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PCR for specific detection of haemorrhagic enteritis virus of turkeys, an avian adenovirus
Journal of Virological Methods 81 (1999) 199 – 203 www.elsevier.com/locate/jviromet
Short communication
PCR for specific detection of haemorrhagic enteritis virus of turkeys, an avian adenovirus M. Hess *, R. Raue, H.M. Hafez Institut fu¨r Geflu¨gelkrankheiten, Freie Uni6ersita¨t Berlin, Koserstraße 21, 14195 Berlin, Germany Received 5 March 1999; received in revised form 23 April 1999; accepted 23 April 1999
Abstract A hexon gene based PCR was developed for specific amplification of DNA sequences from the haemorrhagic enteritis virus (HEV) of turkeys. The hexon genes of different avian adenoviruses were compared for primer construction. Two regions with low sequence homology between HEV and fowl adenovirus (FAV) hexon genes were selected for primer localisation. In correlation with the known sequence data a fragment of 1647 bp was amplified from a live vaccine and spleens of turkeys suffering from haemorrhagic enteritis (HE). All other avian adenoviruses which are able to infect turkeys, i.e. FAV and turkey adenoviruses (TAV), were negative. This is the first PCR for specific detection of HEV DNA which should be useful for rapid diagnosis and epidemiological investigations of HEV infections in turkeys. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Avian adenoviruses; Haemorrhagic enteritis virus; PCR; Spleen samples
Avian adenoviruses are classified routinely into three different groups. The first group combines avian adenoviruses from several species, e.g. chickens, turkeys and geese which share a common group specific antigen. The egg drop syndrome (EDS) virus is the sole member of group III. Group II includes the haemorrhagic enteritis virus (HEV) of turkeys, the marble spleen disease virus (MSDV) pathogenic for pheasants and the * Corresponding author. Tel.: +49-30-8383882; fax: + 4930-8385824. E-mail address: [email protected] (M. Hess)
avian adenovirus splenomegaly virus (AASV) which was observed in young broilers (Domermuth et al., 1980). The group II avian adenoviruses are closely related serologically but can be differentiated with monoclonal antibodies (van den Hurk, 1988). HEV is the only group II avian adenovirus with economic impact, whereas AASV infections are not very common and pheasant populations suffering from MSD are very small. Haemorrhagic enteritis (HE) is a disease of turkeys 4 weeks of age and older (Pierson and Domermuth, 1997). Clinical outbreaks are characterised by intestinal haemorrhages accompanied
0166-0934/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 0 9 3 4 ( 9 9 ) 0 0 0 6 7 - 1
200
M. Hess et al. / Journal of Virological Methods 81 (1999) 199–203
with immunosuppression. The spleen is the major target of the virus resulting in an enlarged, marbled organ (Suresh and Sharma, 1996). Live vaccines are used routinely to prevent HE (Domermuth et al., 1977). Detection of HEV is mostly carried out by agar gel precipitin (AGP) test or ELISA technique (Domermuth et al., 1972; van den Hurk, 1986). Immunohistochemistry for HEV identification was also described but is not used routinely (Fitzgerald et al., 1992; Saunders et al., 1993). The restricted growth of the virus which is usually performed on a lymphoblastoid B-cell line of turkeys (MDTC-RP19) limits the isolation of HEV. Only Zhang and Nagaraja (1989) used restriction enzyme analysis after virus isolation for differentiation of group II avian adenovirus genomes. Detection of HEV DNA by in situ hybridisation and PCR were also described (Suresh and Sharma, 1996). All avian adenoviruses which are able to infect turkeys should be included for the development of a diagnostic PCR specific for HEV detection. Consequently, the data bank was checked for relevant nucleotide sequences of avian adenoviruses. So far, the genomes of three avian adenoviruses representing each group were sequenced completely, namely the CELO virus (FAV1) with 43.8 kb belonging to group I (Chiocca et al., 1996), the EDS virus with a viral genome of 33.2 kb as the sole member of group III (Hess et al., 1997) and recently the HEV with only 26.3 kb (Pitcovski et al., 1998). Based on the experience with a PCR for FAV and EDS virus detection, the HEV hexon gene was chosen for primer construction. In addition to FAV1, EDS virus and HEV, the hexon sequence of FAV10 was published (Sheppard et al., 1995). Therefore the nucleotide sequences of the FAV1, FAV10 and HEV hexon genes were aligned (data not shown). The conserved (pedestal (P)) and variable (loop (L)) regions of the HEV hexon gene were determined according to the proposed model of the human adenovirus 2 hexon protein (Roberts et al., 1986; Athappilly et al., 1994). Primers HEV1F and HEV2R, were constructed and are able to hybridise in the loop regions L1 and L4 of the HEV hexon gene.
