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Application of polymerase chain reaction and fluorescent antibody technique for the diagnosis of inclusion body hepatitis-hydropericardium syndrome (IBH-HPS) caused by fowl adenovirus serotype-4 Rahul S., Kataria J.M., Kumar N. Senthil, Dhama K., Dash B.B., Uma R., Praveen B.N. Division of Avian Diseases, Indian Veterinary Research Institute, Izatnagar - 243122 (U.P.) Abstract The suitability of polymerase chain reaction (PCR) and fluorescent antibody technique (FAT) for the detection of fowl adenovirus serotype 4 (FAV-4) in various tissues viz. liver, spleen, bursa of Fabricius, thymus and kidneys, from cases of experimentally induced inclusion body hepatitis-hydropericardium syndrome (IBH-HPS) in specific pathogen free (SPF) layer chicks was evaluated. Briefly, fifteen (15) SPF chicks at 14 days of age were inoculated orally with an Indian strain of FAV-4 at the dose rate of 104.5 EID50 per chick. Another fifteen chicks served as uninfected control. Both the groups were observed for the development of clinical symptoms and lesions at different intervals up to 28 days post infection. Mortality rate of 13.33 per cent was recorded in infected chicks with characteristic signs and lesions of IBH-HPS viz. typical hydropericardium with accumulation of clear watery straw-coloured fluid in the pericardium, swollen, discoloured and friable livers and congested kidneys. On histopathological examinations basophilic intra-nuclear inclusions were observed in hepatocytes. Various tissues viz. liver, spleen, bursa, thymus and kidneys, were collected from chicks showing characteristic lesions and uninfected controls. Detection of FAV-4 DNA and antigen in these tissues was attempted by PCR and FAT, respectively. Cryosections of the tissues when stained with anti FAV-4 FITC conjugate at optimized working dilution of 1: 20 gave distinct specific fluorescence in all the samples examined. DNA extracted from the different tissues, on testing by PCR for the presence of specific viral DNA gave an FAV-4 specific amplified product of 728 bp size. The tissues from control chicks were found negative for FAV-4 DNA and antigen. Both PCR and FAT detected the presence of FAV-4 in tissues. This confirmed the establishment of experimental infection in chicks, thus proving their utility as tests for the rapid and confirmatory diagnosis of FAV-4 infection in poultry. Top | Inclusion body hepatitis-hydropericardium syndrome (IBH-HPS), also known as "litchi disease", is a common disease of broiler chicken (Cheema et al., 1989) caused by fowl adenoviruses of serotype 4. The disease mostly affects young birds between 3 and 6 weeks of age (Niazi et al., 1989) and occasionally layer and breeder pullets also. Rarely the disease has been reported from older broilers, (Asrani et al., 1997) or in other species of birds like pigeon (Naeem and Akram, 1995). |
IBH/HPS is an acute infectious disease characterized by typical hydropericardium, severe anaemia, necrotic hepatitis and high mortality. In natural outbreaks, the affected birds may not exhibit any clinical signs (Jaffery, 1988) except sudden heavy mortality (Ravikumar et al., 1997). Mortality rates in various outbreaks range from 15- 60% (Asrani et al., 1997). |
