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Bull Vet Inst Pulawy 54, 451-455, 2010 INCIDENCE AND DETECTION OF AVIADENOVIRUSES OF SEROTYPES 1 AND 5 IN POULTRY BY PCR AND DUPLEX PCR JOWITA SAMANTA NICZYPORUK, ELŻBIETA SAMOREK-SALAMONOWICZ, AND HANNA CZEKAJ Department of Poultry Viral Diseases, National Veterinary Research Institute, 24-100 Pulawy, Poland [email protected] Received for publication July 19, 2010 Abstract The aim of the study was to develop and optimise a single PCR and duplex PCR for the detection and differentiation of avian adenovirus serotypes 1 and 5 in poultry. The methods were based on sequence of hexon gene of the serotypes. The PCR and duplex PCR DNA products were 227 pb and 178 bp for serotypes 1 and 5, respectively, and were highly specific. The duplex-PCR method was found to be a highly specific, sensitive, and efficient rapid tool for the detection of serotypes 1 and 5 of adenoviruses, which could be detected in one reaction mixture. During this study, 30 of the isolates obtained from internal organs of healthy birds contained both serotypes of the adenovirus. In the case of six isolates, both serotypes were detected in one bird. Key words: avian adenoviruses, PCR, duplex PCR. Adenoviruses are icosahedral non-enveloped dsDNA viruses with capsid of 74-80 nm in diameter, constructed by 252 capsomers, which surround the core of 60-65 nm in diameter. The main protein of the adenovirus capsid is the hexon protein, which creates 240 trimers and is responsible for the serological differentiation. This protein is 2,800-2,900 bp long with the molecular weight of 103 kDa. It is characterised by a great changeability and is the main protein playing a role in determination of virus antigenic state. Hexon consists of conservative and variable domains. The conservative domains are responsible for creating basement of the molecules and are responsible for the trimer formation. The highly variable domains are placed mainly outside of the virion and are responsible for antigenic variation of adenovirus (5, 11, 15). Aviadenoviruses are divided into three groups. Group I is additionally divided in to five subgroups (AE), containing together 12 serotypes. This group is most numerous and the viruses belonging to it cause IBH inclusion body hepatitis in chickens. Group II contains viruses, which are responsible for haemorrhagic enteritis in turkeys, marble spleen disease in pheasants, and hydropericardium hepatitis syndrome (4), whereas group III includes virus inducing egg drop syndrome 76 (3, 5). Adenoviral infections can be found as asymptomatic and can be a complication factor in the course of different diseases (11). Nowadays, molecular diagnostic methods applied to detect and identify adenoviral infections are based on the virus isolation and serological methods such as: AGID, ELISA, and SN (12). Because of the nucleotide sequence variability of hexon gene, there is a possibility to differentiate several serotypes of adenoviruses from group I by PCR and its modifications (5, 8). The aim of the study was to evaluate the PCR and duplex PCR methods of detection and differentiation of aviadenoviruses, and to estimate the occurrence of these viruses in chicken flocks in Poland. Material and Methods Virus standards. Standard aviadenoviruses (FAdV), belonging to serotypes 1 and 5 were obtained as lyophilisates from the Charles River Laboratory (USA). Strains were grown in chicken embryo kidney (CEK) cultures. CEK cultures. CEK cultures were prepared from 18-19-d old SPF chicken embryos (Lohman, Germany) according to standard procedure. Growth medium constituted Eagle’a medium (MEM) with addition of 10% of bovine serum and 0.1% of antibiotic mixture (Antibiotic–Antimycotic, Gibco). Maintenance medium consisted of MEM with 0.1% of antibiotic mixture. Monolayer of chicken embryo kidney culture was obtained after 48 h incubation period at 37.5°C. Isolation of adenoviruses from birds. Blood and tissues from the liver and intestines were collected during anatomopathological examination of 162 healthy birds originating from 54 reproductive and slaughter flocks. After