Precise location of the primers on the HEV hexon gene is: HEV1F: 5%-TACTGCTGCTATTTGTTGTG-3%, nt 729-748 and HEV2R: 5%-TCATTAACTCCAGCAATTGG-3%, nt 2375-2356. The two regions were selected because of their low sequence identity with the corresponding regions of the FAV1 and FAV10 hexon genes. In this article, all 12 fowl adenoviruses (FAV1– 12) were used together with two group I turkey adenoviruses (TAV1,2). The FAV reference strains used, the propagation of viruses and the extraction of the DNAs from purified viruses was carried out according to methods recently described (Raue and Hess, 1998). The Dindoral® SPF HE-vaccine (Merial, France) was used as a positive control. DNA extraction was performed in the same way as the reference strains. Spleens from turkeys with different clinical histories were tested for HEV DNA to verify the PCR for practical use. All clinical samples which were investigated are listed in Table 1. The QIA amp® tissue kit (Qiagen, Germany) was used for DNA extraction from the spleens according to the manufacturer’s instructions. For PCR 50 ng of purified viral DNA or 1 mg of DNA extracted from the spleens were incubated together with 125 pmol of each primer in a total volume of 25 ml using ‘Ready To Go®’ PCR Beads (Amersham Pharmacia Biotech, Germany). The PCR was carried out on a TRIO-Thermoblock (Biometra, Germany) with the following conditions for 30 cycles: 94°C for 1 min, 64°C for 1 min and 72°C for 1.5 min. Of the resulting PCR mixture, 5 ml was analysed by 0.5% agarose gel electrophoresis and stained with ethidium bromide. The primary aim was to investigate the specificity of the PCR for detection of HEV. For this purpose all avian adenoviruses which could infect turkeys were included in the PCR. After separation of 5 ml of the PCR reaction on an 0.5% agarose gel only the HEV DNA was positive (Fig. 1). Neither FAV- nor TAV-strains reacted with the used primers. With : 1600 bp the estimated size of the amplified fragment was in agreement with the 1647 bp calculated from the HEV hexon sequence.
M. Hess et al. / Journal of Virological Methods 81 (1999) 199–203
201
Table 1 Description of the HEV samples included in this study Lane on agarose gel (Fig. 2)
Sample no.
Material
Flock description, clinical signs
1
Vaccine strain Dindoral® SPF HEV No. 1 GGD 671/93 GGD 581/94 GB 418/98 HEV 157 HEV 001 Control
Spleen Spleen Spleen Spleen Spleen Spleen Spleen
–––a 10-week-old turkeys (non vaccinated) with signs and gross lesions of HE 12-week-old turkeys (non vaccinated) with signs and gross lesions of HE 8-week-old turkeys vaccinated with retarded growth ––– 9-week-old turkeys (non vaccinated) with signs and gross lesions of HE 1-day-old turkey
2 3 4 5 6 7 8 a
no data available.
