Analysis of pathogenicity and immune efficacy of fowl adenovirus serotype 4 isolates

Analysis of pathogenicity and immune efficacy of fowl adenovirus serotype 4 isolates Abstract In the present study, the pathogenicity of fowl adenovirus (FAdV) serotype 4 strain HB1501 was systematically determined by using different doses and routes of inoculation in chickens and assessed the immune efficacy induced by fowl adenovirus serotype 4 (FAdV-4) strain SB15 in a vaccination trial. The results revealed that strain HB1501 was highly virulent in chickens, inflicting 30 to 100% mortality when administered by intramuscular and intravenous routes, and its pathogenicity was influenced markedly by the inoculation route. The inactivated oil-emulsion FAdV-4 vaccine derived from strain SB15 completely protected specific pathogen-free chickens against morbidity and mortality following infection with the virulent FAdV-4 strain HB1501. Importantly, no obvious clinical signs or gross lesions were observed and high antibody levels against FAdV were measured in the SB15-vaccinated chickens, indicating the potential of this strain to become an alternative vaccine candidate. This study provides detailed information on the pathogenicity of FAdV-4, which should be of great value to the control of hepatitis-hydropericardium syndrome. INTRODUCTION Hepatitis-hydropericardium syndrome (HHS) was first identified in Pakistan in 1987 (Anjum et al., 1989). As a severe disease in poultry, it is caused mainly by fowl adenovirus serotype 4 (FAdV-4), which is characterized by the accumulation of transparent or straw-colored fluid with aqueous or gelatinous consistency in the pericardial sac, nephritis and hepatitis, and has a high mortality rate usually ranging from 30 to 70% (Kim et al., 2008; Schachner et al., 2014). Fowl adenoviruses (FAdV) belong to the Aviadenovirus genus, which together with 4 other genera (Mastadenovirus, Atadenovirus, Siadenovirus, and Ichtadenovirus) are Adenoviridae family members. Fowl adenoviruses have been classified into 5 species (A to E) and further divided into 12 serotypes (FAdV 1 to 8a, and 8b to 11) (Hess, 2000). Besides HHS, FAdV causes a variety of diseases, such as inclusion body hepatitis and adenoviral gizzard erosion. Each of the 12 FAdV serotypes is associated with inclusion body hepatitis, but some FAdV-1 strains are responsible for adenoviral gizzard erosion (Ono et al., 2003). Fowl adenoviruses have been reported in many countries, such as the USA (Mendelson et al., 1995), Canada (Grgić et al., 2014; Schachner et al., 2014), Korea (Park et al., 2017), Poland (Niczyporuk, 2016), Japan (Mase et al., 2009), India (Singh et al., 2002), and Chile (Toro et al., 1999), and they have a severe economic impact on the global poultry industry. Furthermore, the number of HHS cases has increased since 2013 in China (Zhao et al., 2015), and the FAdV-4 strains have been mainly Chinese isolates (Li et al., 2016; Ye et al., 2016). Various vaccines such as inactivated liver homogenate, live or inactivated oil-emulsion vaccines, and new generation vaccines have been developed to control HHS in some countries (Afzal and Ahmad, 1990; Toro et al., 2002; Kim et al., 2014; Schachner et al., 2014), and these vaccines are efficient in protecting birds from FAdV-4. Two FAdV strains, SB15 and HB1501, were isolated from different flocks during HHS outbreaks in China in 2015. Both of them belong to FAdV-4 with a close genetic relationship. Here, the pathogenicity of HB1501 was systematically determined by inoculating chickens with this virus using different doses and inoculation routes. In the vaccination trial, the immune efficacy induced by strain SB15, which was plaque-purified 3 times in the chicken hepatocellular carcinoma cell line (LMH) and inactivated with formaldehyde, was assessed by challenging birds with the virulent HB1501 strain, and then evaluating them by clinical observation, clinical sign scores, gross autopsy, and serological methodologies. MATERIALS AND METHODS Animals and ethics statement Specific pathogen-free (SPF) white leghorn chickens were obtained from the Beijing Merial Vital Laboratory Animal Technology Co., Ltd, China. All the chickens were kept in isolators throughout the experiment at China Agricultural University. The experimental protocols and the housing facility were approved by the Animal Welfare and Ethical Censor Committee at China Agricultural University and Beijing Administration Committee of Laboratory Animals under the leadership of the Beijing Association for Science and Technology. The approval ID is XYXK (Jing) 2013–0013. Cells and Viruses The LMH cells were grown in Dulbecco's Modified Eagle's Medium/F12 (DMEM/F12) (ThermoFischer, NY, USA) containing 10% fetal bovine serum (FBS) (ThermoFischer) and 1% penicillin–streptomycin (Macgene, Beijing, China) at 37°C with 5% CO2. FAdV-4 SB15 and HB1501 strains were isolated from the liver samples of suspected FAdV-infected chickens collected in 2015. The highly virulent HB1501 isolate was used as the challenge strain to determine the immune efficacy induced by the inactivated FAdV SB15 vaccine. Two FAdV strains were cultured in LMH cells. The 2 strains were plaque-purified three times as described previously (Alexander et al., 1998) and then titrated by median tissue culture infective doses (TCID50) and plaque-forming units (PFU). The TCID50 was determined in 96-well titration plates, according to the endpoint method of Reed and Muench (1938). PFU was determined as described previously (Alexander et al., 1998). Cytopathic Effects (CPE) of FAdV in LMH Cells The chicken hepatocellular cells in 24-well plates were infected with SB15 or HB1501 at 106.0 TCID50, and then incubated in DMEM/F12 containing 1% FBS. The CPE of each FAdV strain in LMH cells was observed at 12 h intervals until 48 h post-infection (hpi). Uninfected cells served as the control. Optimal Harvest Time Determination The growth dynamics of the FAdV-4 strain SB15 was evaluated in LMH cells to determine the optimal harvest time. Cells in triplicate wells of 24-well plates were infected with the SB15 strain containing 106.0 TCID50, and then incubated in DMEM/F12 containing 1% FBS. The cells and culture supernatants were harvested at 8 h intervals until 64 hpi. The viral titers were quantified in LMH cells and expressed as TCID50. Pathogenicity Testing of FAdV-4 HB1501 in SPF Chickens Grouping and Viral Challenge One hundred 3-wk-old SPF chickens were randomly divided into 10 groups of 10 each and housed separately in isolators. Birds were inoculated with 0.2 mL of FAdV-4 HB1501 using different doses (107.0TCID50/bird, 106.0TCID50/bird, and 105.0TCID50/bird) and routes of inoculation (oral; intramuscular, I.M.; intravenous, I.V.) except for the control group (group 10) (Table 1). Table 1. Experimental design in pathogenicity test of FAdV strain HB1501. Challenge1 Observation and sampling Groups Virus Route2 Dose (TCID50/bird) Observation time (d) Serology3 1 HB1501 Oral 107.0 14 10 2 HB1501 Oral 106.0 14 10 3 HB1501 Oral 105.0 14 10 4 HB1501 I.M. 107.0 14 10 5 HB1501 I.M. 106.0 14 10 6 HB1501 I.M. 105.0 14 10 7 HB1501 I.V. 107.0 14 10 8 HB1501 I.V. 106.0 14 10 9 HB1501 I.V. 105.0 14 10 10 - - - 14 10 Challenge1 Observation and sampling Groups Virus Route2 Dose (TCID50/bird) Observation time (d) Serology3 1 HB1501 Oral 107.0 14 10 2 HB1501 Oral 106.0 14 10 3 HB1501 Oral 105.0 14 10 4 HB1501 I.M. 107.0 14 10 5 HB1501 I.M. 106.0 14 10 6 HB1501 I.M. 105.0 14 10 7 HB1501 I.V. 107.0 14 10 8 HB1501 I.V. 106.0 14 10 9 HB1501 I.V. 105.0 14 10 10 - - - 14 10 1Three-wk-old SPF chickens were infected with FAdV strain HB1501. 2I.M., intramuscular; I.V., intravenous. 3The sera were collected at 21 dpc in all survived chickens and tested with ELISA. View Large Table 1. Experimental design in pathogenicity test of FAdV strain HB1501. Challenge1 Observation and sampling Groups Virus Route2 Dose (TCID50/bird) Observation time (d) Serology3 1 HB1501 Oral 107.0 14 10 2 HB1501 Oral 106.0 14 10 3 HB1501 Oral 105.0 14 10 4 HB1501 I.M. 107.0 14 10 5 HB1501 I.M. 106.0 14 10 6 HB1501 I.M. 105.0 14 10 7 HB1501 I.V. 107.0 14 10 8 HB1501 I.V. 106.0 14 10 9 HB1501 I.V. 105.0 14 10 10 - - - 14 10 Challenge1 Observation and sampling Groups Virus Route2 Dose (TCID50/bird) Observation time (d) Serology3 1 HB1501 Oral 107.0 14 10 2 HB1501 Oral 106.0 14 10 3 HB1501 Oral 105.0 14 10 4 HB1501 I.M. 107.0 14 10 5 HB1501 I.M. 106.0 14 10 6 HB1501 I.M. 105.0 14 10 7 HB1501 I.V. 107.0 14 10 8 HB1501 I.V. 106.0 14 10 9 HB1501 I.V. 105.0 14 10 10 - - - 14 10 1Three-wk-old SPF chickens were infected with FAdV strain HB1501. 2I.M., intramuscular; I.V., intravenous. 