Development of a new method for sperm RNA purification in the chicken.

Currently RNA transcripts are being used as male fertility biomarker for many mammalian species, but research work on chicken is at halt because classical RNA isolation methods are not effective for chicken spermatozoa. Hence, attempts have been made to optimize RNA isolation protocol from chicken sperm by using different methods, and to confirm the presence of sperm-specific transcripts of PRM and PLCZ1. Semen samples were centrifuged at low speed for removing debris like uric acid. Further, 1mL diluted semen was gently placed over 40% PureSperm or 45%/90% Percoll, and centrifuged to remove somatic cells and immature diploid spermatocytes. RNA was isolated from sperm by using RNAzol or TRIzol reagent or RNeasy Micro kit with certain modification, and RNA quantity and quality were evaluated. RNA isolated by using RNAzol or RNeasy Micro Kit yielded good quantity and quality of RNA for downstream applications compared to TRIzol. 40% PureSperm was found effective in removing somatic cells. RT-PCR results showed that sperm RNA samples were negative for CD4 and PTPRC. All the sperm RNA samples were positive for PRM and PLCZ1, markers of sperm RNA.

Anti-neoplastic effect of avian reovirus σ C protein on Rous sarcoma virus-induced tumors in chicken.

This study investigated the anti-neoplastic potential of avian reovirus σC (sigma C) protein on Rous sarcoma virus-induced fibrosarcoma in chicken. The recombinant vector expressing σC protein was injected intra-tumorally into specific pathogen free chicken with fibro-sarcoma at the dose 100µg per bird, while control birds were mock-treated with 100µg of empty vector per bird. Recombinant σC protein induced apoptosis in tumors of treated birds resulting in progressive tumor regression, while similar changes were absent in tumors of mock-treated controls. The σC protein-induced apoptosis in tumors was quantified by flow cytometry and the mean level of apoptosis up to 66% was observed in treated tumors, whereas any significant level of apoptosis was absent in mock-treated controls.

Complete genome sequence of Newcastle disease virus mesogenic vaccine strain R2B from India.

Mesogenic vaccine strains of Newcastle disease virus (NDV) are widely used in many countries of Asia and Africa to control the Newcastle disease of poultry. In India, the mesogenic strain R2B was introduced in 1945; it protects adult chickens that have been preimmunized with a lentogenic vaccine virus and provides long-lasting immunity. In this article, we report the complete genome sequence of the hitherto unsequenced Indian vaccine virus strain R2B. The viral genome is 15,186 nucleotides in length and contains the polybasic amino acid motif in the fusion protein cleavage site, indicating that this vaccine strain has evolved from a virulent virus. Phylogenetic analysis of this mesogenic vaccine virus classified it with the viruses belonging to genotype III of the class cluster II of NDV.

Prevalence of chicken infectious anaemia virus (CIAV) in commercial poultry flocks of northern India: a serological survey.

Globally, the chicken infectious anaemia virus (CIAV) has gained much importance as an immunosuppressive and economically important emerging pathogen of poultry. In recent years, the virus has been detected and isolated from poultry flocks of India. The present study reports the first sero-epidemiological investigation of the presence of CIAV infection in poultry flocks of the country. A total of 404 serum samples were collected from chicken flocks of eleven poultry farms, which contain a total of 0.34 million birds from four Northern states, suspected of having chicken infectious anaemia (CIA). Screening of the sera samples using a commercially available enzyme-linked immunosorbent assay (ELISA) kit revealed 351 serum samples (86.88%) to be positive for CIAV antibodies. A high CIAV prevalence rate recorded in the present investigation, along with earlier virus detection reports, indicates the widespread distribution of the virus and that CIAV should be considered an economically important poultry pathogen affecting poultry industry of India. Extensive nationwide epidemiological studies are suggested for revealing the economic impact of CIA and to initiate further research along with devising and adapting suitable prevention and control strategies especially the use of suitable vaccines for safeguarding poultry health and production in the country.