The disease may be suspected based on characteristic gross lesions, with high mortality, among broiler chicks of 3-6 weeks of age (Ravi Kumar et al., 1997). A characteristic gross lesion is hydropericardium, occurring in more than 90% of the affected birds (Anjum et al., 1989). The liver may be pale yellow, swollen, friable/mottled with large areas of focal necrotic patches (Ravikumar et al., 1997). Abdul-Aziz and Hasan (1995) reported the presence of gross lesions in liver and kidneys and microscopic lesions in liver, kidneys, bursa and spleen. Histopathological lesions, particularly the demonstration of characteristic basophilic intranuclear inclusion bodies in hepatocytes, necrotic hepatitis, oedematous and congested lungs, interstitial nephritis with urate deposition and focal haemorrhages in kidneys help in tentative diagnosis. The demonstration of adenoviral particles in the nucleus of infected cells by transmission electron microscopy (Chandra et al., 1997) can conclusively prove the aetiology. Immunodiagnosis can be performed by employing serological tests such as gel diffusion, indirect haemagglutination test, immunoperoxidase and ELISA (Noor-ul-Hassan et al., 1994; Oberoi et al., 1996; Nagal et al., 1990; Saifuddin and Wilks, 1991), which are considered to be specific and reliable. Group specificity can be confirmed with an immuno-fluorescence assay (Adair et al., 1980) whereas neutralization test has been employed to determine the exact serotype (Grimes and King, 1977; Monreal et al., 1980). |
The diagnosis of FAV-4 infection by isolation and identification of virus is cumbersome and time consuming. Molecular techniques like polymerase chain reaction, nucleic acid hybridization, restriction enzyme analysis and DNA sequencing can be used for early and quick diagnosis of the disease. PCR based amplification of the FAV-4 hexon gene (Ganesh et al., 2002) from infected liver, has been proved a highly reliable and sensitive technique for the diagnosis of FAV-4 infection but, the suitability of other tissues for this purpose needs to be validated. Detection of adenoviral DNA in tissue samples by in situ hybridization, using probes based on nucleic acid sequence of FAV-10 penton base and the virus-associated RNA of FAV-1 has also been reported (Goodwin et al., 1996; Latimer et al., 1997). |
Hence, the present study evaluated the suitability of PCR and FAT for the detection of FAV-4 in various tissues viz. liver, spleen, bursa, thymus and kidneys, from cases of experimentally induced IBH-HPS in specific pathogen free (SPF) layer chicks. |
Top Materials and Methods Virus The Fowl adenovirus (FAV-4) strain used in this study was obtained from the Division of Avian Diseases, Indian Veterinary Research Institute (IVRI), Izatnagar. Primary chicken embryo liver cell culture was prepared as per the method of Adair et al. (1979) and used for the propagation of the FAV-4 strain. Antigen, Anti-sera and Conjugate The standard antigens and positive antisera against NDV, Marek’s disease virus (MDV), infectious bursal disease virus (IBDV), reovirus, FAV-4, EDS-76 virus and avian mycoplasma, required for the screening of experimental specific pathogen free (SPF) chickens, were obtained from the Division of Avian Diseases, IVRI. FITC conjugated rabbit anti-FAV-4 globulin was also obtained from the Division of Avian Diseases. Specific-Pathogens-Free Eggs Thirty specific pathogen free (SPF) embryonated eggs, certified free from various important poultry pathogens, were kindly supplied by M/S Venkateshwara Hatcheries Group Limited (VHL), Pune. These eggs were incubated in a clean environment at Hatchery Unit of Central Avian Research Institute (CARI), Izatnagar and the chickens that hatched were raised under strict isolation to be maintained free from any infection. All biosecurity, sanitary and hygienic measures were followed to provide a pathogen free environment to chicks. The day-old SPF chicks were used for the experimental study and these 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 FAV-4 by AGPT. Primers Oligonucleotide primers corresponding to the hypervariable region of hexon gene region of FAV used in the study were as reported by Ganesh et al. (2001). The primer sequences as given below were obtained commercially synthesized (Q-Biogene, France).
