Indian Journal of Comparative Microbiology, Immunology and Infectious Diseases
Year : 2003, Volume : 24, Issue : 2
First page : ( 125) Last page : ( 131)
Print ISSN : 0970-9320.

Detection of chicken anaemia virus and avian reovirus by polymerase chain reaction and fluorescent antibody test in various tissues from experimentally co-infected chicks

Bhardwaj Nitin, Kataria J.M.*, Dhama K., Arthur Sylvester S., Senthilkumar N.

Division of Avian Diseases, Indian Veterinary Research Institute, Izatnagar–243122 (U.P.)

*Corresponding author

Received:  27  August,  2004.

Abstract

Avian reovirus (ARV) is an economically important emerging avian pathogen associated with a variety of disease manifestations mainly viral arthritis, malabsorption syndrome and infectious proventriculitis, which are commonly observed in broilers. Chicken infectious anaemia (CIA) caused by chicken anaemia virus (CAV) has recently been reported in the country. These disease conditions many a times go unnoticed in field due to masking of pathognomic symptoms by secondary/mixed infections and nutritional disorders, which are commonly observed. Therefore, the present study was formulated with the objective to detect CAV and ARV specific antigen/nucleic acid by indirect immunofluorescent test (IIFT)/fluorescent antibody test (FAT) and polymerase chain reaction (PCR)/reverse transcription–polymerase chain reaction (RT-PCR), respectively. Detection of CAV and ARV infections by PCR/RT-PCR and IIFT/FAT in various tissues of infected chicks proved to be rapid and reliable tests for confirmatory disease diagnosis and could well be used for the detection of these agents at an early stage.

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Materials and Methods

Viruses

The ARV (VA-1) isolated from Chicken (Gallus domesticus) at 9th passage level in CEF and an Indian isolate of CAV at 7th passage level in MSB1 tissue culture used in the present study were obtained from Division of Avian Diseases, Indian Veterinary Research Institute (IVRI), Izatnagar.

Embryonated chicken eggs and chickens

Unvaccinated dayold broiler chicks of both sex, and seven-day-old embryonated chicken eggs were obtained from Experimental Hatchery Unit of Central Avian Research Institute (CARI), Izatnagar. All biosecurity, sanitary and hygienic measures were followed to provide a pathogen free environment to chicks. The day-old broiler chicks used for this experimental study were monitored weekly for NDV, IBDV, MDV, reovirus, Salmonella Gallinarum, E. coli and avian mycoplasma infections following conventional standard procedures viz. agar gel precipitation test (AGPT), haemagglutination inhibition test (HI), plate agglutination and bacterial cultures. Chicks were also screened before experimental infection for the presence of maternal antibodies in their sera against avian reovirus by agar gel precipitation test (AGPT) carried out as described by Kawamura and Tsubahara (1966) and against CAV by indirect immunofluorescent test (IIFT) carried out as per the method described by McNulty et al. (1988).

Primers

For CAV

For PCR amplification, the primers amplifying the conserved region were used as described by Dhama et al., 2002. These primers flanked a 454 base pair (bp) DNA sequence in size and were synthesized from Gibco BRL, USA (Table 1)

For ARV

Oligonucleotide primers from the S1 gene region of ARV used in the study were as reported by Koti (2000). The primer sequences were commercially synthesized (Gibco BRL, USA) and are as given (Table 2)

Propagation and titration of virus strain in CEF cultures

Chicken embryo fibroblast (CEF) cell cultures were prepared from 10-11 day-old chicken embryos as per the method of Merchant et al. (1960) with slight modifications. Once formed the monolayer was infected by inoculating 0.5 ml of 1:10 diluted virus and flasks were incubated at 37°C. Cytopathic effects (CPE) started after 48 h and after development of complete CPE, the flasks were transferred to deep freeze (−40°C). Rapid freezethawing was done at 37°C and −40°C for three times. For further passaging, the virus material was thawed, centrifuged and the supernatant after dilution in 1:10 in HBSS was inoculated on to fresh cultures. Titration of VA-1 isolates was carried out in CEF cell cultures in micro culture plates (96-well microtitre plates, Nunc, Denmark) and the 50% tissue culture infective dose (TCID50) was calculated using the method of Reed and Muench (1938).

Titration of CAV

A pretitrated (10^4.5 TCID50/0.1 ml) CAV isolate (at 7th passage level) maintained in MSB1 cell culture was used in this study.