Anti-neoplastic effect of chicken anemia virus VP3 protein (apoptin) in Rous sarcoma virus-induced tumours in chicken.

The anti-neoplastic effect of chicken anemia virus VP3 protein (apoptin) was investigated in vitro in Rous sarcoma virus (RSV)-transformed chicken embryo fibroblast (CEF) cells and in RSV-induced tumours of specific-pathogen-free (SPF) chicks in vivo. The apoptin gene was cloned in the pVAX expression vector and in vitro expression of the recombinant vector pVAX-CAV-VP3 was confirmed. Two groups of SPF chicks, each containing ten chicks, were used. Chicks in groups I and II were inoculated with RSV at 1 day old. Group I served as the control, receiving pVAX vector without insert, and group II received recombinant vector pVAX-CAV-VP3 containing the apoptin gene, on day 10. An in vitro study confirmed that apoptin induced apoptosis in RSV-transformed CEF cells, which was demonstrated by observation of the characteristic changes of apoptosis using the indirect immunofluorescence technique and acridine orange/ethidium bromide staining. In vivo study also indicated that apoptin induced apoptosis and caused tumour regression by an intratumoral-delivery method. Apoptotic changes, such as nuclear condensation, fragmentation of the chromatin and formation of apoptic bodies in the tumour cells, were demonstrated by histopathology and acridine orange/ethidium bromide staining. No apoptotic changes were seen in the tumours of the control group. The results of the present study showed that apoptin had an anti-neoplastic effect in vivo and in vitro in RSV-induced tumours. The anti-neoplastic effect is due to apoptin-induced apoptosis. Further improvements in the dose, delivery method and delivery frequency of the apoptin-expressing recombinant vector could help to develop apoptin as an anti-neoplastic drug.

Recombinant haemagglutinin neuraminidase antigen-based single serum dilution ELISA for rapid serological profiling of Newcastle disease virus.

A recombinant haemagglutinin neuraminidase (HN) antigen-based single serum dilution enzyme linked immuno-sorbent assay (ELISA) was developed to measure the specific antibody in sera of chickens against Newcastle disease virus. A linear relationship was found between the predicted antibody titres at a single working dilution of 1:1000 and the corresponding observed serum titres as determined by the standard serial dilution method. Regression analysis was used to determine a standard curve from which an equation was derived that allowed the demonstration of this correlation. The equation was then used to convert the corrected absorbance readings of the single working dilution directly into the predicted ELISA antibody titres. The assay proved to be sensitive, specific and accurate as compared to the standard haemagglutination inhibition (HI) test.

Economically important non-oncogenic immunosuppressive viral diseases of chicken--current status.

Immunosuppressive viral diseases threaten the poultry industry by causing heavy mortality and economic loss of production, often as a result of the chickens' increased susceptibility to secondary infections and sub-optimal response to vaccinations. This paper aimed to present an up-to-date review of three specific economically important non-oncogenic immunosuppressive viral diseases of chickens, viz. chicken infectious anaemia (CIA), infectious bursal disease (IBD) and hydropericardium syndrome (HPS), with emphasis on their immunosuppressive effects. CIA and IBD causes immunosuppression in chickens and the socio-economic significance of these diseases is considerable worldwide. CIA occurs following transovarian transmission of chicken anaemia virus and has potential for inducing immunosuppression alone or in combination with other infectious agents, and is characterized by generalized lymphoid atrophy, increased mortality and severe anemia. The virus replicates in erythroid and lymphoid progenitor cells, causing inapparent, sub-clinical infections that lead to depletion of these cells with consequent immunosuppressive effects. The IBD virus replicates extensively in IgM(+) cells of the bursa and chickens may die during the acute phase of the disease, although IBD virus-induced mortality is highly variable and depends, among other factors, upon the virulence of the virus strain. The sub-clinical form is more common than clinical IBD because of regular vaccination on breeding farms. Infection at an early age significantly compromises the humoral and local immune responses of chickens because of the direct effect of B cells or their precursors. HPS is a recently emerged immunosuppressive disease of 3-6-weeked broilers, characterized by sudden onset, high mortality, typical hydropericardium and enlarged mottled and friable livers, with intranuclear inclusion bodies in the hepatocytes. The agent, fowl adenovirus-4, causes immunosuppression by damaging lymphoid tissues; the presence of IBD and CIA viruses may predispose for HPS or HPS may predispose for other viral infections. Synergism with CIA or other virus infections or prior immunosuppression is necessary to produce IBH-HPS in chickens and the susceptibility of chickens infected with fowl adenovirus varies throughout the course of CIA infection. The mechanism of immunosuppression has been studied in detail for certain chicken viruses at molecular levels, which will provides new opportunities to control these diseases by vaccination.