3The sera were collected at 21 dpc in all survived chickens and tested with ELISA. View Large Clinical Observations and Sampling All the birds were monitored daily for clinical signs (e.g., depression, ruffled feathers, reluctance to move) or death for 14 d. The clinical signs were assessed as daily clinical scores: 0 for normal, 1 for mild depression, 2 for severe depression, 3 for paralysis/prostration, and 4 for death (Zhao et al., 2015). Serum samples were collected at 21 d post-inoculation (dpi) and antibody levels against FAdV-4 were determined by a recombinant fiber-2 protein-based indirect enzyme-linked immune-sorbent antibody assay (ELISA), whose cut-off value was 0.130 (OD450nm value). The optimal dilution level of the serum or secondary antibodies were 1:200 and 1:6,000, respectively. All the birds were then humanely euthanized and necropsied. Immune Efficacy Trial of FAdV strain SB15 Oil-Adjuvant Inactivated FAdV Vaccine Preparation The LMH-derived SB15 virus was inactivated with formaldehyde at 4°C at a final concentration of 0.1% for 24 h. Inactivated viral antigen was mixed with the oil emulsion adjuvant, Marcol 52 (Seppic, Paris, France), at a ratio of 1:1 (V/V). Chicken Immunizations Forty 1-wk-old SPF chickens were randomly divided into 4 groups of 10 each and housed separately in isolators. Two groups of chickens were inoculated subcutaneously with the SB15 vaccine at a dose of 106.5TCID50/bird or 107.5TCID50/bird. Another 20 birds served as the unvaccinated control group (Table 2). To confirm the antibody responses against the inactivated FAdV-4 vaccine, sera from the vaccinated chickens were collected at 21 dpi and the antibody levels were determined by ELISA. Table 2. Experimental design in immune efficacy trial of FAdV strain SB15. Inoculation1 Challenge2 Observation and sampling Groups Virus Route3 Dose (TCID50/bird) Virus Route4 Dose (TCID50/bird) Observation time (d) Serology5 1 SB15 S.C. 107.5 HB1501 I.V. 106.0 14 10 2 SB15 S.C. 106.5 HB1501 I.V. 106.0 14 10 3 - - - HB1501 I.V. 106.0 14 10 4 - - - - - - 14 10 Inoculation1 Challenge2 Observation and sampling Groups Virus Route3 Dose (TCID50/bird) Virus Route4 Dose (TCID50/bird) Observation time (d) Serology5 1 SB15 S.C. 107.5 HB1501 I.V. 106.0 14 10 2 SB15 S.C. 106.5 HB1501 I.V. 106.0 14 10 3 - - - HB1501 I.V. 106.0 14 10 4 - - - - - - 14 10 1One-wk-old SPF chickens were inoculated with FAdV strain SB15. 2The chickens were challenged at 21 dpi. 3S.C., subcutaneous. 4I.V., intravenous. 5Ten sera were collected at 21 dpi and 14 dpc and tested with ELISA. View Large Table 2. Experimental design in immune efficacy trial of FAdV strain SB15. Inoculation1 Challenge2 Observation and sampling Groups Virus Route3 Dose (TCID50/bird) Virus Route4 Dose (TCID50/bird) Observation time (d) Serology5 1 SB15 S.C. 107.5 HB1501 I.V. 106.0 14 10 2 SB15 S.C. 106.5 HB1501 I.V. 106.0 14 10 3 - - - HB1501 I.V. 106.0 14 10 4 - - - - - - 14 10 Inoculation1 Challenge2 Observation and sampling Groups Virus Route3 Dose (TCID50/bird) Virus Route4 Dose (TCID50/bird) Observation time (d) Serology5 1 SB15 S.C. 107.5 HB1501 I.V. 106.0 14 10 2 SB15 S.C. 106.5 HB1501 I.V. 106.0 14 10 3 - - - HB1501 I.V. 106.0 14 10 4 - - - - - - 14 10 1One-wk-old SPF chickens were inoculated with FAdV strain SB15. 2The chickens were challenged at 21 dpi. 3S.C., subcutaneous. 4I.V., intravenous. 5Ten sera were collected at 21 dpi and 14 dpc and tested with ELISA. View Large Challenge Tests The birds were challenged with 0.2 mL (106.0 TCID50/bird) of FAdV-4 HB1501 via the intravenous route at 21 dpi (Table 2). All the birds were monitored daily and scored for clinical signs for 14 d. The clinical signs were scored as previously described. Serum samples were collected on d 14 post challenge and antibody levels were determined by ELISA. All birds were euthanized and necropsied on d 14 post challenge. RESULTS Cytopathic Effects of FAdV in LMH Cells LMH cells are normally polygonal with a dendritic-like appearance. Uninfected cells served as the control and no CPE was observed with them. In cells infected with both FAdV strains, a visible CPE was observed at 24 hpi, as characterized by cell rounding and cell aggregation. When the incubation time was increased, the spacing between the cells widened and particles were observed in the cells at high magnification (400×). At 36 hpi, there was a severe CPE, and complete detachment of the cell monolayer was observed at 48 hpi. Virus Purification and Titration The 2 FAdV strains used in our study were plaque-purified 3 times and titrated by TCID50 and PFU. Both strains produced visible plaques 4 d after inoculation, which developed progressively to diameters of 1 to 3 mm within 8 dpi. The number of plaques decreased as the dilution increased. The TCID50 for HB1501 and SB15 were 108.0 TCID50/0.2 mL and 107.67 TCID50/0.2 mL, respectively. The PFU calculation for SB15 was 2.06 × 108.0 PFU/mL. Optimal Harvest Time Determination The growth dynamics of SB15 were evaluated in LMH cells at 8 h intervals until 64 hpi to determine the optimal harvest time. The values obtained represent averages of the results from 3 independent experiments (Figure 1). The virus titer showed an increasing trend, reached a maximum at 48 hpi, gradually declining thereafter. The optimal harvest time for maximizing the virus yield was determined at 32 to 48 hpi. Figure 1. View largeDownload slide Growth properties of FAdV-4 strain SB15 in LMH cells. The cells and culture supernatants were harvested at 8 h intervals until 64 hpi. The viral titers were quantified in LMH cells and expressed as TCID50. Values represent averages of the results from 3 independent experiments. The error bars show the standard deviations. Figure 1. View largeDownload slide Growth properties of FAdV-4 strain SB15 in LMH cells. The cells and culture supernatants were harvested at 8 h intervals until 64 hpi. The viral titers were quantified in LMH cells and expressed as TCID50. Values represent averages of the results from 3 independent experiments. The error bars show the standard deviations. Pathogenicity Testing of FAdV-4 HB1501 in SPF Chickens Clinical Signs No clinical signs or deaths were observed in the oral infection and control groups during the experiment. Chickens inoculated with HB1501 by the I.M. and I.V. route showed obvious clinical signs of depression, and also ruffled feathers, decreased feeding and water consumption, reluctance to move, paralysis, and death (Figure 2A). Figure 2. View largeDownload slide Clinical scores (A) and survival rates (B) in chickens experimentally challenged with FAdV-4 strain HB1501. (A) Clinical scoring: 0 for normal, 1 for mild depression, 2 for severe depression, 3 for paralysis/prostration, and 4 for death. The mean scores per group per day are shown. The error bars show the standard deviations. (B) Birds were infected with 0.2 mL of strain HB1501 by different doses and routes except for the control group. Figure 2. View largeDownload slide Clinical scores (A) and survival rates (B) in chickens experimentally challenged with FAdV-4 strain HB1501. (A) Clinical scoring: 0 for normal, 1 for mild depression, 2 for severe depression, 3 for paralysis/prostration, and 4 for death. The mean scores per group per day are shown. The error bars show the standard deviations. (B) Birds were infected with 0.2 mL of strain HB1501 by different doses and routes except for the control group. In group 4 (I.M., 107.0TCID50/bird), the first clinical signs of the disease were observed at 2 dpi. Mortality started at 3 dpi, and at 5 dpi all the animals were found dead (Figure 2B). The mortality rate was 100%. In group 5 (I.M., 106.0TCID50/bird), the first clinical signs of the disease were observed at 3 dpi. Mortality started at 3 dpi, 8 birds died in all, and the mortality rate reached 80%. The surviving birds gradually recovered from 6 dpi. In group 6 (I.M., 105.0TCID50/bird), the first clinical signs of the disease were observed at 3 dpi. Mortality started at 4 dpi, 7 birds died in all, and the mortality rate was 70%. The surviving birds gradually recovered from 10 dpi. Similar results were found in the I.V. groups. Mortality was observed between 2 and 7 dpi, when 10, 10, and 3 birds from groups 7, 8, and 9 died, respectively. The mortality rates for groups 7, 8, and 9 reached 100, 100, and 30%, respectively. Gross Lesions Obvious lesions were observed in all the dead birds at necropsy, including hydropericardium, swollen and friable liver with multi focal areas of necrosis and petechial hemorrhage, and swollen and pale kidneys. No obvious lesions were observed in the oral infection groups or in any birds that recovered from infection with HB1501. No gross lesions were identified in any control group birds. Antibody Responses Sera were collected at 21 dpi from all the surviving chickens and tested by ELISA. Most of the birds inoculated with HB1501 showed positive antibody responses, regardless of the infection routes and doses (Figure 3). In groups 1, 2, and 3, the antibody response against FAdV was detected in most of the surviving birds, with positivity rates of 90, 50, and 90%, respectively. In groups 5 and 6, the antibody response against FAdV was detected in all the surviving birds, with positivity