The second aim was to test if the presented PCR was suitable for practical purposes (Fig. 2). Therefore HEV DNA was extracted from spleens of turkeys showing clinical symptoms of HE (Table 1). In addition, the spleen from a bird was included which came from a vaccinated flock (lane 5). The Dindoral® SPF live vaccine was taken as positive control (lane 1) and the spleen from a one-day-old turkey was included as negative control (lane 8) together with a negative reagent control (c). HEV DNA was detected from all clinical samples and the vaccine strain (Fig. 2). No band was detected in the negative controls (lane 8 and c).
In this article a PCR is described for selective detection of HEV DNA. Together with a PCR for detection and separation of FAV and EDS (Raue and Hess, 1998) all avian adenoviruses with economic importance for poultry production can now be detected and differentiated by hexon gene based PCRs. The variety of avian adenoviruses and the respective host systems should be considered for the establishment of a PCR which detects only HEV DNA. Beside HEV, turkeys are the primary host of turkey adenoviruses (TAV1,2) which share the group specific antigen of group I avian adenoviruses (McFerran et al., 1975). These viruses
Fig. 1. Electrophoresis of the PCR products from purified HEV vaccine strain (lane 1), FAV1 – 12 (lanes 2 – 13) and TAV1,2 (lanes 14 and 15). C, negative reagent control. M, DNA size marker (1 kb DNA-ladder, Life Technologies).
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M. Hess et al. / Journal of Virological Methods 81 (1999) 199–203
Acknowledgements The authors wish to thank Dr J.B. McFerran (Stormont, Belfast, Northern Ireland) for donating of TAV1,2. We also thank Dr J. Cook (Intervet International, UK) and Professor U. Neumann (Klinik fu¨r Geflu¨gel, Tiera¨rztliche Hochschule Hannover, Germany) for providing some of the spleens which were used for HEV DNA extraction.
References
Fig. 2. Electrophoresis of the PCR products received from the HEV samples described in Table 1. Lane 1, HEV vaccine strain. Lanes 2 – 7, field samples listed in Table 1. Lane 8, 1-day-old turkey as negative control. C, negative reagent control. M, DNA size marker (1 kb DNA-ladder, Life Technologies).
were isolated from young turkeys with inclusion body hepatitis and respiratory problems (Easton and Simmons, 1977; Guy and Barnes, 1997). Contrary to this Scott and McFerran (1972) concluded that TAV1 should be regarded as a natural parasite of turkeys based on the widespread incidence of antibodies. In addition to these species specific avian adenoviruses, FAV1 (CELO virus) was also isolated from turkeys (Cho, 1976). With respect to these mixed infections, which seemed to be very common, the presented PCR is a rapid diagnostic method to detect specifically HEV DNA. The detection of HEV DNA from spleen samples underlines the usefulness of the presented PCR under practical conditions. Spleen samples were not only positive from diseased birds but also from an 8-week-old turkey vaccinated in the 4th week of age. It cannot be excluded that this bird had a field challenge with HEV. However, no clinical signs of HE were found. In conclusion, the PCR described above has several advantages in diagnosing HE in turkeys compared to traditional techniques with respect to specificity and sensitivity.