Experimental infection

One hundred twenty (120) clinically healthy day-old broiler chicks hatched at CARI hatchery, Izatnagar, from the embryonated chicken eggs were used in the experimental studies. These were divided randomly into four groups A-D, having equal number of chicks. All the sixty dayold broiler chicks of Group A and C were inoculated intramuscularly (I/M) with 0.5 ml of 10^4.5 TCID50/0.1ml of MSB1 cell culture passaged CIAV isolate (at 7th passage level). Chicks in-group D were inoculated I/M with 0.5 ml PBS. Ten days post-CAV infection, the chicks in groups B and C were infected with 10^6.5 EID50/1ml of ARV, VA-1 isolate. The various groups were reared separately under strict isolated conditions and fed autoclaved feed and water, supplemented with vitamins and minerals, ad libitum. They were monitored for signs of disease and mortality, if any. Dead chicks were subjected to necropsy and tissues collected for polymerase chain reaction (PCR), fluorescent staining for CAV and ARV nucleic acid, antigen detection and histopathology at various intervals.

Detection of virus in tissues of experimentally infected chicks

Detection of CAV antigen by indirect fluorescent antibody technique (IFAT)

Tissues viz. liver, spleen, bursa and thymus collected from experimentally infected chicks were employed for detection of CAV antigen at different intervals. These tissues were tested for detection of virus specific antigen by IFAT (Dhama et al., 2002). Sections from uninfected chicks included as controls were stained and examined similarly for IF studies. The sections in triplicate were fixed on clean microscopic slides by keeping in chilled acetone for 10 min. The sections were washed thrice in PBS and 1:50 diluted anti-CAV chicken hyper immune serum was added. The slides were incubated for 1 h at 37°C in a moist chamber. Then, these were washed thrice with PBST and 1:30 diluted anti-chicken FITC conjugate was added and incubated for 1 h at 37°C in a dark moist chamber. After washing thrice, the slides were mounted with 50% glycerol saline and examined under the UV microscope (Nikon, Japan) for characteristic granular bright green fluorescence, in the absence of reactions in uninfected controls, was considered positive for the presence of CAV antigen.

Detection of CAV DNA by PCR

Extraction of total DNA from CAV-infected tissues

For extraction of viral DNA from infected tissues method of Todd et al. (1992) was followed with slight modifications. Approximately 100 mg of pooled tissue comprising thymus, bone marrow, liver, spleen and bursa were homogenized thoroughly into a suspension in 1 ml of sterile TNE buffer. To 540 µl suspension, 30 µl of 10% SDS (0.5% final concentration) and 30 µl of 20 mg/ml proteinase-K (1 mg/ml final concentration) were added and the reaction mixture was incubated overnight at 370C for lysing the cells. For DNA extraction from the lysate, equal volume (600 µl) of phenol-chloroform-isoamyl alcohol (25:24:1) was added, vortex mixed gently and centrifuged at 10,000 rpm for 10 min at 4°C. After centrifugation, the supernatant (aqueous phase) was collected carefully and phenol-chloroform extraction procedure repeated once again. To the aquous phase collected, equal volume of chloroform-isoamyl alcohol (24:1) was added, vortex mixed gently and centrifuged at 10,000 rpm for 10 min at 40C. 360 µl of the above supernatant was collected carefully (aqueous phase) to which 40 µl of 3 M sodium acetate (1/10th volume, 0.3M final concentration, pH 5.2) was added along with 1ml (2.5 volumes) of chilled absolute ethanol. The reaction mixture was mixed thoroughly and kept overnight at −20°C. The precipitated DNA was centrifuged at 12,000 rpm for 10 min at 4°C and the supernatant discarded gently. The pellet obtained was dissolved gently in 1 ml of 70% ethanol, centrifuged at 12,000 rpm for 15 min at 4°C for washing of DNA and the supernatant discarded. DNA pellet was air dried completely, resuspended in 20 µl of Tris EDTA (TE) buffer and stored at −20°C for further use.

Detection of CAV DNA in infected tissues by PCR

All DNA amplifications were performed in an automatic thermal cycler (PTC 200 MJ Research, USA) in 25 µl reaction mixture volumes using primer pair CA-1 and CA-2 in 0.2 ml thin walled PCR tubes as per Dhama et al. (2002). The amplified PCR products were confirmed for their expected size (454 bp) by agarose gel electrophoresis on 1.5% (w/v) agarose gel in 0.5X tris borate EDTA (TBE) buffer as per the method of Sambrook et al. (1989).