rates of 100 and 100%, respectively. In group 7, the antibody response against FAdV was detected in all the surviving birds, with a positivity rate of 100%. Chickens infected by the I.V. route had higher titers than those in the other groups. The antibody response against FAdV was not detected in any control group birds. Figure 3. View largeDownload slide The antibody response against FAdV in chickens experimentally challenged with FAdV-4 strain HB1501. The sera from the surviving birds were collected at 21 dpi and tested by ELISA. The antibody titers of the individual birds are shown by black circles, and short lines represent the averages of each group. Cut-off titer = 0.130. Figure 3. View largeDownload slide The antibody response against FAdV in chickens experimentally challenged with FAdV-4 strain HB1501. The sera from the surviving birds were collected at 21 dpi and tested by ELISA. The antibody titers of the individual birds are shown by black circles, and short lines represent the averages of each group. Cut-off titer = 0.130. Immune Efficacy Trial of FAdV SB15 Clinical Signs No clinical signs or deaths attributable to FAdV-4 were identified in any birds in the control group or the SB15-vaccinated groups during the experiment. Birds in the unvaccinated group challenged with strain HB1501 showed clinical signs of depression, ruffled feathers, and reluctance to move as early as 2 dpi, (Figure 4A). The mean scores per group per day for the clinical signs in the challenge group were much higher than those of the vaccination groups and the control group. Mortality was observed between 3 and 5 dpi, when 6 birds died, and the mortality rate reached 60% (Figure 4B). The surviving birds gradually recovered from 7 dpi. Figure 4. View largeDownload slide Clinical scores (A), survival rates (B), and antibody response against FAdV (C) in vaccinated SPF chickens following HB1501 challenge. (A) Clinical scoring: 0 for normal, 1 for mild depression, 2 for severe depression, 3 for paralysis/prostration, and 4 for death. The mean scores per group per day are shown. The error bars show the standard deviations. (B) Birds were inoculated with strain SB15 and challenged with strain HB1501 via I.V. routes at 21 dpi. (C) Sera, collected at 21 dpi and 10 dpc, were tested by ELISA. The antibody titers of the individual birds are shown. Cut-off titer = 0.130. Figure 4. View largeDownload slide Clinical scores (A), survival rates (B), and antibody response against FAdV (C) in vaccinated SPF chickens following HB1501 challenge. (A) Clinical scoring: 0 for normal, 1 for mild depression, 2 for severe depression, 3 for paralysis/prostration, and 4 for death. The mean scores per group per day are shown. The error bars show the standard deviations. (B) Birds were inoculated with strain SB15 and challenged with strain HB1501 via I.V. routes at 21 dpi. (C) Sera, collected at 21 dpi and 10 dpc, were tested by ELISA. The antibody titers of the individual birds are shown. Cut-off titer = 0.130. Gross Lesions No gross lesions were identified in any control group birds or the SB15 vaccinated groups during the experiment. Obvious lesions were observed in all the dead birds in the unvaccinated group challenged with HB1501, including hydropericardium, swollen and friable liver with multifocal areas of necrosis and petechial hemorrhage, and swollen and pale kidneys. These results suggest that both vaccination doses (107.5TCID50/bird, 106.5TCID50/bird) provided complete protection against HB1501. Antibody Response Sera were collected at 21 dpi and 14 d post-challenge (dpc) and tested by ELISA (Figure 4C). All birds inoculated with SB15 showed positive antibody responses at 21 dpi. Birds vaccinated with higher doses of the virus had higher titers than those in the low dose group. The mean ELISA titers for birds in the vaccination groups that received 107.5TCID50/bird or 106.5TCID50/bird were 1.14 and 0.57 at 21 dpi, respectively. Furthermore, the values increased to 1.31 and 1.33 at 14 dpc, respectively. The mean ELISA titer for the surviving birds in the unvaccinated challenge group was 1.28. Antibody responses against FAdV were not detected in any control group birds. DISCUSSION Hepatitis-hydropericardium syndrome, the most severe disease induced by FAdV infection, has significant economic impact on the global poultry industry (Toro et al., 1999; Singh et al., 2002; Mase et al., 2009; Grgić et al., 2014; Schachner et al., 2014; Niczyporuk, 2016; Park et al., 2017). Various vaccines have been made to control HHS (Afzal and Ahmad, 1990; Toro et al., 2002; Kim et al., 2014; Schachner et al., 2014). Two FAdV-4 strains, SB15 and HB1501, were isolated from HHS-affected flocks during FAdV outbreaks in China in 2015. Here, the pathogenecity of HB1501 was systematically determined by different doses and routes of inoculation in chickens. We then further assessed the immune efficacy of SB15 with a challenge-protection study. LMH is a continuous cell line appropriate for the growth of FAdV (Alexander et al., 1998), and is more convenient for research than primary chicken embryo liver cells and chick kidney cells, as they are fairly tedious to prepare. Therefore, the two FAdV strains used in this study were adapted to growth in LMH cells. With both strains, visible CPE characterized by cell rounding and cell aggregation was observed. Granules were observed in the cells at high magnification (400×), which is consistent with the description for primary chick kidney cell and primary chicken embryo liver cell cultures (Adair et al., 1979; Li et al., 2016). Our results support many others detailing that most FAdV strains can be propagated efficiently in LMH cells (Alexander et al., 1998; Grgić et al., 2011). The pathogenicity of the FAdV-4 HB1501 was systematically determined using different doses and routes of inoculation. These data revealed that HB1501 was highly virulent in SPF chickens infected by I.M. and I.V. routes, inflicting 30–100% mortality. Obvious clinical signs and gross lesions were observed, and those caused by HB1501 were identical to those described in previous studies of pathogenicity for an isolate of FAdV-4 (Zhao et al., 2015; Niu et al., 2016). It should be noted that the pathogenicity of HB1501 was markedly influenced by the route of inoculation. When the virus was inoculated orally, which is the natural route for infection with FAdV, HB1501 did not display pathogenicity, and no obvious clinical signs, deaths, or gross lesions were observed; however, it was highly virulent when inoculated by the I.M. and I.V. routes. Similar results were observed in several previous studies on FAdV-4 pathogenicity (Reece et al., 1987; Ojkic and Nagy, 2003). Additionally, we showed that increasing the dose of the virus accelerated the disease course and severity. The mortality rate also gradually increased, reaching 100% when the injection dose was higher than 107.0TCID50 by the I.M. route or 106.0TCID50 by the I.V. route. To the best of our knowledge, this is the first study to systematically compare the influence of three different routes (oral, I.M. and I.V.) and doses of inoculation on the pathogenicity of a strain of FAdV. To confirm that the infection was effectively established, serum samples were collected for detection of FAdV antibodies during pathogenicity testing of HB1501. Our results showed that most of the birds inoculated with HB1501 tested positive for FAdV antibodies at 21 dpi, except for a small number of chickens inoculated by the oral route. It was found that chickens infected by the I.V. route had higher ELISA titers than those in the other groups in the pathogenicity test of strain HB1501, which was identical to those described in previous studies (Schonewille et al., 2010; Grgić et al., 2011). The reason might be that natural route of viral infection may be unable to ensure that all birds receive the same amount of virus (Grgić et al., 2011). In this study, the I.V. route (106.0TCID50/bird) was chosen for challenge infections with strain HB1501 in the immune efficacy trial, and the results were consistent with a study on the evaluation of protection efficacy induced by an inactivated oil-emulsion FAdV-4 vaccine (Kim et al., 2014). Many studies have been conducted with the aim of protecting birds from infection with FAdV-4 in some countries (Mansoor et al., 2011; Kim et al., 2014). Broiler chickens free from maternal antibodies against FAdV were immunized with a chicken embryo-adapted FAdV-4 isolate, which was attenuated by serially passaging, and the vaccination gave 94.73% protection (Mansoor et al., 2011). A vaccine containing inactivated oil-emulsion FAdV-4 was shown to provide broad cross-protection against various FAdV serotypes (Kim et al., 2014). In this study, the immune efficacy of SB15 was assessed using a challenge-protection approach. Our results suggest that SB15 completely protected SPF birds against morbidity and mortality when challenged with the