Athappilly, F.K., Murali, R., Rux, J.J., Cai, Z., Burnett, R.M., 1994. The refined crystal structure of hexon, the major coat protein of adenovirus type 2, at 2.9 A, resolution. J. Mol. Biol. 242, 430 – 455. Chiocca, S., Kurzbauer, R., Schaffner, G., Baker, A., Mautner, V., Cotten, M., 1996. The complete DNA sequence and genomic organization of the avian adenovirus CELO. J. Virol. 70, 2939 – 2949. Cho, B.R., 1976. An adenovirus from a turkey pathogenic to both chicks and turkey poults. Avian Dis. 20, 714 – 723. Domermuth, C.H., Gross, W.B., Douglass, C.S., DuBose, R.T., Harris, J.R., Davis, R.B., 1977. Vaccination for haemorrhagic enteritis of turkeys. Avian Dis. 21, 557 – 565. Domermuth, C.H., Gross, W.B., DuBose, R.T., Douglass, C.S., Reubush, C.B., Jr., 1972. Agar gel diffusion precipitin test for haemorrhagic enteritis of turkeys. Avian Dis. 16, 852 – 857. Domermuth, C.H., Weston, C.R., Cowen, B.S., Colwell, W.M., Gross, W.B., DuBose, R.T., 1980. Incidence and distribution of avian adenovirus group II splenomegaly of chickens. Avian Dis. 24, 591 – 594. Easton, G.D., Jr., Simmons, D.G., 1977. Antigenic analysis of several turkey respiratory adenoviruses by reciprocal-neutralization kinetics. Avian Dis. 21, 605 – 611. Fitzgerald, S.D., Reed, W.M., Burnstein, T., 1992. Detection of type II avian adenoviral antigen in tissue sections using immunohistochemical staining. Avian Dis. 36, 341 – 347. Guy, J.S., Barnes, H.J., 1997. Characterization of an avian adenovirus associated with inclusion body hepatitis in dayold turkeys. Avian Dis. 41, 726 – 731. Hess, M., Blo¨cker, H., Brandt, P., 1997. The complete nucleotide sequence of the egg drop syndrome virus: an intermediate between mastadenoviruses and aviadenoviruses. Virology 238, 145 – 156. McFerran, J.B., Adair, B.M., Connor, T.J., 1975. Adenoviral antigens (CELO, QBV, GAL). Am. J. Vet. Res. 36, 527 – 529. Pierson, F.W., Domermuth, C.H., 1997. Hemorrhagic enteritis, marble spleen disease, and related infections. In: Calnek, B.W., Barnes, H.J., Beard, C.W., McDougald, L.R.,
M. Hess et al. / Journal of Virological Methods 81 (1999) 199–203 Saif, Y.M. (Eds.), Diseases of Poultry. Iowa State University Press, Ames, Iowa, pp. 624–663. Pitcovski, J., Mualem, M., Rei-Koren, Z., Krispel, S., Shmueli, E., Peretz, Y., Gutter, B., Gallili, G.E., Michael, A., Goldberg, D., 1998. The complete DNA sequence and genome organization of the avian adenovirus, haemorrhagic enteritis virus. Virology 249, 307–315. Raue, R., Hess, M., 1998. Hexon based PCRs combined with restriction enzyme analysis for rapid detection and differentiation of fowl adenoviruses and egg drop syndrome virus. J. Virol. Methods 73, 211–217. Roberts, M.M., White, J.L., Grutter, M.G., Burnett, R.M., 1986. Three-dimensional structure of the adenovirus major coat protein hexon. Science 232, 1148–1151. Saunders, G.K., Pierson, F.W., van den Hurk, J.V., 1993. Haemorrhagic enteritis virus infection in turkeys: a comparison of virulent and avirulent virus infections, and a proposed pathogenesis. Avian Pathol. 22, 47–58.
203
Scott, M., McFerran, J.B., 1972. Isolation of adenoviruses from turkeys. Avian Dis. 16, 413 – 420. Sheppard, M., McCoy, R.J., Werner, W., 1995. Genomic mapping and sequence analysis of the fowl adenovirus serotype 10 hexon gene. J. Gen. Virol. 76, 2595 – 2600. Suresh, M., Sharma, J.M., 1996. Pathogenesis of type II avian adenovirus infection in turkeys: in vivo immune cell tropism and tissue distribution of the virus. J. Virol. 70, 30 – 36. van den Hurk, J.V., 1986. Quantitation of haemorrhagic enteritis virus antigen and antibody using enzyme-linked immunosorbent assays. Avian Dis. 30, 662 – 671. van den Hurk, J.V., 1988. Characterisation of group II avian adenoviruses with a panel of monoclonal antibodies. Can. J. Vet. Res. 52, 458 – 467. Zhang, C., Nagaraja, K.V., 1989. Differentiation of avian adenovirus type-II strains by restriction endonuclease fingerprinting. Am. J. Vet. Res. 50, 1466 – 1470.
