Pathotyping of Newcastle Disease Virus: a Novel Single BsaHI Digestion Method of Detection and Differentiation of Avirulent Strains (Lentogenic and Mesogenic Vaccine Strains) from Virulent Virus.

We provide a novel single restriction enzyme (RE; BsaHI) digestion approach for detecting distinct pathotypes of Newcastle disease virus (NDV). After scanning 4,000 F gene nucleotide sequences in the NCBI database, we discovered a single RE (BsaHI) digestion site in the cleavage site. APMV-I "F gene" class II-specific primer-based reverse transcriptase PCR was utilized to amplify a 535-bp fragment, which was then digested with the RE (BsaHI) for pathotyping avian NDV field isolates and pigeon paramyxovirus-1 isolates. The avirulent (lentogenic and mesogenic strains) produced 189- and 346-bp fragments, respectively, but the result in velogenic strains remained undigested with 535-bp fragments. In addition, 45 field NDV isolates and 8 vaccine strains were used to confirm the approach. The sequence-based analysis also agrees with the data obtained utilizing the single RE (BsaHI) digestion approach. The proposed technique has the potential to distinguish between avirulent and virulent strains in a short time span, making it valuable in NDV surveillance and monitoring research. IMPORTANCE The extensive use of the NDV vaccine strain and the existence of avirulent NDV strains in wild birds makes it difficult to diagnose Newcastle Disease virus (NDV). The intracerebral pathogenicity index (ICPI) and/or sequencing-based identification, which are required to determine virulent NDV, are time-consuming, costly, difficult, and cruel techniques. We evaluated 4,000 F gene nucleotide sequences and discovered a restriction enzyme (RE; BsaHI) digestion technique for detecting NDV and vaccine pathotypes in a short time span, which is cost-effective and useful for field cases as well as for large-scale NDV monitoring and surveillance. The data acquired using the single RE BsaHI digestion technique agree with the sequence-based analysis.

A Comparative Study of Pathology and Host Immune Response Induced by Very Virulent Infectious Bursal Disease Virus in Experimentally Infected Chickens of Aseel and White Leghorn Breeds.

Indigenous breeds of young chickens in India are believed to be resistant to the classical strain of infectious bursal disease virus (IBDV). However, the mechanism underlying this resistance is obscure. Innate immunity is a key factor in defining the clinical course and pathology of microbial infections. The present study is aimed to compare the pathology of very virulent IBDV (vvIBDV) and immunological host response in experimentally infected - vaccinated and unvaccinated indigenous Aseel and commercial White Leghorn chickens. The viral loads and innate immune gene expression profiles of MDA-5, Mx, IFN-α, and IFN-ß in different lymphoid organs were analyzed by quantitative PCR. The histopathological scores in Aseel birds were lower than in White Leghorns despite comparable viral loads. The degrees of histopathological lesions were fewer in vaccinated birds than in unvaccinated birds of both breeds. Analysis of innate immune response genes revealed that the cytoplasmic pattern recognition receptor MDA-5 gene was overexpressed mainly in the cecal tonsils of both vaccinated and nonvaccinated White Leghorn chickens. An increase in the expression of the IFN-α gene was seen in the cecal tonsils of Aseels, and an increase in IFN-ß gene expression was seen in the thymuses of White Leghorns following vvIBDV challenge both in vaccinated and nonvaccinated birds. In addition, we observed that the Mx gene plays a minimal role, if any, in vvIBDV infection of the breeds under study. It remains interesting and important that although vvIBDV causes disease in indigenous Aseel birds, the faster clearance and reduced pathology of the virus in Aseel birds compared to White Leghorn chicken indicate some unidentified innate immune factors that are limiting IBDV in this breed. Further studies will be required to correlate kinetics of humoral and cellular immune response in relation to the virus load in different organs to illuminate the mechanism of genetic resistance in native breeds of chicken.

Molecular characterization, isolation, pathology and pathotyping of peafowl (Pavo cristatus) origin Newcastle disease virus isolates recovered from disease outbreaks in three states of India.