virulent FAdV-4 HB1501 strain via the I.V. route. Neither clinical signs nor gross lesions at necropsy were observed and the antibody levels of the vaccinated chickens were high. These results indicate that this strain can be used in vaccination procedures to reduce the economic losses caused by FAdV-4 infections. The mechanism of FAdV infection is very complex in chickens (Li et al., 2016). It has been suggested that there is a synergism between FAdV and chicken infectious anemia virus or other viruses (Toro et al., 2000). Therefore, besides preventing FAdV infections, vaccination and good animal management practices for the control of infectious immunosuppressive agents such as infectious bursal disease virus and chicken infectious anemia virus are necessary (Shivachandra et al., 2003; Balamurugan amd Kataria, 2006). In conclusion, our data confirm that FAdV-4 HB1501 is highly virulent in chickens and its pathogenicity was markedly influenced by the route of inoculation. FAdV-4 SB15 provided effective protection against challenge with HB1501, indicating its potential as an alternative vaccine candidate for controlling FAdV-4 infections and HHS in China. Acknowledgements This study was supported by the National Key Research and Development Program of China (2017YFD0500700). REFERENCES Adair B. M. , Curran W. L. , Mcferran J. B. . 1979 . Ultrastructural studies of the replication of fowl adenoviruses in primary cell cultures . Avian Pathol. 8 : 133 – 144 . Google Scholar CrossRef Search ADS PubMed Afzal M. , Ahmad I. . 1990 . Efficacy of an inactivated vaccine against hydropericardium syndrome in broilers . Vet. Rec. 126 : 59 – 60 . Google Scholar PubMed Alexander H. S. , Huber P. , Cao J. , Krell P. J. , Nagy E. . 1998 . Growth characteristics of fowl adenovirus type 8 in a chicken hepatoma cell line . J. Virol. Methods 74 : 9 – 14 . Google Scholar CrossRef Search ADS PubMed Anjum A. D. , Sabri M. A. , Iqbal Z. . 1989 . Hydropericarditis syndrome in broiler chickens in Pakistan . Vet. Rec. 124 : 247 – 248 . Google Scholar CrossRef Search ADS PubMed Balamurugan V. , Kataria J. M. . 2006 . Economically important non-oncogenic immunosuppressive viral diseases of chicken-current status . Vet. Res. Commun. 5 : 541 – 566 . Google Scholar CrossRef Search ADS Grgić H. , Krell P. J. , Nagy E. . 2014 . Comparison of fiber gene sequences of inclusion body hepatitis (IBH) and non-IBH strains of serotype 8 and 11 fowl adenoviruses . Virus Genes 48 : 74 – 80 . Google Scholar CrossRef Search ADS PubMed Grgić H. , Yang D. H. , Nagy E. . 2011 . Pathogenicity and complete genome sequence of a fowl adenovirus serotype 8 isolate . Virus Res. 156 : 91 – 97 . Google Scholar CrossRef Search ADS PubMed Hess M. 2000 . Detection and differentiation of avian adenoviruses: a review . Avian Pathol. 29 : 195 – 206 . Google Scholar CrossRef Search ADS PubMed Kim J. N. , Byun S. H. , Min J. K. , Kim J. J. , Sung H. W. , Mo I. P. . 2008 . Outbreaks of hydropericardium syndrome and molecular characterization of Korean fowl adenoviral isolates . Avian Dis. 52 : 526 – 530 . Google Scholar CrossRef Search ADS PubMed Kim M. S. , Lim T. H. , Lee D. H. , Youn H. N. , Yuk S. S. , Kim B. Y. , Choi S. W. , Jung C. H. , Han J. H. , Song C. S. . 2014 . An inactivated oil-emulsion fowl adenovirus serotype 4 vaccine provides broad cross-protection against various serotypes of fowl adenovirus . Vaccine 32 : 3564 – 3568 . Google Scholar CrossRef Search ADS PubMed Li H. , Wang J. , Qiu L. , Han Z. , Liu S. . 2016 . Fowl adenovirus species c serotype 4 is attributed to the emergence of hepatitis-hydropericardium syndrome in chickens in China . Infect. Genet. Evol. 45 : 230 – 241 . Google Scholar CrossRef Search ADS PubMed Mansoor M. K. , Hussain I. , Arshad M. , Muhammad G. . 2011 . Preparation and evaluation of chicken embryo-adapted fowl adenovirus serotype 4 vaccine in broiler chickens . Trop. Anim. Health Prod. 43 : 331 – 338 . Google Scholar CrossRef Search ADS PubMed Mase M. , Chuujou M. , Inoue T. , Nakamura K. , Yamaguchi S. , Imada T. . 2009 . Genetic characterization of fowl adenoviruses isolated from chickens with hydropericardium syndrome in Japan . J. Vet. Med. Sci. 71 : 1455 – 1458 . Google Scholar CrossRef Search ADS PubMed Mendelson C. , Nothelfer H. B. , Monreal G. . 1995 . Identification and characterization of an avian adenovirus isolated from a -spiking mortality syndrome- field outbreak in broilers on the Delmarva Peninsula, USA . Avian Pathol. 24 : 693 – 706 . Google Scholar CrossRef Search ADS PubMed Niczyporuk J. S. 2016 . Phylogenetic and geographic analysis of fowl adenovirus field strains isolated from poultry in Poland . Arch. Virol. 161 : 33 – 42 . Google Scholar CrossRef Search ADS PubMed Niu Y. J. , Sun W. , Zhang G. H. , Qu Y. J. , Wang P. F. , Sun H. L. , Xiao Y. H. , Liu S. D. . 2016 . Hydropericardium syndrome outbreak caused by fowl adenovirus serotype 4 in China in 2015 . J. Gen. Virol. 97 : 2684 – 2690 . Google Scholar CrossRef Search ADS PubMed Ojkic D. , Nagy E. . 2003 . Antibody response and virus tissue distribution in chickens inoculated with wild-type and recombinant fowl adenoviruses . Vaccine 22 : 42 – 48 . Google Scholar CrossRef Search ADS PubMed Ono M. , Okuda Y. , Yazawa S. , Imai Y. , Shibata I. , Sato S. , Okada K. . 2003 . Adenoviral gizzard erosion in commercial broiler chickens . Vet. Pathol. 40 : 294 – 303 . Google Scholar CrossRef Search ADS PubMed Park H. S. , Lim I. S. , Kim S. K. , Kim T. K. , Park C. K. , Yeo S. G. . 2017 . Molecular analysis of the hexon, penton base, and fiber-2 genes of Korean fowl adenovirus serotype 4 isolates from hydropericardium syndrome-affected chickens . Virus Genes 53 : 111 – 116 . Google Scholar CrossRef Search ADS PubMed Reece R. L. , Barr D. A. , Grix D. C. . 1987 . Pathogenicity studies with a strain of fowl adenovirus serotype 8 (vri-33) in chickens . Australian Vet J 64 : 365 – 367 . Google Scholar CrossRef Search ADS Reed L. J. , Muench H. . 1938 . A simple method of estimating fifty per cent endpoints . Am. J. Epidemiol. 27 : 493 – 497 . Google Scholar CrossRef Search ADS Schachner A. , Marek A. , Jaskulska B. , Bilic I. , Hess M. . 2014 . Recombinant FAdV-4 fiber-2 protein protects chickens against hepatitis-hydropericardium syndrome (HHS) . Vaccine 32 : 1086 – 1092 . Google Scholar CrossRef Search ADS PubMed Schonewille E. , Jaspers R. , Paul G. , Hess M. . 2010 . Specific-pathogen-free chickens vaccinated with a live FAdV-4 vaccine are fully protected against a severe challenge even in the absence of neutralizing antibodies . Avian Dis . 54 : 905 – 910 . Google Scholar CrossRef Search ADS PubMed Shivachandra S. B. , Sah R. L. , Singh S. D. , Kataria J. M. , Manimaran K. . 2003 . Immunosuppression in broiler chicks fed aflatoxin and inoculated with fowl adenovirus serotype-4 (FAV-4) associated with hydropericardium syndrome . Vet. Res. Commun. 27 : 39 – 51 . Google Scholar CrossRef Search ADS PubMed Singh A. , Oberoi M. S. , Grewal G. S. , Hafez H. M. , Hess M. . 2002 . The use of PCR combined with restriction enzyme analysis to characterize fowl adenovirus field isolates from northern India . Vet. Res. Commun. 26 : 577 – 585 . Google Scholar CrossRef Search ADS PubMed Toro H. , Gonzalez C. , Cerda L. , Hess M. , Reyes E. , Geisse C. . 2000 . Chicken anemia virus and fowl adenoviruses: association to induce the inclusion body hepatitis/hydropericardium syndrome . Avian Dis. 44 : 51 – 58 . Google Scholar CrossRef Search ADS PubMed Toro H. , González C. , Cerda L. , Morales M. A. , Dooner P. , Salamero M. . 2002 . Prevention of inclusion body hepatitis/hydropericardium syndrome in progeny chickens by vaccination of breeders with fowl adenovirus and chicken anemia virus . Avian Dis. 46 : 547 – 554 . Google Scholar CrossRef Search ADS PubMed Toro H. , Prusas C. , Raue R. , Cerda L. , Geisse C. , González C. , Hess M. . 1999 . Characterization of fowl adenoviruses from outbreaks of inclusion body hepatitis/hydropericardium syndrome in Chile . Avian Dis. 43 : 262 – 270 . Google Scholar CrossRef Search ADS PubMed Ye J. , Liang G. , Zhang J. , Wang W. , Song N. , Wang P. , Zheng W. , Xie Q. , Shao H. , Wan Z. , Wang C. , Chen H. , Gao W. , Qin A. . 2016 . Outbreaks of serotype 4 fowl adenovirus with novel genotype, China , Emerg Microbes Infect 5 : e50 – e50 . Google Scholar CrossRef Search ADS PubMed Zhao J. , Zhong Q. , Zhao Y. , Hu Y. X. , Zhang G. Z. . 2015 . Pathogenicity and complete genome characterization of fowl adenoviruses isolated from chickens associated with inclusion body hepatitis and hydropericardium syndrome in China . PLoS One 10 : e0133073 . Google Scholar CrossRef Search ADS PubMed © 2018 Poultry Science Association Inc. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Poultry Science Oxford University Press

Analysis of pathogenicity and immune efficacy of fowl adenovirus serotype 4 isolates

Poultry Science , Volume Advance Article (8) – Jul 11, 2018

Loading next page...