. .
Short communication
PCR for specific detection of haemorrhagic enteritis virus of turkeys, an avian adenovirus M. Hess *, R. Raue, H.M. Hafez Institut fu¨r Geflu¨gelkrankheiten, Freie Uni6ersita¨t Berlin, Koserstraße 21, 14195 Berlin, Germany Received 5 March 1999; received in revised form 23 April 1999; accepted 23 April 1999
Abstract A hexon gene based PCR was developed for specific amplification of DNA sequences from the haemorrhagic enteritis virus (HEV) of turkeys. The hexon genes of different avian adenoviruses were compared for primer construction. Two regions with low sequence homology between HEV and fowl adenovirus (FAV) hexon genes were selected for primer localisation. In correlation with the known sequence data a fragment of 1647 bp was amplified from a live vaccine and spleens of turkeys suffering from haemorrhagic enteritis (HE). All other avian adenoviruses which are able to infect turkeys, i.e. FAV and turkey adenoviruses (TAV), were negative. This is the first PCR for specific detection of HEV DNA which should be useful for rapid diagnosis and epidemiological investigations of HEV infections in turkeys. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Avian adenoviruses; Haemorrhagic enteritis virus; PCR; Spleen samples
Avian adenoviruses are classified routinely into three different groups. The first group combines avian adenoviruses from several species, e.g. chickens, turkeys and geese which share a common group specific antigen. The egg drop syndrome (EDS) virus is the sole member of group III. Group II includes the haemorrhagic enteritis virus (HEV) of turkeys, the marble spleen disease virus (MSDV) pathogenic for pheasants and the * Corresponding author. Tel.: +49-30-8383882; fax: + 4930-8385824. E-mail address: [email protected] (M. Hess)
avian adenovirus splenomegaly virus (AASV) which was observed in young broilers (Domermuth et al., 1980). The group II avian adenoviruses are closely related serologically but can be differentiated with monoclonal antibodies (van den Hurk, 1988). HEV is the only group II avian adenovirus with economic impact, whereas AASV infections are not very common and pheasant populations suffering from MSD are very small. Haemorrhagic enteritis (HE) is a disease of turkeys 4 weeks of age and older (Pierson and Domermuth, 1997). Clinical outbreaks are characterised by intestinal haemorrhages accompanied
0166-0934/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 0 9 3 4 ( 9 9 ) 0 0 0 6 7 - 1
200
M. Hess et al. / Journal of Virological Methods 81 (1999) 199–203
with immunosuppression. The spleen is the major target of the virus resulting in an enlarged, marbled organ (Suresh and Sharma, 1996). Live vaccines are used routinely to prevent HE (Domermuth et al., 1977). Detection of HEV is mostly carried out by agar gel precipitin (AGP) test or ELISA technique (Domermuth et al., 1972; van den Hurk, 1986). Immunohistochemistry for HEV identification was also described but is not used routinely (Fitzgerald et al., 1992; Saunders et al., 1993). The restricted growth of the virus which is usually performed on a lymphoblastoid B-cell line of turkeys (MDTC-RP19) limits the isolation of HEV. Only Zhang and Nagaraja (1989) used restriction enzyme analysis after virus isolation for differentiation of group II avian adenovirus genomes. Detection of HEV DNA by in situ hybridisation and PCR were also described (Suresh and Sharma, 1996). All avian adenoviruses which are able to infect turkeys should be included for the development of a diagnostic PCR specific for HEV detection. Consequently, the data bank was checked for relevant nucleotide sequences of avian adenoviruses. So far, the genomes of three avian adenoviruses representing each group were sequenced completely, namely the CELO virus (FAV1) with 43.8 kb belonging to group I (Chiocca et al., 1996), the EDS virus with a viral genome of 33.2 kb as the sole member of group III (Hess et al., 1997) and recently the HEV with only 26.3 kb (Pitcovski et al., 1998). Based on the experience with a PCR for FAV and EDS virus detection, the HEV hexon gene was chosen for primer construction. In addition to FAV1, EDS virus and HEV, the hexon sequence of FAV10 was published (Sheppard et al., 1995). Therefore the nucleotide sequences of the FAV1, FAV10 and HEV hexon genes were aligned (data not shown). The conserved (pedestal (P)) and variable (loop (L)) regions of the HEV hexon gene were determined according to the proposed model of the human adenovirus 2 hexon protein (Roberts et al., 1986; Athappilly et al., 1994). Primers HEV1F and HEV2R, were constructed and are able to hybridise in the loop regions L1 and L4 of the HEV hexon gene.