Experimental Infection Thirty clinically healthy SPF chicks of fourteen days age were used in the experimental studies. These were divided randomly into two groups viz.A and B, having equal number of chicks. Fifteen SPF chicks of group A were inoculated orally with an Indian strain of FAV-4 at the dose rate of 104.5 EID50 per chick, while another fifteen chicks of group B served as uninfected control. Both the groups were reared separately under strict isolated conditions and fed autoclaved balanced feed and water, supplemented with vitamins and minerals, ad libitum. Infected chicks were also given antibiotic Lixen (Cephalexin, Glaxo India Ltd.) for six days in prophylactic dose to prevent secondary bacterial infections. They were observed for the development of clinical symptoms and lesions at different intervals up to 28 days post infection.Dead chicks were subjected to systematic necropsy examination and tissues viz. liver, spleen, bursa of Fabricius, thymus and kidneys, were collected for histopathology, fluorescent antibody technique (FAT) based detection of FAV-4 antigen and polymerase chain reaction based detection of FAV-4 DNA. Three chicks from uninfected group were sacrificed at 7th, 14th, 21st and 28th day of experiment and tissues similarly collected for histopathology, FAT and PCR. Histopathological Examination For studying the histopathological changes, pieces of various tissues were collected in 10% neutral buffered formalin from each of the dead chick and those sacrificed from the uninfected control group. The formalin fixed tissues were processed as per standard procedure and examined microscopically. Detection of Viral Antigen by Fluorescent Antibody Technique Detection of FAV-4 antigen in tissues of experimentally infected chicks was carried out by FAT as described by Deepak (1998) with some modifications. Different dilutions of rabbit anti-FAV-4 conjugate were made in PBS (pH 7.4). checker board titration method was employed for optimizing the working dilutions of the conjugate. Tissues viz. liver, spleen, bursa, thymus and kidneys collected from experimentally infected chicks found positive by PCR for FAV-4 DNA were employed for standardization of FAT. Tissues were cryosectioned at -20 oC with a cryotome (Minotome, IEC, USA) 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- FAV-4 globulin (0.2 ml), available at the Division of Avian Diseases, was poured over the sections and incubated for 30 min at 37 oC 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% glycerin-PBS (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 SPF chicks, included as controls were also stained and examined similarly for FAV-4 antigen. Extraction of DNA from Tissues For extraction of DNA from infected tissues, the method of Sambrook et al. (1989) was followed. Approximately 100 mg of infected tissue viz. liver, spleen, bursa, thymus and spleen was homogenized thoroughly into a suspension in 1 ml of sterile TNE buffer. To 540 µl of 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 37 oC for lysing the cells. For DNA extraction from the lysate, equal volume (600 µl) of tris saturated phenol (pH-7.8) was added, vortex mixed gently and centrifuged at 10,000 rpm for 10 min at 4oC. After centrifugation, the supernatant (aqueous phase) was collected carefully and equal volume of phenol: chloroform (25:24) added. Mixed gently by vortexing and centrifuged as above. Then 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 1 ml (2.5 volumes) of chilled absolute ethanol. The mixture was mixed thoroughly and kept overnight at -20 oC. The precipitated DNA was centrifuged at 12,000 rpm for 10 min at 4oC 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 4oC for washing of DNA, and the supernatant discarded. DNA pellet was air dried completely, resuspended in 20 µl of TE buffer and stored at -20 oC for further use. Tissues from healthy control chicks (SPF) were also similarly treated for DNA extraction. Detection of FAV-4 DNA in Tissues DNA samples extracted from tissues of experimentally infected chicks and uninfected controls were tested by the PCR technique for the presence of FAV-4 DNA by checking specific amplification of the hexon gene. The technique as described by Ganesh et al. (2002) was followed by with slight modifications and the amplified PCR products were confirmed by agarose gel electrophoresis on 1.5% gel in 0.5X TBE buffer. Top RESULTS Propagation of Viruses The FAV-4 virus strain was passaged in primary chicken embryo liver cell culture. CPE was characterized by cell degeneration and rounding within 48 to 96 hours, after which the virus was harvested with at least three freezing and thawing cycles and stored at -20 oC until further use. Screening of Experimental Chicks Pre-infection sera and the sera collected at weekly intervals from experimental day-old SPF chicks were negative for antibodies to common viral pathogens viz. IBDV, MDV, reovirus and to Salmonella Gallinarum and Mycoplasmagallisepticum. Bacterial cultures for isolation of E. coli and Salmonella Gallinarum, performed before the experimental infection and monitored during the course of the study were found negative. Pre-infection sera were also negative for antibodies to FAV-4, indicating that chicks were sero-negative for maternal antibodies against FAV-4 infection. No evidence of other diseases was detected in SPF chicks during the experimental period. Clinical Signs of Mortality Subsequent to FAV-4 infection a mortality rate of 13.3 per cent (two out of fifteen) was recorded in FAV-4 infected SPF chicks (group A), while none of the birds in groups B died. The mortality in group A was observed on the 3rd and 5th days post FAV-4 infection and the dead birds did not exhibit overt clinical signs before death except for ruffled feathers, dullness and lack of appetite. Gross Lesions FAV-4 infected SPF chicks (group A) that died during the course of the experiment exhibited characteristic gross lesions of IBH-HPS like typical hydropericardium (Fig. 1), inflammation and congestion of liver, spleen and kidneys. Kidneys revealed petechial haemorrhages and urate deposition in renal tubules.