 
/lp/ou_press/analysis-of-pathogenicity-and-immune-efficacy-of-fowl-adenovirus-iwpZgJ8pHU
Publisher
Oxford University Press
Copyright
© 2018 Poultry Science Association Inc.
ISSN
0032-5791
eISSN
1525-3171
D.O.I.
10.3382/ps/pey113
Publisher site
See Article on Publisher Site

Abstract

Abstract In the present study, the pathogenicity of fowl adenovirus (FAdV) serotype 4 strain HB1501 was systematically determined by using different doses and routes of inoculation in chickens and assessed the immune efficacy induced by fowl adenovirus serotype 4 (FAdV-4) strain SB15 in a vaccination trial. The results revealed that strain HB1501 was highly virulent in chickens, inflicting 30 to 100% mortality when administered by intramuscular and intravenous routes, and its pathogenicity was influenced markedly by the inoculation route. The inactivated oil-emulsion FAdV-4 vaccine derived from strain SB15 completely protected specific pathogen-free chickens against morbidity and mortality following infection with the virulent FAdV-4 strain HB1501. Importantly, no obvious clinical signs or gross lesions were observed and high antibody levels against FAdV were measured in the SB15-vaccinated chickens, indicating the potential of this strain to become an alternative vaccine candidate. This study provides detailed information on the pathogenicity of FAdV-4, which should be of great value to the control of hepatitis-hydropericardium syndrome. INTRODUCTION Hepatitis-hydropericardium syndrome (HHS) was first identified in Pakistan in 1987 (Anjum et al., 1989). As a severe disease in poultry, it is caused mainly by fowl adenovirus serotype 4 (FAdV-4), which is characterized by the accumulation of transparent or straw-colored fluid with aqueous or gelatinous consistency in the pericardial sac, nephritis and hepatitis, and has a high mortality rate usually ranging from 30 to 70% (Kim et al., 2008; Schachner et al., 2014). Fowl adenoviruses (FAdV) belong to the Aviadenovirus genus, which together with 4 other genera (Mastadenovirus, Atadenovirus, Siadenovirus, and Ichtadenovirus) are Adenoviridae family members. Fowl adenoviruses have been classified into 5 species (A to E) and further divided into 12 serotypes (FAdV 1 to 8a, and 8b to 11) (Hess, 2000). Besides HHS, FAdV causes a variety of diseases, such as inclusion body hepatitis and adenoviral gizzard erosion. Each of the 12 FAdV serotypes is associated with inclusion body hepatitis, but some FAdV-1 strains are responsible for adenoviral gizzard erosion (Ono et al., 2003). Fowl adenoviruses have been reported in many countries, such as the USA (Mendelson et al., 1995), Canada (Grgić et al., 2014; Schachner et al., 2014), Korea (Park et al., 2017), Poland (Niczyporuk, 2016), Japan (Mase et al., 2009), India (Singh et al., 2002), and Chile (Toro et al., 1999), and they have a severe economic impact on the global poultry industry. Furthermore, the number of HHS cases has increased since 2013 in China (Zhao et al., 2015), and the FAdV-4 strains have been mainly Chinese isolates (Li et al., 2016; Ye et al., 2016). Various vaccines such as inactivated liver homogenate, live or inactivated oil-emulsion vaccines, and new generation vaccines have been developed to control HHS in some countries (Afzal and Ahmad, 1990; Toro et al., 2002; Kim et al., 2014; Schachner et al., 2014), and these vaccines are efficient in protecting birds from FAdV-4. Two FAdV strains, SB15 and HB1501, were isolated from different flocks during HHS outbreaks in China in 2015. Both of them belong to FAdV-4 with a close genetic relationship. Here, the pathogenicity of HB1501 was systematically determined by inoculating chickens with this virus using different doses and inoculation routes. In the vaccination trial, the immune efficacy induced by strain SB15, which was plaque-purified 3 times in the chicken hepatocellular carcinoma cell line (LMH) and inactivated with formaldehyde, was assessed by challenging birds with the virulent HB1501 strain, and then evaluating them by clinical observation, clinical sign scores, gross autopsy, and serological methodologies. MATERIALS AND METHODS Animals and ethics statement Specific pathogen-free (SPF) white leghorn chickens were obtained from the Beijing Merial Vital Laboratory Animal Technology Co., Ltd, China. All the chickens were kept in isolators throughout the experiment at China Agricultural University. The experimental protocols and the housing facility were approved by the Animal Welfare and Ethical Censor Committee at China Agricultural University and Beijing Administration Committee of Laboratory Animals under the leadership of the Beijing Association for Science and Technology. The approval ID is XYXK (Jing) 2013–0013. Cells and Viruses The LMH cells were grown in Dulbecco's Modified Eagle's Medium/F12 (DMEM/F12) (ThermoFischer, NY, USA) containing 10% fetal bovine serum (FBS) (ThermoFischer) and 1% penicillin–streptomycin (Macgene, Beijing, China) at 37°C with 5% CO2. FAdV-4 SB15 and HB1501 strains were isolated from the liver samples of suspected FAdV-infected chickens collected in 2015. The highly virulent HB1501 isolate was used as the challenge strain to determine the immune efficacy induced by the inactivated FAdV SB15 vaccine. Two FAdV strains were cultured in LMH cells. The 2 strains were plaque-purified three times as described previously (Alexander et al., 1998) and then titrated by median tissue culture infective doses (TCID50) and plaque-forming units (PFU). The TCID50 was determined in 96-well titration plates, according to the endpoint method of Reed and Muench (1938). PFU was determined as described previously (Alexander et al., 1998). Cytopathic Effects (CPE) of FAdV in LMH Cells The chicken hepatocellular cells in 24-well plates were infected with SB15 or HB1501 at 106.0 TCID50, and then incubated in DMEM/F12 containing 1% FBS. The CPE of each FAdV strain in LMH cells was observed at 12 h intervals until 48 h post-infection (hpi). Uninfected cells served as the control. Optimal Harvest Time Determination The growth dynamics of the FAdV-4 strain SB15 was evaluated in LMH cells to determine the optimal harvest time. Cells in triplicate wells of 24-well plates were infected with the SB15 strain containing 106.0 TCID50, and then incubated in DMEM/F12 containing 1% FBS. The cells and culture supernatants were harvested at 8 h intervals until 64 hpi. The viral titers were quantified in LMH cells and expressed as TCID50. Pathogenicity Testing of FAdV-4 HB1501 in SPF Chickens Grouping and Viral Challenge One hundred 3-wk-old SPF chickens were randomly divided into 10 groups of 10 each and housed separately in isolators. Birds were inoculated with 0.2 mL of FAdV-4 HB1501 using different doses (107.0TCID50/bird, 106.0TCID50/bird, and 105.0TCID50/bird) and routes of inoculation (oral; intramuscular, I.M.; intravenous, I.V.) except for the control group (group 10) (Table 1). Table 1. Experimental design in pathogenicity test of FAdV strain HB1501. Challenge1 Observation and sampling Groups Virus Route2 Dose (TCID50/bird) Observation time (d) Serology3 1 HB1501 Oral 107.0 14 10 2 HB1501 Oral 106.0 14 10 3 HB1501 Oral 105.0 14 10 4 HB1501 I.M. 107.0 14 10 5 HB1501 I.M. 106.0 14 10 6 HB1501 I.M. 105.0 14 10 7 HB1501 I.V. 107.0 14 10 8 HB1501 I.V. 106.0 14 10 9 HB1501 I.V. 105.0 14 10 10 - - - 14 10 Challenge1 Observation and sampling Groups Virus Route2 Dose (TCID50/bird) Observation time (d) Serology3 1 HB1501 Oral 107.0 14 10 2 HB1501 Oral 106.0 14 10 3 HB1501 Oral 105.0 14 10 4 HB1501 I.M. 107.0 14 10 5 HB1501 I.M. 106.0 14 10 6 HB1501 I.M. 105.0 14 10 7 HB1501 I.V. 107.0 14 10 8 HB1501 I.V. 106.0 14 10 9 HB1501 I.V. 105.0 14 10 10 - - - 14 10 1Three-wk-old SPF chickens were infected with FAdV strain HB1501. 2I.M., intramuscular; I.V., intravenous. 3The sera were collected at 21 dpc in all survived chickens and tested with ELISA. View Large Table 1. Experimental design in pathogenicity test of FAdV strain HB1501. Challenge1 Observation and sampling Groups Virus Route2 Dose (TCID50/bird) Observation time (d) Serology3 1 HB1501 Oral 107.0 14 10 2 HB1501 Oral 106.0 14 10 3 HB1501 Oral 105.0 14 10 4 HB1501 I.M. 107.0 14 10 5 HB1501 I.M. 106.0 14 10 6 HB1501 I.M. 105.0 14 10 7 HB1501 I.V. 107.0 14 10 8 HB1501 I.V. 106.0 14 10 9 HB1501 I.V. 105.0 14 10 10 - - - 14 10 Challenge1 Observation and sampling Groups Virus Route2 Dose (TCID50/bird) Observation time (d) Serology3 1 HB1501 Oral 107.0 14 10 2 HB1501 Oral 106.0 14 10 3 HB1501 Oral 105.0 14 10 4 HB1501 I.M. 107.0 14 10 5 HB1501 I.M. 106.0 14 10 6 HB1501 I.M. 105.0 14 10 7 HB1501 I.V. 107.0 14 10 8 HB1501 I.V. 106.0 14 10 9 HB1501 I.V. 105.0 14 10 10 - - - 14 10 1Three-wk-old SPF chickens were infected with FAdV strain HB1501. 