Disease outbreak investigations were carried out in three states of Northern India namely Haryana (Rewari), Uttar Pradesh (Noida) and Delhi, where a total of 110 Indian peafowls (Pavo cristatus) showed sudden onset of nervous signs and died within a period of two weeks during June, 2012. The F (fusion) gene-based RT-PCR detection of Newcastle disease virus (NDV) in affected tissues confirmed the presence of the virus. Three NDV isolates were selected (one from each area under investigation) and further characterized. They were found to be of virulent pathotype (velogenic NDV) based on both pathogenicity assays (MDT, ICPI and IVPI) and partial F gene sequence analysis. Additionally, the phylogenetic analysis revealed that the isolates belonged to the genotype VIIi and XIII of class II avian Paramyxovirus serotype1 (APMV-1) and related closely to new emerging sub-genotypes. This is the first report regarding the presence of the fifth panzootic vNDV genotype VIIi from India. In this scenario, extensive epidemiological studies are suggested for surveillance of NDV genotypes in wild birds and poultry flocks of the country along with adopting suitable prevention and control measures.

Baruscapillaria obsignata: a serious cause of enteropathy and high mortality in turkeys (meleagris gallopavo).

BACKGROUND: Capillariasis, an important parasitic disease of birds is caused at least by seven different genera of trichurid nematodes with clinical outcome ranging from mild enteritis to high mortality. OBJECTIVE: This study was aimed to investigate the causative agent involved in high mortality associated with severe enteric illness among turkey flocks in an organized commercial poultry farm at Bareilly, India. MATERIALS AND METHODS: Turkey carcasses (n = 119) and fecal samples from the affected deep litter pen constituted as the study materials. The disease was investigated by systematic necropsy, direct microscopy and histopathology. Representative samples were screened for other enteric pathogens. RESULTS: Microscopic examination of mucosal scraping revealed capillarid worms and their eggs in all the samples. The morphological features of adult worms were typically consistent to Baruscapillaria obsignata. Histopathology exhibited thickened muscular and mucosal layers, mononuclear and heterophilic infiltration in the lamina propria, blunting and clubbing of villi, epithelial denudation and sections of capillarid worms. Administration of levamisole at 80 ppm in drinking water reduced the mortality, clinical illness and worm load after three days of therapy. CONCLUSIONS: The capillarid worms in different avian hosts can cause different clinical manifestations and outcomes. From India, this is the first report describing intestinal pathology caused by B. obsignata in turkeys. We conclude that the B. obsignata infection is capable of causing life threatening enteropathy in turkeys and, hence, routine screening, scheduled deworming and good litter management are crucial to control the infection and its associated loss.

A sensitive haemadsorption technique based RT-PCR for concentration and detection of Newcastle disease virus from clinical samples and allantoic fluid.

The present study describes the exploitation of haemadsorption (HAd) property of the Newcastle disease virus (NDV) for the development of a novel sensitive HAd technique based RT-PCR for detection of NDV from clinical samples of virus infected experimental birds. The NDV propagated allantoic fluid from the infected embryonated chicken eggs or supernatant of the processed clinical samples (tissue triturate, cloaca and tracheal swabs) from the experimentally infected birds were added with chicken red blood cells (RBC) to adsorb the virus on RBC's surface. The virus adsorbed RBCs were subjected to trizol method of RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR) for detection of NDV. The HAd based RNA extraction showed better yield of 700-900 ng RNA and when subjected to RT-PCR detection revealed a 100 times higher sensitivity than the conventional RNA extraction and RT-PCR detection system. This could be an alternate technique which can be exploited in low NDV load situations in clinical samples.

Development of a DNA vaccine for chicken infectious anemia and its immunogenicity studies using high mobility group box 1 protein as a novel immunoadjuvant indicated induction of promising protective immune responses.