Histopathological Changes Liver of infected chicks that died with IBH-HPS revealed characteristic basophilic INIB’s in hepatocytes (Fig. 2) along with fatty infiltration of hepatocytes, congestion of blood vessels and haemorrhages in some cases. Lymphoid organs like bursa, spleen and thymus revealed depletion of lymphoid elements and congestion.
In uninfected controls (group B), none of these changes were noted at different post FAV-4 infection intervals through out the experiment. Detection of Viral Antigens in Tissues Cryosections of various tissues viz. liver, thymus, bursa, spleen and kidneys, collected from SPF chicks experimentally infected with FAV-4 and dead with IBH-HPS were stained with anti FAV-4 FITC conjugate for the presence of specific fluorescence. The optimized working dilution of conjugate (1: 20) gave distinct viral specific fluorescence (Fig. 3-7) in all the above-mentioned tissues. Tissues collected from uninfected control birds (group D) were negative for specific fluorescence on FAT.
Extraction of DNA from Tissues Total genomic DNA was extracted from tissues following the SDS- proteinase K, phenol- chloroform extraction and ethanol precipitation method. The OD260/280 ratio of the extracted DNAs ranged from 1.72 to 1.86, indicating adequate purity and the concentration of DNAs ranged from 0.62- 0.76 µg/ml. Detection of FAV-4 DNA in Tissues The 728 bp FAV-4 hexon gene specific product was amplified by PCR with DNA extracted from all the tissues viz. liver, spleen, bursa, thymus and kidneys, of SPF chicks dead with IBH-HPS (Fig. 8). DNA extracted from tissues of uninfected controls was negative for the amplification of FAV-4 DNA by PCR.
Top DISCUSSION IBH/HPS is an acute infectious disease especially of broiler poultry, resulting in heavy mortality and serious economic loss to the industry. The disease manifestations, besides a high mortality rate, include a characteristic hydropericardium, with the heart simulating a de-shelled litchi fruit (Litchi chinensis.) and hepatitis with basophilic intranuclear inclusion bodies in hepatocytes. IBH/HPS has been described primarily as a disease of broilers (Cheema et al., 1989) of 3 to 6 weeks of age (Niazi et al., 1989) and occasionally of layer and breeder pullets aged between 2 and 20 weeks (Shukla et al., 1997). |
Clinical diagnosis of the disease before the occurrence of mortality is difficult since the birds do not show specific clinical signs. Clinical diagnosis of HPS is, therefore, hardly possible in spontaneous outbreaks owing to its acute nature (Balamurugan and Kataria, 2004). Diagnosis of IBH-HPS infection has been carried out on the basis of gross lesions, histopathological lesions, particularly of INIBs in hepatocytes (Gowda and Satyanarayana, 1994), demonstration of adenovirus particles in the nuclei of infected liver cells by transmission electron microscopy (Cheema et al., 1989; Chandra et al., 1997) or isolation of virus, either in cell culture or in embryonated eggs (Kataria et al., 1995). Further confirmation is done by neutralization tests using serotype-specific sera (Jadhao et al., 1997). Diagnosis of the condition based on gross lesions and techniques like histopathological examination or agar gel immuno diffusion, which are of low sensitivity and/or specificity, is not desirable in case of severe outbreaks. The various methods described for confirmatory diagnosis of FAV-4 infection like antigen capture ELISA, electron microscopy and virus neutralization test are labour-intensive or cumbersome. |