Precise location of the primers on the HEV hexon gene is: HEV1F: 5%-TACTGCTGCTATTTGTTGTG-3%, nt 729-748 and HEV2R: 5%-TCATTAACTCCAGCAATTGG-3%, nt 2375-2356. The two regions were selected because of their low sequence identity with the corresponding regions of the FAV1 and FAV10 hexon genes. In this article, all 12 fowl adenoviruses (FAV1– 12) were used together with two group I turkey adenoviruses (TAV1,2). The FAV reference strains used, the propagation of viruses and the extraction of the DNAs from purified viruses was carried out according to methods recently described (Raue and Hess, 1998). The Dindoral® SPF HE-vaccine (Merial, France) was used as a positive control. DNA extraction was performed in the same way as the reference strains. Spleens from turkeys with different clinical histories were tested for HEV DNA to verify the PCR for practical use. All clinical samples which were investigated are listed in Table 1. The QIA amp® tissue kit (Qiagen, Germany) was used for DNA extraction from the spleens according to the manufacturer’s instructions. For PCR 50 ng of purified viral DNA or 1 mg of DNA extracted from the spleens were incubated together with 125 pmol of each primer in a total volume of 25 ml using ‘Ready To Go®’ PCR Beads (Amersham Pharmacia Biotech, Germany). The PCR was carried out on a TRIO-Thermoblock (Biometra, Germany) with the following conditions for 30 cycles: 94°C for 1 min, 64°C for 1 min and 72°C for 1.5 min. Of the resulting PCR mixture, 5 ml was analysed by 0.5% agarose gel electrophoresis and stained with ethidium bromide. The primary aim was to investigate the specificity of the PCR for detection of HEV. For this purpose all avian adenoviruses which could infect turkeys were included in the PCR. After separation of 5 ml of the PCR reaction on an 0.5% agarose gel only the HEV DNA was positive (Fig. 1). Neither FAV- nor TAV-strains reacted with the used primers. With : 1600 bp the estimated size of the amplified fragment was in agreement with the 1647 bp calculated from the HEV hexon sequence.
M. Hess et al. / Journal of Virological Methods 81 (1999) 199–203
201
Table 1 Description of the HEV samples included in this study Lane on agarose gel (Fig. 2)
Sample no.
Material
Flock description, clinical signs
1
Vaccine strain Dindoral® SPF HEV No. 1 GGD 671/93 GGD 581/94 GB 418/98 HEV 157 HEV 001 Control
Spleen Spleen Spleen Spleen Spleen Spleen Spleen
–––a 10-week-old turkeys (non vaccinated) with signs and gross lesions of HE 12-week-old turkeys (non vaccinated) with signs and gross lesions of HE 8-week-old turkeys vaccinated with retarded growth ––– 9-week-old turkeys (non vaccinated) with signs and gross lesions of HE 1-day-old turkey
2 3 4 5 6 7 8 a
no data available.
The second aim was to test if the presented PCR was suitable for practical purposes (Fig. 2). Therefore HEV DNA was extracted from spleens of turkeys showing clinical symptoms of HE (Table 1). In addition, the spleen from a bird was included which came from a vaccinated flock (lane 5). The Dindoral® SPF live vaccine was taken as positive control (lane 1) and the spleen from a one-day-old turkey was included as negative control (lane 8) together with a negative reagent control (c). HEV DNA was detected from all clinical samples and the vaccine strain (Fig. 2). No band was detected in the negative controls (lane 8 and c).