Biological and molecular characterization of chicken anaemia virus isolates of Indian origin.

In the present study, four chicken anaemia virus (CAV) isolates (CAV-A, -B, -E and -P) recovered from different geographical regions of India were characterized. CAV genome of 1,766 bp nucleotide region containing the complete coding region of VP2 and VP3 proteins, and partial coding region of VP1 protein were sequenced. The nucleotide and deduced amino acid sequence of the Indian CAV isolates were aligned and compared with CAV isolates of European, Asian, American and Australian origin. Phylogenetic analysis of the Indian CAV isolates were also carried out based on the nucleotide and deduced amino acid sequences. The results indicated that Indian isolates were genetically evolved from different parts of the world. Indian isolate, CAV-A was found closely related to European Cux-1 strain, CAV-B and -P were closely related to Bangladesh BD-3 strain and CAV-E was closely related to Australian 704 strain. The pathogenicity of the four CAV isolates was studied in day-old specific pathogen free (SPF) chicks. Day-old SPF chicks (n=50) were divided into five groups comprised of 10 chicks in each group. Group 1 was kept as control and groups 2-5 were infected with each CAV isolate separately. The chicks were infected at a dose rate of 1 ml cell culture fluid (10(4.5)TCID(50)/0.1 ml) per bird intramuscularly. The clinical signs, mortality and packed cell volume (PCV) and body weight gain were recorded on 5, 10 and 15 days post-infection. At 15th day, all the birds were sacrificed and various organs, viz., thymus, bone marrow, spleen, liver and bursa were examined for gross and microscopic changes. The pathogenicity study indicated that all the CAVs except CAV-B were able to produce clinical disease and immunosuppression in young chicks whereas the isolate CAV-B produced no clinical disease but only induced immunosuppression, which was revealed by microscopic examination of the lymphoid organs. The study showed valuable information on molecular epidemiological status of CAV isolates prevalent in India for the first time.

Association of fowl adenovirus serotype 12 with hydropericardium syndrome of poultry in India.

Eight fowl adenovirus (FAdV) isolates obtained from different geographical regions of India were typed by a virus-neutralization test (VNT) using rabbit antisera against all the 12 serotypes of FAdV and by PCR for the hyper-variable region of hexon gene combined with restriction fragment length polymorphism (RFLP) analysis using AluI and MboI restriction enzymes. It was found that six isolates belonged to FAdV-4, one to FAdV-12 and one to both of them. This study revealed the involvement of FAdV-12 alone or in association with FAdV-4 in precipitating inclusion body hepatitis--hydropericardium syndrome (IBH-HPS) among poultry flocks in the country.

Use of multiple antigenic peptides related to antigenic determinants of infectious bursal disease virus (IBDV) for detection of anti-IBDV-specific antibody in ELISA--quantitative comparison with native antigen for their use in serodiagnosis.