Results of the study prove that FAT using suitable dilution of rabbit anti-FAV-4 FITC conjugate can be used to detect FAV-4 antigen in cryosections of liver, spleen, bursa of Fabricius, thymus and kidneys. FAT can de used for quick diagnosis of the condition, while being satisfactorily sensitive and specific in comparison to serological techniques like AGID or counter immuno electrophoresis. Deepak (1998), while studying the pathogenesis of HPS in 2-week-old chickens, had detected an immunofluorescent FAV antigen in the thymus, spleen and bursa from 3 to 10 DPI, in the liver from 3 to 14 DPI and in the heart up to 5 DPI. |
Polymerase chain reaction could amplify FAV-4 hexon gene specific product from DNA samples of various tissues screened. The simple DNA extraction procedure followed ensures that results could be obtained quickly enough. Many workers have reported polymerase chain reaction based diagnosis of HPS (Dahiya et al., 2002; Ganesh et al., 2002). The DNA isolated from either infected liver tissue or purified virus was subjected to PCR using hexon gene specific primers (Ganesh et al., 2002). In this study the direct screening of tissues by PCR for FAV-4 was found to be equally sensitive as FAT technique. Both techniques detected FAV-4 in all the tissues screened from chicks dead with IBH-HPS. |
The rapid and confirmatory detection of FAV-4 antigen and DNA in tissues viz. liver, spleen, bursa, thymus and kidneys, of chicks with experimentally induced IBH-HPS prove the suitability of these techniques for the early diagnosis of the condition and identification of the aetiological agent in the event of an outbreak. This will help in arriving quickly at a definite prevention and control strategy for IBH-HPS in commercial poultry production units across the country. |
Top Figures | Fig. 1: Experimentally infected SPF chick with IBH/HPS showing characteristic hydropericardium and hepatitis. 3 DPI
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| | Fig. 2: Liver: Hepatocytes showing basophilic intranuclear inclusion bodies. (H & E × 400)
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| | Fig. 3: Intranuclear immunofluorescent antigen in hepatocytes. FA stain × 400
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| | Fig. 4: Intranuclear immunofluorecsent antigen in splenic lymphocytes. FA stain × 400
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| | Fig. 5: Intranuclear immunofluorescent antigen in bursal lymphocytes. FA stain × 400
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| | Fig. 6: Intranuclear immunofluorescent antigen in thymic lymphocytes. FA stain × 400
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| | Fig. 7: Intranuclear immunofluorescent antigen in kidney parenchyma. FA stain × 400
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| | Fig. 8: Screening of tissue samples from experimentally infected SPF chicks for FAV-4 DNA by PCR.
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| References | | Abdul-AzizT.A., HassanS.Y.
(1995).
Res. Vet. Sci.,
59:
219-221. TopBack | | AdairB.M., CurranW.L., McFerranJ.B.
(1979).
Avian Pathol.,
8:
133. TopBack | | AdairB.M., McFerranJ.B, CalvertV.M.
(1980).
Development of a microtitre fluorescent antibody test for serological detection of adenovirus infection in birds.
Avian Pathol.,
9:
291. TopBack | | AkhtarS.
(1995).
Vet. Rec.,
136:
118. TopBack | | AnjumA,D., SabbriM.A., IqbalZ.
(1989).
Vet. Rec.,
124:
247. TopBack | | AsraniR.K., GuptaV.K., SharmaS.K., SinghS.P., KatochR.C.
(1997).
Vet. Rec.,
144:
271. TopBack | | ChandraR., ShuklaS.K., KumarM., GargS.K.
(1997).
Vet. Rec.,
138:
70. TopBack | | CheemaA.H., AhmadJ., AfzalM.
(1989).