2I.M., intramuscular; I.V., intravenous. 3The sera were collected at 21 dpc in all survived chickens and tested with ELISA. View Large Clinical Observations and Sampling All the birds were monitored daily for clinical signs (e.g., depression, ruffled feathers, reluctance to move) or death for 14 d. The clinical signs were assessed as daily clinical scores: 0 for normal, 1 for mild depression, 2 for severe depression, 3 for paralysis/prostration, and 4 for death (Zhao et al., 2015). Serum samples were collected at 21 d post-inoculation (dpi) and antibody levels against FAdV-4 were determined by a recombinant fiber-2 protein-based indirect enzyme-linked immune-sorbent antibody assay (ELISA), whose cut-off value was 0.130 (OD450nm value). The optimal dilution level of the serum or secondary antibodies were 1:200 and 1:6,000, respectively. All the birds were then humanely euthanized and necropsied. Immune Efficacy Trial of FAdV strain SB15 Oil-Adjuvant Inactivated FAdV Vaccine Preparation The LMH-derived SB15 virus was inactivated with formaldehyde at 4°C at a final concentration of 0.1% for 24 h. Inactivated viral antigen was mixed with the oil emulsion adjuvant, Marcol 52 (Seppic, Paris, France), at a ratio of 1:1 (V/V). Chicken Immunizations Forty 1-wk-old SPF chickens were randomly divided into 4 groups of 10 each and housed separately in isolators. Two groups of chickens were inoculated subcutaneously with the SB15 vaccine at a dose of 106.5TCID50/bird or 107.5TCID50/bird. Another 20 birds served as the unvaccinated control group (Table 2). To confirm the antibody responses against the inactivated FAdV-4 vaccine, sera from the vaccinated chickens were collected at 21 dpi and the antibody levels were determined by ELISA. Table 2. Experimental design in immune efficacy trial of FAdV strain SB15. Inoculation1 Challenge2 Observation and sampling Groups Virus Route3 Dose (TCID50/bird) Virus Route4 Dose (TCID50/bird) Observation time (d) Serology5 1 SB15 S.C. 107.5 HB1501 I.V. 106.0 14 10 2 SB15 S.C. 106.5 HB1501 I.V. 106.0 14 10 3 - - - HB1501 I.V. 106.0 14 10 4 - - - - - - 14 10 Inoculation1 Challenge2 Observation and sampling Groups Virus Route3 Dose (TCID50/bird) Virus Route4 Dose (TCID50/bird) Observation time (d) Serology5 1 SB15 S.C. 107.5 HB1501 I.V. 106.0 14 10 2 SB15 S.C. 106.5 HB1501 I.V. 106.0 14 10 3 - - - HB1501 I.V. 106.0 14 10 4 - - - - - - 14 10 1One-wk-old SPF chickens were inoculated with FAdV strain SB15. 2The chickens were challenged at 21 dpi. 3S.C., subcutaneous. 4I.V., intravenous. 5Ten sera were collected at 21 dpi and 14 dpc and tested with ELISA. View Large Table 2. Experimental design in immune efficacy trial of FAdV strain SB15. Inoculation1 Challenge2 Observation and sampling Groups Virus Route3 Dose (TCID50/bird) Virus Route4 Dose (TCID50/bird) Observation time (d) Serology5 1 SB15 S.C. 107.5 HB1501 I.V. 106.0 14 10 2 SB15 S.C. 106.5 HB1501 I.V. 106.0 14 10 3 - - - HB1501 I.V. 106.0 14 10 4 - - - - - - 14 10 Inoculation1 Challenge2 Observation and sampling Groups Virus Route3 Dose (TCID50/bird) Virus Route4 Dose (TCID50/bird) Observation time (d) Serology5 1 SB15 S.C. 107.5 HB1501 I.V. 106.0 14 10 2 SB15 S.C. 106.5 HB1501 I.V. 106.0 14 10 3 - - - HB1501 I.V. 106.0 14 10 4 - - - - - - 14 10 1One-wk-old SPF chickens were inoculated with FAdV strain SB15. 2The chickens were challenged at 21 dpi. 3S.C., subcutaneous. 4I.V., intravenous. 5Ten sera were collected at 21 dpi and 14 dpc and tested with ELISA. View Large Challenge Tests The birds were challenged with 0.2 mL (106.0 TCID50/bird) of FAdV-4 HB1501 via the intravenous route at 21 dpi (Table 2). All the birds were monitored daily and scored for clinical signs for 14 d. The clinical signs were scored as previously described. Serum samples were collected on d 14 post challenge and antibody levels were determined by ELISA. All birds were euthanized and necropsied on d 14 post challenge. RESULTS Cytopathic Effects of FAdV in LMH Cells LMH cells are normally polygonal with a dendritic-like appearance. Uninfected cells served as the control and no CPE was observed with them. In cells infected with both FAdV strains, a visible CPE was observed at 24 hpi, as characterized by cell rounding and cell aggregation. When the incubation time was increased, the spacing between the cells widened and particles were observed in the cells at high magnification (400×). At 36 hpi, there was a severe CPE, and complete detachment of the cell monolayer was observed at 48 hpi. Virus Purification and Titration The 2 FAdV strains used in our study were plaque-purified 3 times and titrated by TCID50 and PFU. Both strains produced visible plaques 4 d after inoculation, which developed progressively to diameters of 1 to 3 mm within 8 dpi. The number of plaques decreased as the dilution increased. The TCID50 for HB1501 and SB15 were 108.0 TCID50/0.2 mL and 107.67 TCID50/0.2 mL, respectively. The PFU calculation for SB15 was 2.06 × 108.0 PFU/mL. Optimal Harvest Time Determination The growth dynamics of SB15 were evaluated in LMH cells at 8 h intervals until 64 hpi to determine the optimal harvest time. The values obtained represent averages of the results from 3 independent experiments (Figure 1). The virus titer showed an increasing trend, reached a maximum at 48 hpi, gradually declining thereafter. The optimal harvest time for maximizing the virus yield was determined at 32 to 48 hpi. Figure 1. View largeDownload slide Growth properties of FAdV-4 strain SB15 in LMH cells. The cells and culture supernatants were harvested at 8 h intervals until 64 hpi. The viral titers were quantified in LMH cells and expressed as TCID50. Values represent averages of the results from 3 independent experiments. The error bars show the standard deviations. Figure 1. View largeDownload slide Growth properties of FAdV-4 strain SB15 in LMH cells. The cells and culture supernatants were harvested at 8 h intervals until 64 hpi. The viral titers were quantified in LMH cells and expressed as TCID50. Values represent averages of the results from 3 independent experiments. The error bars show the standard deviations. Pathogenicity Testing of FAdV-4 HB1501 in SPF Chickens Clinical Signs No clinical signs or deaths were observed in the oral infection and control groups during the experiment. Chickens inoculated with HB1501 by the I.M. and I.V. route showed obvious clinical signs of depression, and also ruffled feathers, decreased feeding and water consumption, reluctance to move, paralysis, and death (Figure 2A). Figure 2. View largeDownload slide Clinical scores (A) and survival rates (B) in chickens experimentally challenged with FAdV-4 strain HB1501. (A) Clinical scoring: 0 for normal, 1 for mild depression, 2 for severe depression, 3 for paralysis/prostration, and 4 for death. The mean scores per group per day are shown. The error bars show the standard deviations. (B) Birds were infected with 0.2 mL of strain HB1501 by different doses and routes except for the control group. Figure 2. View largeDownload slide Clinical scores (A) and survival rates (B) in chickens experimentally challenged with FAdV-4 strain HB1501. (A) Clinical scoring: 0 for normal, 1 for mild depression, 2 for severe depression, 3 for paralysis/prostration, and 4 for death. The mean scores per group per day are shown. The error bars show the standard deviations. (B) Birds were infected with 0.2 mL of strain HB1501 by different doses and routes except for the control group. In group 4 (I.M., 107.0TCID50/bird), the first clinical signs of the disease were observed at 2 dpi. Mortality started at 3 dpi, and at 5 dpi all the animals were found dead (Figure 2B). The mortality rate was 100%. In group 5 (I.M., 106.0TCID50/bird), the first clinical signs of the disease were observed at 3 dpi. Mortality started at 3 dpi, 8 birds died in all, and the mortality rate reached 80%. The surviving birds gradually recovered from 6 dpi. In group 6 (I.M., 105.0TCID50/bird), the