Multiple antigenic peptides (MAPs) prepared for the predicted antigenic determinants on the VP2 protein of infectious bursal disease virus (IBDV) were used as antigens in enzyme-linked immunosorbent assay (ELISA)--an alternative to whole viral antigen to detect anti-IBDV antibodies in the chicken sera. Two MAPs were synthesized, which could specifically detect the anti-IBDV antibodies in serum samples by ELISA. The optimum quantity of MAP1 and MAP2 required to coat the wells of the ELISA plate was 5 ng/ml, whereas the amount of purified IBDV whole viral antigen was 500 ng/ml, indicating the high efficiency of MAPs. In this study, we mainly focused on the antigenicity of two eight-branched MAPs to detect anti-IBDV antibodies in ELISA, which would serve as safe, chemically defined, noninfectious alternative antigens to whole virus in serodiagnosis. The specificity and sensitivity of both MAP1 and MAP2 were found to be relatively better than the whole viral antigen.

Restriction enzyme analysis of Indian isolates of egg drop syndrome 1976 virus recovered from chicken, duck and quail.

Egg drop syndrome 1976 (EDS-76) is caused by a haemagglutinating adenovirus belonging to group III of the genus Aviadenovirus in the family Adenoviridae. All isolates are serologically identical, but have been divided into three groups based on restriction endonuclease (RE) analysis. In this study the viral DNA of various Indian EDS-76 viral isolates (CEDS-A, CEDS-B, EDS-M, EDS-ML, EDS-1/AD/86, EDS-KC and QEDS) obtained from different avian species and different geographical regions were digested with restriction endonucleases viz., EcoRI, BamHI, HindIII and PstI. The results showed that one Indian isolate obtained from duck (DEDS-KC) was different from all other chicken and quail counterparts. All other isolates were identical to the reference viral strain BC-14, which belong to group I of EDS-76 viruses. The duck isolate EDS-KC could not be placed in any of the three groups reported earlier.

Detection of Infectious bursal disease virus by ELISA using an antipeptide antibody raised against VP3 region.

Antigenic determinant analysis was carried out on VP3, one of major immunogenic proteins of Infectious bursal disease virus (IBDV) using computer algorithms. Altogether 17 peptides were synthesized for predicted putative regions and were tested for their reactivity with IBDV-positive polyclonal sera as well as with antisera to other common avian viruses to confirm specificity and to rule out cross reactivity. Of 17 peptides tested, three were selected and synthesized in multiple antigenic peptide (MAP) format. The immunization of rabbits with the three MAPs resulted in high humoral immune response. The purified antipeptide antibodies were screened against native IBDV antigen and the respective titers were determined. Out of the three antisera to MAPs that raised against the MAP3, spanning the amino acids (aa) 974-995 region on the VP3 protein had a very high titer (2048) and reacted specifically with IBDV. Thus, the antiserum to MAP3 detected native virus in enzyme-linked immunosorbent assay (ELISA), revealing the presence of a potential antigenic determinant on the C-terminus of the protein. This study proved that an antipeptide antibody could be used as a safe and specific tool for the diagnosis of IBD in chickens.

The hydropericardium syndrome in poultry--a current scenario.

Inclusion-body hepatitis hydropericardium syndrome (IBH-HPS) is an important, recently emerged, disease of poultry, particularly of 3- to 6-week-old broiler chicks, characterized by its sudden onset, with high mortality ranging from 20% to 70%, typical hydropericardium and enlarged mottled and friable livers, with intranuclear inclusion bodies in the hepatocytes. The causative agent is a non-enveloped icosahedral fowl adenovirus (FAV) serotype 4, belonging to the Adenovirus genus of the family Adenoviridae, which can be propagated or cultivated in chicken embryo liver and kidney primary cell cultures. The transmission of disease occurs vertically and laterally by the oral-faecal route. The liver of infected birds shows necrotic foci and basophilic intranuclear inclusion bodies in the hepatocytes. The disease can be diagnosed from its gross and histopathological changes in the liver and by various serological tests, such as agar gel immunodiffusion, counterimmunoelectrophoresis, indirect haemagglutination, the fluorescent antibody technique, enzyme-linked immunosorbent assay and the polymerase chain reaction. The disease has been brought under control by the use of formalin-inactivated vaccines, prepared from infected liver homogenate, and of inactivated cell culture vaccines. The vaccines are effective in the face of natural outbreaks or experimental challenge and significantly reduce the mortality.