the homogenisation, triple freeze and thaw, centrifugation, and filtering through the Millipore 452 filter with pores of 450 nm in diameter, the CEK monolayer was infected with the collected materials. The infected cultures were incubated at 37.5°C and observed under the microscope day by day. Three passages of isolated viruses were obtained. Agar gel precipitation (AGP). The sera and the virus material from the 3rd passage in CEK by micro method in 1.5% agar with addition of 8% of NaCl, according to standard procedure, were examined using immunoreagents (Charles River Laboratories, USA). As a negative control, serum from SPF chickens and noninfected CEK cells were used. After 24-48 h incubation at 18°C-25°C, the presence of precipitation lines between the examined samples and antigen confirmed positive results of this method. DNA isolation. Total DNA was isolated from chicken embryo kidney cells infected with standard or field serotypes of adenoviruses. The isolation was performed according to commercial procedure using DNA Mini Kit (Qiagen, Germany). The DNA samples were also collected from the liver and intestines of healthy birds. Isolates of DNA were preserved in -20°C for the next step of the study. PCR positive control. Total DNA positive control was obtained from the reference serotypes 1 and 5 of adenovirus propagated in CEK cell cultures. The negative control was the total DNA isolated from noninfected CEK cells. Primers. The sequences of nucleotide primers for serotype 1 were: FAdV 1A (sense primer): 5’TTCGAGATCAAGAGGCCAGT3’ and FAdV 1B (antisense primer) 5’GGTCGAAGTTGCGTAGGAAG’ 3. In order to detect serotype 5, sequences of the used primers were as follows: FAdV 5A (sense primer) 5’TACTGCCGTTTCCACATTCA 3’ and FAdV 5B (antisense primer): 5’ AGCTGATTGCTGGTGTTGTG 3’. The oligonucleotide primers were designed in Primer 3 programme according to Genebank, and were synthesised in the Institute of Biochemistry and Biophysics PAN in Warsaw. PCR. The reaction of amplification was conducted in Basic gradient thermocycler (Biometra, Germany) in the final volume of 25 µl of reaction mix. The mixture contained: 2.5 µl of PCR buffer, 1 µl of dNTP (10 mM), 1.5 µl of each pair of primers, 4 µl of total DNA isolated from serotype 1, and 11.5 µl of sterile water. After the pre-denaturation at 95°C for 5 min, the denaturation was performed at 94°C for 45 s, the primers annealing was at 61°C for 1 min, the chain elongation was at 72°C for 2 min, and the final elongation at 72°C for 10 min. In total 35 replication cycles were performed. PCR product analysis. After the reaction of amplification, the electrophoresis was conducted in 2% agarose gel with 1µg/mL of ethidium bromide in Mini Sub-Cell (Biorad, USA). Electrophoresis process was conducted in Tris-borate-EDTA buffer, pH 8.2, (150 V and 80 mA) for 50 min. After the electrophoresis, the size of the amplification products was compared with the DNA Mass Ruller 1,031 bp (Fermatas). The results were visualised using transiluminator UV, and then photographed and analysed. The results were positive when the obtained product had predicted size for pair of nucleotide primers. Specificity of reaction. Total DNA from standard serotypes of Marek’s disease virus (MDV), infectious laryngotracheitis virus (ILTV), and chicken anaemia virus (CAV) derived from the commercial vaccines were used to determine the specificity of the reaction. Method sensitivity. Method sensitivity was defined using tenfold dilutions of whole cell DNA isolated from CEK cells infected with standard serotypes of adenoviruses, which correspond to DNA concentrations of 10 up to 0.0001 ng/µL. Results PCR. The appropriate PCR parameters were chosen for serotypes 1 and 5 of adenoviruses. The optimal concentration of polymerase was detected in volume of 2.0 µl (5 u/µL). The volume of the whole cell DNA was optimal at 2.0 µl of DNA for serotypes 1 and 5, with 10 ng/µL concentration. The primers were the most effective at 1.5 µl with 10 ng/µL concentration. At 61°C, PCR products of standard serotypes 1 and 5 corresponded with the predicted size of 178 bp for serotype 1 and 