Res. Sci. Tech. Off. Int. Epi.,
8:
789. TopBack | | CowenB.S.
(1992).
World’s Poult. Sci. J.,
48:
247. TopBack | | DahiyaS., SrivastavaR.N., HessM., GulatiB.R.
(2002).
Avian Diseases,
46:
230. TopBack | | DeepakJ.N.
(1998).
Studies on pathogenicity and immunosuppressive effects of fowl adenovirus serotype-4 isolated from hydropericardium syndrome in chicken.
M. V. Sc. Thesis submitted to Deemed University, IVRI, Izatnagar. TopBack | | GaneshK., SuryanarayanaV., RaghavanR., GowdaS.
(2001).
Vety. Microbiol.,
78:
1. TopBack | | GaneshK., SuryanarayanaV.V.S., RaghavanR.
(2002).
Vet. Res. Comm.,
26:
73. TopBack | | GoodwinM.A., LatimerK.S., ResurreccionR.S., MillerP.G., CampagnoliR.P.
(1996).
Avian Dis.,
40:
828. TopBack | | GowdaR.N.S., SatyanarayanaM.L.
(1994).
Indian J. Vet. Pathol.,
18:
159. TopBack | | GrimesT.M., KingD.J.
(1977).
Am. J. Vet. Res.,
38:
317. TopBack | | JadhaoS.J., KatariaJ.M., VermaK.C, SahR.L.
(1997).
Indian J. Comp. Micrbiol.Immunol.Infect Dis.,
18 (1):
33. TopBack | | JafferyM.S.
(1988).
“A treatise on Angara disease (Hydropericardium- Pulmorary disease-hepatonephritis syndrome).
Pakistan Vet. Med. Assoc. Pub.,
1-34. TopBack | | KatariaJ.M., SahR.L., VermaK.C, BansalM.P.
(1995).
Studies on inclusion body hepatitis in chickens.
Presented in Natl. Symp. on impact of diseases on Livestock / poultry production and wildlife conservation. IAVP, Orissa Vety. College, Bhubaneswar, 9-11 Nov, 1995. p.
75. TopBack | | KhawajaD.A., AhmedS., RaufA.M., ZulfigarM., MahmoodS.M., HassanM.
(1988).
Pak J. Vet. Res.,
1:
2. TopBack | | LatimerK.S., NiagroF.D., WilliamsO.C., RamisA., GoodwinM.A., RitchieB.W., CampagnoliR.P.
(1997).
Avian Dis.,
41:
773. TopBack | | MonrealG., DornR., KassimM.
(1980).
Berliner Minchner Tierarztliche Wochenschrift,
93:
125. TopBack | | NaeemK., AkramH.S.
(1995).
Vet. Rec.,
136:
296. TopBack | | NagalK.B., MaitiN.K., OberoiM.S., SharmaS.N.
(1990).
Indian J. Anim. Sci.,
69:
1023. TopBack | | NiaziA.K., KhanM.Z, SiddiqueM.
(1989).
Vet. Rec.,
125:
400. TopBack | | Noor-ul-Hassan, AfzalM., HameedA., KhanR.A.R.
(1994).
Pak. Vet. J.,
14:
5. TopBack | | OberoiM.S., SinghA., SinghB.
(1996).
Indian J. Virol.,
12:
123. TopBack | | KumarRavi, ChandraR., ShuklaS.K., AgarwalD.K., KumarM.
(1997).
Trop. Anim. Hlth. Prod.,
29:
158. TopBack | | SaifuddinM, WilksC.R.
(1991).
Arch. Virol.,
116:
33. TopBack | | SambrookJ., FritschE.F., ManiatisT.
(1989).
Molecular cloning. A laboratory manual.
2nd ed.
Cold Spring Harbor Laboratory Press. TopBack | | ShuklaS.K., ChandraR., KumarM., DixitV.P.
(1997).
Indian J. Anim. Sci.,
67:
766. TopBack |
| |
|
|
|