Detection of egg drop syndrome 1976 virus by polymerase chain reaction and study of its persistence in experimentally infected layer birds.

Polymerase chain reaction (PCR) assay was developed for the detection of Egg drop syndrome 1976 (EDS-76) virus in tissues, namely in the uterus, spleen and buffy coat. It was also used to study the persistence of the virus in tissues of experimentally infected layer birds. The PCR assay could detect as little as 10 fg of purified EDS-76 viral DNA. It also amplified the DNA of Fowl adenovirus serotypes 4 (FAV-4) and 8 (FAV-8). The virus persisted in the uterus up to day 21 post infection (p.i.). Detection of EDS-76 viral DNA in the buffy coat could be useful for studying the occurrence of the respective disease in layer bird flocks.

Immunosuppression in broiler chicks fed aflatoxin and inoculated with fowl adenovirus serotype-4 (FAV-4) associated with hydropericardium syndrome.

A total of 240 unvaccinated day-old broiler chicks, which had been found to be negative for antibodies against FAV-4, were divided into four groups of 60 chicks each. Group A was fed aflatoxin at 1 ppm from 7 days to 7 weeks of age. Group V was infected intra-abdominally at 14 days of age with 0.2 ml of FAV-4, having a titre of 10(5.5) TCID50 per 0.2 ml. The combined group AV was given the aflatoxin and infected with FAV-4. The fourth group C served as the control. More pronounced clinical signs, a higher mortality rate (56.7%), and reductions in body weight gain and in the organ to body weight ratios of the bursa and spleen were recorded in group AV. A significant (p < 0.01) reduction in the HI antibody titre following vaccination against Newcastle disease, and of skin thickness in the delayed hypersensitivity test following sensitization with DNCB, indicated an additive immunosuppressive effect from aflatoxin and FAV-4 on the humoral and cell-mediated immune responses in group AV compared to groups A and V. Microscopically, marked depletion and degeneration of lymphocytes in the thymus, bursa, spleen and caecal tonsils were observed in group AV up to 5 weeks PI.

Avian reovirus induces an inhibitory effect on lymphoproliferation in chickens.

The cellular immune responses of chickens inoculated with the vaccine strain S-1133 and/or a field isolate VA-1 of avian reovirus (ARV) were studied. Both strains of virus caused inhibition of the phytohaemagglutinin (PHA)-induced lymphoproliferative response of peripheral blood mononuclear cells (PBMC) and splenic mononuclear cells (SMC) during the initial stage from day 4 up to day 10 post-inoculation (PI), with a later return to the normal value. The inhibition in the PHA-induced lymphoproliferation of SMC could be partially overcome by depletion of adherent cells. The supernatant of the PHA-stimulated SMC culture was also checked in vitro for the presence of suppressive factor(s) produced in response to ARV infection. The culture supernatant from chickens at day 5 PI caused significant inhibition of the PHA-induced lymphoproliferation of control birds, suggesting the presence of suppressive factor(s). ARV infection also significantly inhibited IL-2 production on day 5. There was a significant increase in nitric oxide production by the splenic mononuclear cells of chickens inoculated with either strain of ARV.

Characterisation of fowl adenoviruses from chickens affected with infectious hydropericardium during 1994-1998 in India.