Detection of ARV antigen by fluorescent antibody technique (FAT)

Distribution of ARV antigen in tissues of experimentally infected chicks was carried out by FAT as described by Menendez et al. (1975) with some modifications. Tissues viz. liver, spleen, bursa and thymus collected from experimentally infected chicks were employed for standardization of FAT for detection of ARV antigen in tissues of experimentally infected chicks.

Tissues were cryosectioned at −20°C and fixed in cold acetone at room temperature for 10 min. Slides were air dried and washed three times in PBS (pH–7.4), each time for 10 min. FITC conjugated anti-ARV globulin (0.2 ml), available at the Division of Avian Diseases, was poured over the sections and incubated for 30 min at 37°C in a dark moist chamber in order to prevent drying during staining. After incubation, the sections were thoroughly washed in PBS and mounted in 50% buffered glycerin (pH–7.4). The slides were then examined for the presence of apple green fluorescence under fluorescent microscope (Nikon Eclipse E200, Japan). Sections from uninfected broiler chicks included as controls were stained and examined similarly for FAT studies.

Detection of ARV RNA by RT-PCR

Extraction of total RNA from reovirus-infected tissues

Extraction of RNA from reovirus-infected tissues was done as per Koti (2000). Approximately 100 mg of spleen, liver, bursal and joint tissues were triturated and a 20% suspension was prepared in sterile PBS. From this, 400 ml suspension was taken and extracted with equal volume of chloroform: isoamyl alcohol (24:1) at 12,000 rpm for 5 min. The aqueous phase was collected and treated with 0.5% SDS and 1 mg/ml proteinase K (final concentration) at 37°C for 2 h. Total RNA from lysed cells was extracted twice with phenol:chloroform:isoamyl alcohol (25:24:1) and once with chloroform:isoamyl alcohol (24:1). Viral RNA was precipitated by 0.3 M sodium acetate (final concentration), pH 5.2 and 2.5 volumes of absolute alcohol. The RNA pellet was obtained by centrifugation at 13,000 rpm for 20 min and washed with 70% ethanol. Dried RNA pellet was dissolved in 10 ml to 20 ml of nuclease-free water and stored at −20°C for further use.

Detection of ARV RNA in infected tissues

Viral RNA extracted from tissues viz. liver, spleen, bursa, thymus and joints, of experimentally infected chicks were tested by the PCR technique as described by Koti (2000) to detect the presence of ARV RNA by checking specific amplification of the S1 gene using oligonucleotide primers ARV SR and SF. The amplified PCR products were confirmed by agarose gel electrophoresis on 1.5% gel in 0.5X TBE buffer.

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Results

Propagation and titration of experimental viruses

The VA-1 isolate of avian reovirus (ARV) used in the present study was propagated and serially passaged 2-3 times in primary chicken embryo fibroblast (CEF) cell culture. The cytopathic effects (CPE) were characterized by cell degeneration, formation of multinucleated cells (syncytia), appearance of intracytoplasmic eosinophilic inclusions that later became basophilic with appearance of small plaques due to sloughing of the infected cells by 72 h PI. By 96 h PI, most of the cells fell off the surface, however, long cytoplasmic extensions were intact and large spaces appeared in cell sheet. The CAV isolate having a titre of 104.5 TCID50/0.1 ml of MSB1 cell culture (at 7th passage level) was obtained. The virus infectivity titres of the infected cell culture fluids, at 9th passage level was found to be 6.25 TCID50/ml.

Screening of experimental chicks

Pre-infection sera collected from day-old chicks before the experimental CAV infection were negative for the presence of antibodies to common viral pathogens viz. NDV, MDV, IBDV, CAV, ARV and also for Salmonella Gallinarum and Mycoplasma gallisepticum. Bacterial cultures for isolation of E. coli and Salmonella Gallinarum monitored during the course of the study were found negative. No evidence of other diseases was detected in chicks during the experimental studies.