227 bp for serotype 5. No PCR product was observed in case of negative control at temperature parameter described above. No products were observed for DNA samples of MDV, CAV, and ILTV. In the next step, the sensitivity of the method was examined. The method sensitivity, described as the highest dilution of DNA in which a positive result was present, was 0.0001 ng/µL DNA for serotype 1 (Fig.1) and for serotype 5 (Fig. 2). Isolation and serological identification of adenoviruses. AGP was performed with serum of 162 healthy birds. The specific antibodies against adenoviruses were detected in 30 (18.5%) blood samples. The samples from the liver and intestines of birds with positive sera in AGP were used for the infection of CEK cells and isolation of adenoviruses. The first changes in the infected cell cultures were observed after 24-36 h of incubation. The cells were bigger, rounder, and filled with granules. During next few days, the number of cells with cytopathic effect (CPE) increased and the cells formed foci. When it was recognised in 80% of the cells, supernatant from cell cultures was collected to prepare next passage. The material from the 3rd passage was treated as an antigen for AGP with positive standard serum (Charles River Laboratories, USA) and was used as detection method for the identification of group antigen of the adenoviruses. Thirty virus isolates were obtained, which caused CPE in CEK cell cultures and showed a positive reaction in AGP test. 453 M 1 2 3 4 5 6 7 Fig. 1. The sensitivity of PCR. M-Mass Ruller 1,031 bp (Fermantas), 1-6 appropriate dilutions (10; 1; 0.1; 0.01; 0.001; and 0.0001 ng/µL of DNA) of 10 ng/µL of DNA of serotype 1, corresponding to concentrations of 10 ng/µL; 1 ng/ µL; 0.1 ng/ µL; 0.01 ng/ µL; 0.001 ng/ µL; and 0.0001 ng/µL of total DNA isolated from infected chicken embryo kidneys (CEK) of serotype 1. M 1 2 3 4 5 6 7 Fig. 2. The sensitivity of single PCR. M-Mass Ruller 1,031 bp (Fermantas), 1-6 appropriate dilutions (10; 1; 0.1 ; 0.01; 0.001; and 0.0001 ng/µL of DNA) of 10 ng/µL DNA of serotype 5, corresponding to concentration of total DNA isolated from infected chicken embryo kidneys (CEK) - 10 ng/µL; 1 ng/ µL; 0.1 ng/ µL; 0.01 ng/ µL; 0.001 ng/ µL; 0.0001 ng/µL of serotype 5. M 1 2 3 4 5 6 7 8 9 10 11 12 M Fig. 3. Duplex PCR field samples. M- Mass Ruller 1,031 bp DNA Ladder (Fermentas). Lanes 1, 2 - field samples as a dual infection with serotypes 1 and 5, lanes 3, 4 - field samples infected with serotype 1, lanes 5, 10 - field samples infected with serotype 5, lanes 11 - K+ positive control, lane 12 - K- negative control. 454 M 1 2 3 4 5 were fully confirmed with the single PCR used for separate serotypes. Discussion Fig. 4. The sensitivity of duplex PCR. M-Mass Ruller 1,031 bp (Fermantas), 1-6 appropriate dilutions (10; 1; 0.1; 0.01; and 0.001 ng/µL of DNA) of 10 ng/µL DNA of serotype 1 and serotype 5, corresponding to concentration of total DNA isolated from infected chicken embryo kidneys (CEK) - 10 ng/µL; 1 ng/ µL; 0.1 ng/ µL; 0.01 ng/ µL; and 0.001 ng/ µL of serotypes 1 and 5. Identification of isolated serotypes by PCR. Thirty virus samples isolated from CEK cell cultures, identified as adenoviruses by serologic method, were used in PCR for the detection of viruses that belong to serotype 1. During electrophoresis, the presence of 178 bp product specific for serotype 1 was indentified in 11 examined samples. In the next step, PCR was performed with specific primers for the detection of serotype 5. In 25 samples, 227 bp product was observed, which was characteristic for viruses representing serotype 5. In six samples, positive results in the first and second reactions were obtained, which indicated simultaneous appearance of both serotypes in one sample. Development of duplex PCR. Because of the presence of two different adenovirus serotypes in one sample, a duplex PCR method, which allows the detection of genetic material of the viruses in one reaction mixture, was developed and optimised. The parameters of the reaction were similar as in case of single reaction of amplification for one serotype. In the study, the