first clinical signs of the disease were observed at 3 dpi. Mortality started at 4 dpi, 7 birds died in all, and the mortality rate was 70%. The surviving birds gradually recovered from 10 dpi. Similar results were found in the I.V. groups. Mortality was observed between 2 and 7 dpi, when 10, 10, and 3 birds from groups 7, 8, and 9 died, respectively. The mortality rates for groups 7, 8, and 9 reached 100, 100, and 30%, respectively. Gross Lesions Obvious lesions were observed in all the dead birds at necropsy, including hydropericardium, swollen and friable liver with multi focal areas of necrosis and petechial hemorrhage, and swollen and pale kidneys. No obvious lesions were observed in the oral infection groups or in any birds that recovered from infection with HB1501. No gross lesions were identified in any control group birds. Antibody Responses Sera were collected at 21 dpi from all the surviving chickens and tested by ELISA. Most of the birds inoculated with HB1501 showed positive antibody responses, regardless of the infection routes and doses (Figure 3). In groups 1, 2, and 3, the antibody response against FAdV was detected in most of the surviving birds, with positivity rates of 90, 50, and 90%, respectively. In groups 5 and 6, the antibody response against FAdV was detected in all the surviving birds, with positivity rates of 100 and 100%, respectively. In group 7, the antibody response against FAdV was detected in all the surviving birds, with a positivity rate of 100%. Chickens infected by the I.V. route had higher titers than those in the other groups. The antibody response against FAdV was not detected in any control group birds. Figure 3. View largeDownload slide The antibody response against FAdV in chickens experimentally challenged with FAdV-4 strain HB1501. The sera from the surviving birds were collected at 21 dpi and tested by ELISA. The antibody titers of the individual birds are shown by black circles, and short lines represent the averages of each group. Cut-off titer = 0.130. Figure 3. View largeDownload slide The antibody response against FAdV in chickens experimentally challenged with FAdV-4 strain HB1501. The sera from the surviving birds were collected at 21 dpi and tested by ELISA. The antibody titers of the individual birds are shown by black circles, and short lines represent the averages of each group. Cut-off titer = 0.130. Immune Efficacy Trial of FAdV SB15 Clinical Signs No clinical signs or deaths attributable to FAdV-4 were identified in any birds in the control group or the SB15-vaccinated groups during the experiment. Birds in the unvaccinated group challenged with strain HB1501 showed clinical signs of depression, ruffled feathers, and reluctance to move as early as 2 dpi, (Figure 4A). The mean scores per group per day for the clinical signs in the challenge group were much higher than those of the vaccination groups and the control group. Mortality was observed between 3 and 5 dpi, when 6 birds died, and the mortality rate reached 60% (Figure 4B). The surviving birds gradually recovered from 7 dpi. Figure 4. View largeDownload slide Clinical scores (A), survival rates (B), and antibody response against FAdV (C) in vaccinated SPF chickens following HB1501 challenge. (A) Clinical scoring: 0 for normal, 1 for mild depression, 2 for severe depression, 3 for paralysis/prostration, and 4 for death. The mean scores per group per day are shown. The error bars show the standard deviations. (B) Birds were inoculated with strain SB15 and challenged with strain HB1501 via I.V. routes at 21 dpi. (C) Sera, collected at 21 dpi and 10 dpc, were tested by ELISA. The antibody titers of the individual birds are shown. Cut-off titer = 0.130. Figure 4. View largeDownload slide Clinical scores (A), survival rates (B), and antibody response against FAdV (C) in vaccinated SPF chickens following HB1501 challenge. (A) Clinical scoring: 0 for normal, 1 for mild depression, 2 for severe depression, 3 for paralysis/prostration, and 4 for death. The mean scores per group per day are shown. The error bars show the standard deviations. (B) Birds were inoculated with strain SB15 and challenged with strain HB1501 via I.V. routes at 21 dpi. (C) Sera, collected at 21 dpi and 10 dpc, were tested by ELISA. The antibody titers of the individual birds are shown. Cut-off titer = 0.130. Gross Lesions No gross lesions were identified in any control group birds or the SB15 vaccinated groups during the experiment. Obvious lesions were observed in all the dead birds in the unvaccinated group challenged with HB1501, including hydropericardium, swollen and friable liver with multifocal areas of necrosis and petechial hemorrhage, and swollen and pale kidneys. These results suggest that both vaccination doses (107.5TCID50/bird, 106.5TCID50/bird) provided complete protection against HB1501. Antibody Response Sera were collected at 21 dpi and 14 d post-challenge (dpc) and tested by ELISA (Figure 4C). All birds inoculated with SB15 showed positive antibody responses at 21 dpi. Birds vaccinated with higher doses of the virus had higher titers than those in the low dose group. The mean ELISA titers for birds in the vaccination groups that received 107.5TCID50/bird or 106.5TCID50/bird were 1.14 and 0.57 at 21 dpi, respectively. Furthermore, the values increased to 1.31 and 1.33 at 14 dpc, respectively. The mean ELISA titer for the surviving birds in the unvaccinated challenge group was 1.28. Antibody responses against FAdV were not detected in any control group birds. DISCUSSION Hepatitis-hydropericardium syndrome, the most severe disease induced by FAdV infection, has significant economic impact on the global poultry industry (Toro et al., 1999; Singh et al., 2002; Mase et al., 2009; Grgić et al., 2014; Schachner et al., 2014; Niczyporuk, 2016; Park et al., 2017). Various vaccines have been made to control HHS (Afzal and Ahmad, 1990; Toro et al., 2002; Kim et al., 2014; Schachner et al., 2014). Two FAdV-4 strains, SB15 and HB1501, were isolated from HHS-affected flocks during FAdV outbreaks in China in 2015. Here, the pathogenecity of HB1501 was systematically determined by different doses and routes of inoculation in chickens. We then further assessed the immune efficacy of SB15 with a challenge-protection study. LMH is a continuous cell line appropriate for the growth of FAdV (Alexander et al., 1998), and is more convenient for research than primary chicken embryo liver cells and chick kidney cells, as they are fairly tedious to prepare. Therefore, the two FAdV strains used in this study were adapted to growth in LMH cells. With both strains, visible CPE characterized by cell rounding and cell aggregation was observed. Granules were observed in the cells at high magnification (400×), which is consistent with the description for primary chick kidney cell and primary chicken embryo liver cell cultures (Adair et al., 1979; Li et al., 2016). Our results support many others detailing that most FAdV strains can be propagated efficiently in LMH cells (Alexander et al., 1998; Grgić et al., 2011). The pathogenicity of the FAdV-4 HB1501 was systematically determined using different doses and routes of inoculation. These data revealed that HB1501 was highly virulent in SPF chickens infected by I.M. and I.V. routes, inflicting 30–100% mortality. Obvious clinical signs and gross lesions were observed, and those caused by HB1501 were identical to those described in previous studies of pathogenicity for an isolate of FAdV-4 (Zhao et al., 2015; Niu et al., 2016). It should be noted that the pathogenicity of HB1501 was markedly influenced by the route of inoculation. When the virus was inoculated orally, which is the natural route for infection with FAdV, HB1501 did not display pathogenicity, and no obvious clinical signs, deaths, or gross lesions were observed; however, it was highly virulent when inoculated by the I.M. and I.V. routes. Similar results were observed in several previous studies on FAdV-4 pathogenicity (Reece et al., 1987; Ojkic and Nagy, 2003). Additionally, we showed that increasing the dose of the virus accelerated the disease course and severity. The mortality rate also gradually increased, reaching 100% when the injection dose was higher than 107.0TCID50 by the I.M. route or 106.0TCID50 by the I.V. route. To the best of our