Pathogenicity of ARV in chicks co-infected with CAV

Chicks experimentally infected with CAV showed acute course of disease with 100% morbidity. Infected chicks exhibited signs of anorexia and weakness at 10 days post CAV infection and developed signs of anaemia and loss in body weight gain. At 2-5 days DPI with ARV, clinical signs became more pronounced as evident by stunting, depression, paleness and ruffled feathers. Chicks infected with ARV alone (group B) and co-infected chicks (group C) exhibited growth retardation, swelling of foot pad and hock, abnormal gait and inability to stand (stand with legs wide apart), which was more intense in chicks of co-infected group (group-C) than the group B infected with ARV alone. Subsequently the chicks became dull and depressed and at 14 DPI showing extensive swelling of foot pad and hock (Fig. 1), which was comparatively mild in ARV alone group (B). Due to abnormal swelling, the normal morphology of the feet was deformed in the co-infected chicks. Gross and histopathological lesions in co-infected chicks sacrificed at various intervals were characterized by atrophied thymus, spleen and bursa as compared to control chicks. These lesions persisted even after 21 DPI in co-infected chicks. In chicks infected only with ARV (group-B), most of the changes were like those of chicks of co-infected group but less in severity. Swelling at the hock and foot pad region started declining at 14 DPI but feet appeared deformed due to uneven swelling.

Detection of viruses in tissues of experimentally infected chicks

Detection of CIAV antigen by FAT

Cryosections of liver and thymus tissues, collected from CAV infected chicks sacrificed at 7 and 10 DPI, were treated with reference serum and rabbit anti-chicken IgG FITC conjugate for the presence of specific apple green fluorescence. The optimized working dilution of reference sera at 1:100 and conjugate at 1:30 in PBS (pH 7.4) gave the specific fluorescence. IIFT stained liver and thymus sections showed CAV specific intra-nuclear fluorescence (Fig. 2). Tissues collected from uninfected control birds (group-D) were negative for specific immunofluorescence.

Detection of CIAV DNA by PCR

Simultaneously the samples of liver and thymus collected from chicks sacrificed at 7 and 10 DPI were screened by PCR for the presence of viral DNA. Total genomic DNA was extracted from these tissues. The 454 bp CAV specific product was amplified by PCR with DNA extracted from tissues of sacrificed chicks (Fig. 3). DNA extracted from tissues of uninfected controls was negative for the amplification of CAV DNA by PCR.

Detection of ARV antigen by FAT

Cryosections of various tissues viz. liver, thymus, bursa, spleen and joints collected from chicks experimentally infected with ARV at 7 DPI were stained with anti-ARV FITC conjugate for the presence of specific fluorescence. The optimized working dilution of conjugate (1:50) gave distinct viral specific intracytoplasmic apple green fluorescence in all the above-mentioned tissues (Fig. 4). Tissues collected from uninfected control birds (group D) were negative for specific fluorescence on FAT.

Detection of ARV RNA by RT-PCR

Samples of liver, thymus, bursa, spleen and joints collected from dead/-sacrificed chicks were screened by RT-PCR for the presence of viral RNA. Total genomic RNA was extracted from tissues following the SDS-proteinase K, phenol-chloroform extraction and ethanol precipitation method. The OD260/280 ratio of the extracted RNA ranged from 1.7 to 1.9 for different tissues indicating that it was free of protein contamination. The 422 bp ARV specific product was amplified by RT-PCR from RNA extracted from tissues of sacrificed chicks (Fig. 5, 6). RNA extracted from tissues of uninfected controls was negative for the amplification by RT-PCR.

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Discussion

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Figures

Fig. 1: A-Extensive swelling of hock joint and foot pad of co-infected chick at 14 DPI B-Control TopBack

Fig. 2: Chick infected with CAV showing intranuclear immuno-fluorescence in hepatocytes (10 DPI) TopBack

Fig. 3: PCR detection of 454 bp CAV antigen in tissue samples Lane M: PCR marker; Lane 1: Liver; Lane 2: Spleen; Lane 3: Thymus TopBack

Fig. 4: Chick infected with ARV showing intracytoplasmic immunofluorescence in joints (7 DPI) TopBack

Fig. 5: Amplified PCR product of VA-1 isolate Lane M: PCR marker; Lane 1: 422 bp PCR product of VA-1 isolate TopBack

Fig. 6: PCR detection of 422 bp ARV antigen in tissue samples Lane M: PCR marker; Lane 1: Liver; Lane 2: Spleen; Lane 3: Bursa; Lane 4: Thymus; Lane 5: Joint TopBack

Tables

Acknowledgements

The authors acknowledge the valuable suggestions and facilities provided by the Director IVRI, Izatnagar. Thanks are due to Hatchery Unit, CARI, Izatnagar for providing day-old broiler chicks. Also the technical assistance rendered by Sh. G.S. Deolia, S.K. Kaw, Kharak Singh, Reza Ali and D.C. Pant during the research work is duly acknowledged.

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