temperature of 61°C for 1min was used for primer annealing and duplex PCR was performed with different amplification cycles: 25, 30, 35, and 40. The bands of most effective PCR products were obtained within 35 cycles of amplification. Characteristic products for positive control, with the size of 178 bp for serotype 1 and 227 bp for serotype 5, as well as both products for the mixture serotypes 1 and 5 were observed. Products of PCR amplification from serotypes 1 and 5 in the examined samples were demonstrated by electrophoresis and photographed. The exemplary examined probes were presented on Fig. 3. The characteristic product of reaction for serotype 1 was observed in 11 samples, and serotype 5 was confirmed in 19 samples. Both serotypes were present in six samples simultaneously. No bands were observed in negative control samples. Results of these reactions Adenovirus infections can cause a serious problem in Poland and in the World’s husbandry. Because of the possibility of existing of simultaneous infection with few adenovirus serotypes, it is necessary to create fast and sensitive diagnostic method (5, 16, 18). Serotype 1 is the most common serotype of adenoviruses and is the model adenovirus, which served as the basis for the description of the genome structure (2, 13). Fatal adenovirus disease in broiler chickens, described as gizzard erosion and ulceration, caused mostly by serotype 1, was reported during the last years (13, 14). In Poland, for the first time, the infection was described in September 2007 (9). In the clinical case of inclusion body hepatitis (IBH), the first isolated serotype was adenovirus serotype 5. However, serotypes 2 and 8 were the most commonly isolated serotypes from the cases of IBH (11, 17). For this reason, a molecular diagnostic technique based on PCR was developed and optimised for useful and reliable detection of adenovirus serotypes 1 and 5. The conservative fragment of the gene coding hexon was used to develop oligonucleotide primers. As the hexon gene is the longest gene in the adenovirus genome and contains conservative and variable sequence, it was possible to create specific oligonucleotide primers to differentiate serotypes 1 and 5. They allow detection of a specific fragment of the hexon gene and create a product size of 178 bp for serotypes 1 and of 227 bp for serotype 5. The differences in the amplification product size allow also creating duplex PCR, useful for the detection and identification of adenoviruses serotypes in one reaction mixture. The developed duplex PCR method was sensitive and allowed the detection of 0.001 ng of viral DNA (Fig. 4). PCR method for the detection of adenoviruses in poultry was described for the first time by Jiang et al. (8). The sensitivity of their method allowed the detection of 0.01 ng of virus DNA. Other researchers described reaction of amplification to detect haemorrhagic enteritis virus in turkeys caused by the virus from group II of adenoviruses (6, 10). Okuda et al. (14) developed and optimised PCR and performed restriction product analysis for the detection of serotype 1. This method provides differentiation of species of adenoviruses from serotype 1, which is the cause of gizzard erosion and ulceration, from the serotypes 1, which are not the reason of these lesions in broilers. PCR and restriction analysis method were designed also to detect adenoviral inclusion body hepatitis in chickens (5). Girgić et al. (7) developed PCR method for identification of serotype 9 of adenoviruses and this method has been used for the determination of vertical transmission of the virus. The multiplex PCR method for the detection and simultaneous identification of aviadenoviruses from 455 group I, avian reovirus, infectious bursal disease virus, and chicken anaemia virus was developed and optimised by Caterina et al. (1).The sensitivity of this method for aviadenoviruses was evaluated as 10 pg (0.01ng) of virus DNA In conclusion, the developed duplex PCR for simultaneous identification of serotypes 1 and 5 is a highly specific and sensitive method. 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