In the present study characterisation has been done for six group I fowl adenoviruses (FAV) isolated from outbreaks of infectious hydropericardium (IHP) of chickens that occurred in different states/regions of India during the years 1994-98. These six viruses were identified as FAV serotype 4 by virus neutralisation and restriction endonuclease analyses. Antigenic analyses of the viruses revealed close relationship (R-values 0.93-0.96). Under the experimental conditions, we have been able to induce IHP using FAV serotype 4 isolate AD: 411 and were also able detect FAV antigens in myocardial tissues by immunofluorescence assay (a new observation), an indication that IHP causing FAV serotype 4 strain replicate in myocardial tissue. Restriction endonuclease analysis of the viral genomes (approximately 46 Kb), using Hind III, Sma I, Xba I, Bam HI, Pst I and Dra I produced identical genetic profiles. Pst I and Bam HI profiles for these six vitus isolates were identical to those published earlier for an IHP causing Pakistani FAV serotype 4 isolate KR31. The identical genetic profiles of viruses, chronology of the outbreaks of IHP in Pakistan during 1989 onward and later in Jammu and Kashmir, India (1994), suggest that FAV serotype 4 isolates involved in outbreaks of IHP in India had probably spread from Pakistan. In order to prevent further spread and economic losses due to IHP in India, based on the antigenic relatedness data in this paper, any one of the six studied FAV serotype 4 isolates can be used as a candidate for mass production of CEH culture based killed vaccine.

Differentiation of classical and very virulent strains/isolates of Infectious bursal disease virus by reverse transcription-polymerase chain reaction.

Reverse transcription-polymerase chain reaction (RT-PCR) is a rapid method for identification and differentiation of viruses. It was used to differentiate very virulent from classical (field/vaccine) strains/isolates of Infectious bursal disease virus (IBDV). RT-PCR products of 552 bp were generated by amplification of variable region of VP2 gene in three field classical isolates, two vaccine strains and two very virulent isolates of IBDV. The PCR products were digested with SacI, HhaI, SspI and StuI. Digestion of the PCR products with SacI and HhaI revealed the presence of a single restriction site in all the field classical isolates and vaccine strains, but no such a restriction site in very virulent strains. On the other hand digestion of these products with SspI and StuI showed the presence of a single restriction site in very virulent strains but no such a restriction site in classical field isolates and vaccine strains. Although the restriction profiles of classical field Indian isolates and vaccine strains were identical, all of these enzymes could differentiate very virulent Indian strains from classical field isolates and vaccine strains.

Characterization of fowl adenovirus serotype-4 associated with hydropericardium syndrome in chicken.

The polypeptides of three fowl adenovirus-4 (FAV-4) field isolates of hydropericardium syndrome from various geographical areas of the country and the standard FAV-1 (CELO virus) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and analysed by protein immunoblotting with polyclonal antibodies to FAV-4 and FAV-1. Protein profile analysis of FAV-4 isolates revealed similarity of all the eight polypeptides with molecular weight ranging from 20 to 107 kDa but differed from CELO, particularly in their 24.2 kDa protein. Subsequent immunoblotting showed relatedness of at least five protein fractions of FAV-4 to CELO virus.

Studies on the pathogenicity of Newcastle disease virus isolates in guinea fowl.

Newcastle disease viruses isolated from chickens and guinea fowl were characterized as viscerotropic, velogenic strains on the basis of their mean death time, intracerebral pathogenicity index, intravenous pathogenicity index and cloacal and conjunctival mean death time. The pathogenesis of the disease caused by both the strains was studied in 4-week-old guinea fowl. Both strains had an incubation period of 5 days and the birds showed dullness, depression, anorexia, diarrhoea and paralysis of the legs. They also exhibited nervous signs such as incoordination, muscle tremors and trembling of the neck at the advanced stage of the disease. Mortality reached 52% in the group infected with the chicken isolate but it was only 8% in the birds infected with the guinea fowl isolate. No specific changes were observed at post-mortem examination except haemorrhages at the tip of the glands of the proventriculus and in the caecal tonsil. Changes in the lymphoid organs and brain were always present in both the groups. Despite the low mortality, the guinea fowl isolated had multiplied in various organs in the birds. In both groups, the frequency of virus isolation increased from 5 to 10 days post infection.