Chicken infectious anaemia (CIA) is an economically important and emerging poultry disease reported worldwide. Current CIA vaccines have limitations like, the inability of the virus to grow to high titres in embryos/cell cultures, possession of residual pathogenicity and a risk of reversion to virulence. In the present study, a DNA vaccine, encoding chicken infectious anaemia virus (CIAV) VP1 and VP2 genes, was developed and co-administered with truncated chicken high mobility group box 1 (HMGB1ΔC) protein in young chicks for the evaluation of vaccine immune response. CIAV VP1 and VP2 genes were cloned in pTARGET while HMGB1ΔC in PET32b vector. In vitro expression of these gene constructs was evaluated by Western blotting. Further, recombinant HMGB1ΔC was evaluated for its biological activity. The CIAV DNA vaccine administration in specific pathogen free chicks resulted in moderately protective ELISA antibody titres in the range of 4322.87 ± 359.72 to 8288.19 ± 136.38, increased CD8(+) cells, and a higher titre was observed by co-administration of novel adjuvant (HMGB1ΔC) and booster immunizations. The use of vaccine with adjuvant showed achieving antibody titres nearly 8500, titre considered as highly protective, which indicates that co-immunization of HMGB1ΔC may have a strong adjuvant activity on CIAV DNA vaccine induced immune responses. The able potential of HMGB1 protein holding strong adjuvant activity could be exploited further with trials with vaccines for other important pathogens for achieving the required protective immune responses.

Emergence of Avian Infectious Bronchitis Virus and its variants need better diagnosis, prevention and control strategies: a global perspective.

Growth in poultry sector is being challenged due to increased incidence and re-emergence of diseases caused due to evolution of several viral pathogens and use of live vaccines. Piles of economic losses are encountered due to these diseases. Avian Infectious Bronchitis (IB), caused by Corona virus, is OIE-listed disease and characterized by respiratory, renal and urogenital involvements, causing high mortality. Economic losses are encountered due to loss of productive performance of both egg and meat-type chickens. Variant viruses evolve due to spontaneous mutations and recombinations, causing disease in vaccinated flocks of all ages. Serotyping and genotyping are the common methods of classification of IBV strains. The virus has 4 clusters, grouped into 7 serotypes and the most important strains are Massachusetts, Connecticut, Arkansas, Gray, Holte and Florida along with numerous others, distributed round the globe. Several conventional and molecular diagnostic methods have been described for the diagnosis of IB in chickens. 'All-in/all-out' operations of rearing along with good biosafety measures forms the basis of prevention, whereas vaccination forms the backbone of IB control programme. Both live and inactivated (oil emulsified) conventional vaccines are available. The new generation vaccines (recombinant and vector-based) developed against locally prevailing IBV strains may be more helpful and avoid the reversion of virulence in live vaccine viruses. The present review deals with all these perspectives of this important emerging poultry pathogen.

Experimental pathological studies of an Indian chicken anaemia virus isolate and its detection by PCR and FAT.

Chicken Infectious Anaemia Virus (CIAV) is one of the potent immunosuppressive and economically important agents affecting poultry industry worldwide. Recent reports indicate the emergence of this virus in the poultry flocks of the country. The present study aimed to investigate the pathogenic potential of a recent isolate of CIAV obtained from poultry flock of Uttaranchal State, India. Twenty first day-old age Specific Pathogen Free (SPF) chicks were inoculated intramuscularly with 10(4.5) median tissue culture infective dose (TCID50) of CIAV passaged in the Marek's disease virus transformed chicken splenic T lymphocyte (MDCC-MSB 1) cell line while 15 chicks were kept as control. The CIAV isolate produced consistent clinical signs, loss in body weight gain, anaemia, low haematocrit values, bone marrow aplasia and generalized lymphoid atrophy. Mean Packed Cell Volume (PCV) value of the infected chicks was significantly low (18.22±2.22) compared to control group (34.12±4.72) at 14 day post infection (dpi). The establishment of virus infection in chicks was confirmed both at molecular and antigenic levels by Polymerase Chain Reaction (PCR) and Indirect Immunofluorescent Test (IIFT), respectively. Characteristic apoptotic pattern was also detected in the affected organs and the virus was re-isolated successfully in MDCC-MSB1 cell cultures. The present results revealed that the virus circulating in poultry flocks of Uttaranchal state is both pathogenic and immunosuppressive in nature. Extensive epidemiological studies are suggested in the poultry flocks of the country along with adaptation of appropriate diagnostic, prevention and control strategies so as to prevent economic losses caused by this important poultry pathogen.

Detection and differentiation of pigeon paramyxovirus serotype-1 (PPMV-1) isolates by RT-PCR and restriction enzyme analysis.