In this article a PCR is described for selective detection of HEV DNA. Together with a PCR for detection and separation of FAV and EDS (Raue and Hess, 1998) all avian adenoviruses with economic importance for poultry production can now be detected and differentiated by hexon gene based PCRs. The variety of avian adenoviruses and the respective host systems should be considered for the establishment of a PCR which detects only HEV DNA. Beside HEV, turkeys are the primary host of turkey adenoviruses (TAV1,2) which share the group specific antigen of group I avian adenoviruses (McFerran et al., 1975). These viruses
Fig. 1. Electrophoresis of the PCR products from purified HEV vaccine strain (lane 1), FAV1 – 12 (lanes 2 – 13) and TAV1,2 (lanes 14 and 15). C, negative reagent control. M, DNA size marker (1 kb DNA-ladder, Life Technologies).
202
M. Hess et al. / Journal of Virological Methods 81 (1999) 199–203
Acknowledgements The authors wish to thank Dr J.B. McFerran (Stormont, Belfast, Northern Ireland) for donating of TAV1,2. We also thank Dr J. Cook (Intervet International, UK) and Professor U. Neumann (Klinik fu¨r Geflu¨gel, Tiera¨rztliche Hochschule Hannover, Germany) for providing some of the spleens which were used for HEV DNA extraction.
References
Fig. 2. Electrophoresis of the PCR products received from the HEV samples described in Table 1. Lane 1, HEV vaccine strain. Lanes 2 – 7, field samples listed in Table 1. Lane 8, 1-day-old turkey as negative control. C, negative reagent control. M, DNA size marker (1 kb DNA-ladder, Life Technologies).
were isolated from young turkeys with inclusion body hepatitis and respiratory problems (Easton and Simmons, 1977; Guy and Barnes, 1997). Contrary to this Scott and McFerran (1972) concluded that TAV1 should be regarded as a natural parasite of turkeys based on the widespread incidence of antibodies. In addition to these species specific avian adenoviruses, FAV1 (CELO virus) was also isolated from turkeys (Cho, 1976). With respect to these mixed infections, which seemed to be very common, the presented PCR is a rapid diagnostic method to detect specifically HEV DNA. The detection of HEV DNA from spleen samples underlines the usefulness of the presented PCR under practical conditions. Spleen samples were not only positive from diseased birds but also from an 8-week-old turkey vaccinated in the 4th week of age. It cannot be excluded that this bird had a field challenge with HEV. However, no clinical signs of HE were found. In conclusion, the PCR described above has several advantages in diagnosing HE in turkeys compared to traditional techniques with respect to specificity and sensitivity.
