HEV or MSDV can be effectively used as live vaccines. Novel vaccines include recombinant hexon protein-based subunit vaccines or recombinant virus-vectored vaccines using fowl poxvirus (FPV) expressing the native hexon of HEV. Notably, subunit vaccines and recombinant virus vectored vaccines altogether offer high protection against challenge or field viruses. Herein, we converse a comprehensive analysis of the HEV genetics, disease pathobiology, advancements in diagnosis and vaccination along with appropriate prevention and control strategies.

Isolation and characterization of genotype XIII Newcastle disease virus from Emu in India.

Newcastle disease virus (NDV) infects at least 241 species of pet and free-living birds in addition to domesticated avian species. Wild, feral and domesticated birds are recognized reservoirs of NDV, and contribute to the epidemiology of NDV in the domesticated poultry. The biological and molecular characterization of velogenic NDV (vNDV) from emus is limited. In this study, 54 tissues were collected from eight Emu flocks between May 2010 and January 2012 from highly poultry-dense areas of India including Namakkal, Hyderabad and Bareilly regions. The presence of vNDV was confirmed by virus isolation, fusion (F)-gene based RT-PCR, sequencing of the cleavage site and the virulence scored. One out of eight flocks received from Hyderabad region was found positive for NDV and the in vivo pathotyping revealed the isolate to be vNDV type. The sequence analysis revealed that the isolate had cleavage site of 112-R-R-R-K-R-F-117, which is typical for vNDV. Sequence and phylogenetic analysis of the partial 'F' gene coding regions suggested that the NDV strain belongs to genotype XIII. The Emu isolate had 98-100 % nucleotide identity with the vNDVs previously reported in poultry flocks of India. The findings of present study based on the biological and molecular characterization of Emu-origin vNDV, highlights the circulation of genotype XIII in Emus for the first time in the country. There is need to understand the possible spill over of these genetically diverse NDV strains into the commercial poultry and their possible implications in disease control strategies.

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.

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.