Detection and pathotyping of Newcastle disease virus (NDV) is extremely important because the appearance of virulent virus has significant economic consequences. During 1981 to 1985, infections of racing and show pigeons with an avian paramyxovirus serotype-1 (APMV-1) hit worldwide, and a panzootic occurred due to a variant form of classical NDV. On the basis of pathogenicity and monoclonal antibody binding studies, the virus was termed 'pigeon PMV-1' (PPMV-1). In the past, number of Newcastle disease outbreaks in poultry and other birds has been attributed to PPMV-1. PPMV-1 viruses are known to present difficulty when assessed by conventional in vivo pathogenicity tests. In this study, the technique of reverse transcription-polymerase chain reaction (RT-PCR) and restriction enzyme (RE) analysis was used to detect and differentiate PPMV-1 isolates of Indian origin. Restriction enzyme digestion analysis of RT-PCR-amplified fusion protein (F) gene, encoding for the cleavage activation sites of fusion protein, was carried out with restriction enzymes BglI, HhaI, HaeIII, HinfI, MboI, MspI, PvuII and StyI. A set of only four enzymes HhaI, MspI or HaeIII, MboI and BglI alone were sufficient to differentially detect APMV-1 and PPMV-1 viruses and their pathotypes. In conclusion, RT-PCR followed by RE analysis proved to be useful for detection and differentiation of APMV-1 and PPMV-1 isolates at genomic level.

Fungal/mycotic diseases of poultry-diagnosis, treatment and control: a review.

Fungal/mycotic diseases cause significant economic losses to the poultry industry either due to their direct infectious nature or due to production of mycotoxins, the secondary fungal metabolites produced in grains or poultry feed. Several fungi have created havoc in the poultry industry and some of them cause direct harm to human health due to their zoonotic implications. They are responsible for high morbidity and mortality, especially in young birds and cause stunted growth and diarrhea; and fatal encephalitis. Mycotic dermatitis is a possible health hazard associated with poultry houses. Mycotoxins are the leading cause of producing immunosuppression in birds, which makes them prone to several bacterial and viral infections leading to huge economic losses to the poultry industry. In comparison to bacterial and viral diseases, advances in diagnosis, treatment, prevention and control of fungal diseases in poultry has not taken much attention. Recently, molecular biological tools have been explored for rapid and accurate diagnosis of important fungal infections. Effective prevention and control measures include: appropriate hygiene, sanitation and disinfection, strict biosecurity programme and regular surveillance/monitoring of fungal infections as well as following judicious use of anti-fungal drugs. Precautionary measures during crop production, harvesting and storing and in feed mixing plants can help to check the fungal infections including health hazards of mycotoxins/mycotoxicosis. The present review describes the fungal pathogens causing diseases in poultry/birds, especially focusing to their diagnosis, prevention and control measures, which would help in formulating appropriate strategies to have a check and control on these unwanted troubles to the poultry producers/farmers.

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.

Recent trends in diagnosis and control of Marek's disease (MD) in poultry.

Marek's Disease (MD), caused by Marek's Disease Virus (MDV) is a highly contagious oncogenic and neuropathic disease of chickens responsible for great economic losses to the poultry industry all around the world and characterized by development of CD4+T cell lymphomas as well as infiltration of nerves and visceral organs by lymphocytes. MD is one of the most common lymphoproliferative diseases of chickens which cause mononuclear cell infiltration in one or more of the following tissues: peripheral nerves, gonads, lymphoid organs, iris, muscle, skin and other visceral organs resulting into development of tumours in visceral organs, paralysis of legs, wings and neck, grey eye (iris) or irregular pupil, vision impairment, blindness, skin lesions and immunosuppression, all of which can be accompanied by non-specific signs such as anorexia, weight loss and poor performance. Today there are evolving highly pathogenic isolates of MDV around the world capable of overwhelming the protection from currently employed vaccines. Thus MD poses a big challenge to the welfare and wellbeing of the poultry with increased condemnation of carcass, loss of productivity and quality products, leading to huge economic losses. It is also an immunosuppressive disease and causes increased susceptibility to other infections. The present review discusses in brief about the Marek's disease, its etiology, conventional and advance tools and techniques being used for its diagnosis, prevention and control strategies in poultry.