Athappilly, F.K., Murali, R., Rux, J.J., Cai, Z., Burnett, R.M., 1994. The refined crystal structure of hexon, the major coat protein of adenovirus type 2, at 2.9 A, resolution. J. Mol. Biol. 242, 430 – 455. Chiocca, S., Kurzbauer, R., Schaffner, G., Baker, A., Mautner, V., Cotten, M., 1996. The complete DNA sequence and genomic organization of the avian adenovirus CELO. J. Virol. 70, 2939 – 2949. Cho, B.R., 1976. An adenovirus from a turkey pathogenic to both chicks and turkey poults. Avian Dis. 20, 714 – 723. Domermuth, C.H., Gross, W.B., Douglass, C.S., DuBose, R.T., Harris, J.R., Davis, R.B., 1977. Vaccination for haemorrhagic enteritis of turkeys. Avian Dis. 21, 557 – 565. Domermuth, C.H., Gross, W.B., DuBose, R.T., Douglass, C.S., Reubush, C.B., Jr., 1972. Agar gel diffusion precipitin test for haemorrhagic enteritis of turkeys. Avian Dis. 16, 852 – 857. Domermuth, C.H., Weston, C.R., Cowen, B.S., Colwell, W.M., Gross, W.B., DuBose, R.T., 1980. Incidence and distribution of avian adenovirus group II splenomegaly of chickens. Avian Dis. 24, 591 – 594. Easton, G.D., Jr., Simmons, D.G., 1977. Antigenic analysis of several turkey respiratory adenoviruses by reciprocal-neutralization kinetics. Avian Dis. 21, 605 – 611. Fitzgerald, S.D., Reed, W.M., Burnstein, T., 1992. Detection of type II avian adenoviral antigen in tissue sections using immunohistochemical staining. Avian Dis. 36, 341 – 347. Guy, J.S., Barnes, H.J., 1997. Characterization of an avian adenovirus associated with inclusion body hepatitis in dayold turkeys. Avian Dis. 41, 726 – 731. Hess, M., Blo¨cker, H., Brandt, P., 1997. The complete nucleotide sequence of the egg drop syndrome virus: an intermediate between mastadenoviruses and aviadenoviruses. Virology 238, 145 – 156. McFerran, J.B., Adair, B.M., Connor, T.J., 1975. Adenoviral antigens (CELO, QBV, GAL). Am. J. Vet. Res. 36, 527 – 529. Pierson, F.W., Domermuth, C.H., 1997. Hemorrhagic enteritis, marble spleen disease, and related infections. In: Calnek, B.W., Barnes, H.J., Beard, C.W., McDougald, L.R.,
M. Hess et al. / Journal of Virological Methods 81 (1999) 199–203 Saif, Y.M. (Eds.), Diseases of Poultry. Iowa State University Press, Ames, Iowa, pp. 624–663. Pitcovski, J., Mualem, M., Rei-Koren, Z., Krispel, S., Shmueli, E., Peretz, Y., Gutter, B., Gallili, G.E., Michael, A., Goldberg, D., 1998. The complete DNA sequence and genome organization of the avian adenovirus, haemorrhagic enteritis virus. Virology 249, 307–315. Raue, R., Hess, M., 1998. Hexon based PCRs combined with restriction enzyme analysis for rapid detection and differentiation of fowl adenoviruses and egg drop syndrome virus. J. Virol. Methods 73, 211–217. Roberts, M.M., White, J.L., Grutter, M.G., Burnett, R.M., 1986. Three-dimensional structure of the adenovirus major coat protein hexon. Science 232, 1148–1151. Saunders, G.K., Pierson, F.W., van den Hurk, J.V., 1993. Haemorrhagic enteritis virus infection in turkeys: a comparison of virulent and avirulent virus infections, and a proposed pathogenesis. Avian Pathol. 22, 47–58.
203
Scott, M., McFerran, J.B., 1972. Isolation of adenoviruses from turkeys. Avian Dis. 16, 413 – 420. Sheppard, M., McCoy, R.J., Werner, W., 1995. Genomic mapping and sequence analysis of the fowl adenovirus serotype 10 hexon gene. J. Gen. Virol. 76, 2595 – 2600. Suresh, M., Sharma, J.M., 1996. Pathogenesis of type II avian adenovirus infection in turkeys: in vivo immune cell tropism and tissue distribution of the virus. J. Virol. 70, 30 – 36. van den Hurk, J.V., 1986. Quantitation of haemorrhagic enteritis virus antigen and antibody using enzyme-linked immunosorbent assays. Avian Dis. 30, 662 – 671. van den Hurk, J.V., 1988. Characterisation of group II avian adenoviruses with a panel of monoclonal antibodies. Can. J. Vet. Res. 52, 458 – 467. Zhang, C., Nagaraja, K.V., 1989. Differentiation of avian adenovirus type-II strains by restriction endonuclease fingerprinting. Am. J. Vet. Res. 50, 1466 – 1470.
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