knowledge, this is the first study to systematically compare the influence of three different routes (oral, I.M. and I.V.) and doses of inoculation on the pathogenicity of a strain of FAdV. To confirm that the infection was effectively established, serum samples were collected for detection of FAdV antibodies during pathogenicity testing of HB1501. Our results showed that most of the birds inoculated with HB1501 tested positive for FAdV antibodies at 21 dpi, except for a small number of chickens inoculated by the oral route. It was found that chickens infected by the I.V. route had higher ELISA titers than those in the other groups in the pathogenicity test of strain HB1501, which was identical to those described in previous studies (Schonewille et al., 2010; Grgić et al., 2011). The reason might be that natural route of viral infection may be unable to ensure that all birds receive the same amount of virus (Grgić et al., 2011). In this study, the I.V. route (106.0TCID50/bird) was chosen for challenge infections with strain HB1501 in the immune efficacy trial, and the results were consistent with a study on the evaluation of protection efficacy induced by an inactivated oil-emulsion FAdV-4 vaccine (Kim et al., 2014). Many studies have been conducted with the aim of protecting birds from infection with FAdV-4 in some countries (Mansoor et al., 2011; Kim et al., 2014). Broiler chickens free from maternal antibodies against FAdV were immunized with a chicken embryo-adapted FAdV-4 isolate, which was attenuated by serially passaging, and the vaccination gave 94.73% protection (Mansoor et al., 2011). A vaccine containing inactivated oil-emulsion FAdV-4 was shown to provide broad cross-protection against various FAdV serotypes (Kim et al., 2014). In this study, the immune efficacy of SB15 was assessed using a challenge-protection approach. Our results suggest that SB15 completely protected SPF birds against morbidity and mortality when challenged with the virulent FAdV-4 HB1501 strain via the I.V. route. Neither clinical signs nor gross lesions at necropsy were observed and the antibody levels of the vaccinated chickens were high. These results indicate that this strain can be used in vaccination procedures to reduce the economic losses caused by FAdV-4 infections. The mechanism of FAdV infection is very complex in chickens (Li et al., 2016). It has been suggested that there is a synergism between FAdV and chicken infectious anemia virus or other viruses (Toro et al., 2000). Therefore, besides preventing FAdV infections, vaccination and good animal management practices for the control of infectious immunosuppressive agents such as infectious bursal disease virus and chicken infectious anemia virus are necessary (Shivachandra et al., 2003; Balamurugan amd Kataria, 2006). In conclusion, our data confirm that FAdV-4 HB1501 is highly virulent in chickens and its pathogenicity was markedly influenced by the route of inoculation. FAdV-4 SB15 provided effective protection against challenge with HB1501, indicating its potential as an alternative vaccine candidate for controlling FAdV-4 infections and HHS in China. Acknowledgements This study was supported by the National Key Research and Development Program of China (2017YFD0500700). REFERENCES Adair B. M. , Curran W. L. , Mcferran J. B. . 1979 . Ultrastructural studies of the replication of fowl adenoviruses in primary cell cultures . Avian Pathol. 8 : 133 – 144 . Google Scholar CrossRef Search ADS PubMed Afzal M. , Ahmad I. . 1990 . Efficacy of an inactivated vaccine against hydropericardium syndrome in broilers . Vet. Rec. 126 : 59 – 60 . Google Scholar PubMed Alexander H. S. , Huber P. , Cao J. , Krell P. J. , Nagy E. . 1998 . Growth characteristics of fowl adenovirus type 8 in a chicken hepatoma cell line . J. Virol. Methods 74 : 9 – 14 . Google Scholar CrossRef Search ADS PubMed Anjum A. D. , Sabri M. A. , Iqbal Z. . 1989 . Hydropericarditis syndrome in broiler chickens in Pakistan . Vet. Rec. 124 : 247 – 248 . Google Scholar CrossRef Search ADS PubMed Balamurugan V. , Kataria J. M. . 2006 . Economically important non-oncogenic immunosuppressive viral diseases of chicken-current status . Vet. Res. Commun. 5 : 541 – 566 . Google Scholar CrossRef Search ADS Grgić H. , Krell P. J. , Nagy E. . 2014 . Comparison of fiber gene sequences of inclusion body hepatitis (IBH) and non-IBH strains of serotype 8 and 11 fowl adenoviruses . Virus Genes 48 : 74 – 80 . Google Scholar CrossRef Search ADS PubMed Grgić H. , Yang D. H. , Nagy E. . 2011 . Pathogenicity and complete genome sequence of a fowl adenovirus serotype 8 isolate . Virus Res. 156 : 91 – 97 . Google Scholar CrossRef Search ADS PubMed Hess M. 2000 . Detection and differentiation of avian adenoviruses: a review . Avian Pathol. 29 : 195 – 206 . Google Scholar CrossRef Search ADS PubMed Kim J. N. , Byun S. H. , Min J. K. , Kim J. J. , Sung H. W. , Mo I. P. . 2008 . Outbreaks of hydropericardium syndrome and molecular characterization of Korean fowl adenoviral isolates . Avian Dis. 52 : 526 – 530 . Google Scholar CrossRef Search ADS PubMed Kim M. S. , Lim T. H. , Lee D. H. , Youn H. N. , Yuk S. S. , Kim B. Y. , Choi S. W. , Jung C. H. , Han J. H. , Song C. S. . 2014 . An inactivated oil-emulsion fowl adenovirus serotype 4 vaccine provides broad cross-protection against various serotypes of fowl adenovirus . Vaccine 32 : 3564 – 3568 . Google Scholar CrossRef Search ADS PubMed Li H. , Wang J. , Qiu L. , Han Z. , Liu S. . 2016 . Fowl adenovirus species c serotype 4 is attributed to the emergence of hepatitis-hydropericardium syndrome in chickens in China . Infect. Genet. Evol. 45 : 230 – 241 . Google Scholar CrossRef Search ADS PubMed Mansoor M. K. , Hussain I. , Arshad M. , Muhammad G. . 2011 . Preparation and evaluation of chicken embryo-adapted fowl adenovirus serotype 4 vaccine in broiler chickens . Trop. Anim. Health Prod. 43 : 331 – 338 . Google Scholar CrossRef Search ADS PubMed Mase M. , Chuujou M. , Inoue T. , Nakamura K. , Yamaguchi S. , Imada T. . 2009 . Genetic characterization of fowl adenoviruses isolated from chickens with hydropericardium syndrome in Japan . J. Vet. Med. Sci. 71 : 1455 – 1458 . Google Scholar CrossRef Search ADS PubMed Mendelson C. , Nothelfer H. B. , Monreal G. . 1995 . Identification and characterization of an avian adenovirus isolated from a -spiking mortality syndrome- field outbreak in broilers on the Delmarva Peninsula, USA . Avian Pathol. 24 : 693 – 706 . Google Scholar CrossRef Search ADS PubMed Niczyporuk J. S. 2016 . Phylogenetic and geographic analysis of fowl adenovirus field strains isolated from poultry in Poland . Arch. Virol. 161 : 33 – 42 . Google Scholar CrossRef Search ADS PubMed Niu Y. J. , Sun W. , Zhang G. H. , Qu Y. J. , Wang P. F. , Sun H. L. , Xiao Y. H. , Liu S. D. . 2016 . Hydropericardium syndrome outbreak caused by fowl adenovirus serotype 4 in China in 2015 . J. Gen. Virol. 97 : 2684 – 2690 . Google Scholar CrossRef Search ADS PubMed Ojkic D. , Nagy E. . 2003 . Antibody response and virus tissue distribution in chickens inoculated with wild-type and recombinant fowl adenoviruses . Vaccine 22 : 42 – 48 . Google Scholar CrossRef Search ADS PubMed Ono M. , Okuda Y. , Yazawa S. , Imai Y. , Shibata I. , Sato S. , Okada K. . 2003 . Adenoviral gizzard erosion in commercial broiler chickens . Vet. Pathol. 40 : 294 – 303 . Google Scholar CrossRef Search ADS PubMed Park H. S. , Lim I. S. , Kim S. K. , Kim T. K. , Park C. K. , Yeo S. G. . 2017 . Molecular analysis of the hexon, penton base, and fiber-2 genes of Korean fowl adenovirus serotype 4 isolates from hydropericardium syndrome-affected chickens . Virus Genes 53 : 111 – 116 . Google Scholar CrossRef Search ADS PubMed Reece R. L. , Barr D. A. , Grix D. C. . 1987 . Pathogenicity studies with a strain of fowl adenovirus serotype 8 (vri-33) in chickens . Australian Vet J 64 : 365 – 367 . Google Scholar CrossRef Search ADS Reed L. J. , Muench H. . 1938 . A simple method of estimating fifty per cent endpoints . Am. J. Epidemiol. 27 : 493 – 497 . Google Scholar CrossRef Search ADS Schachner A. , Marek A. , Jaskulska B. , Bilic I. , Hess M. . 2014 . Recombinant FAdV-4 fiber-2 protein protects chickens against hepatitis-hydropericardium syndrome (HHS) . Vaccine 32 : 1086 – 1092 . Google Scholar CrossRef Search ADS PubMed Schonewille E. , Jaspers R. , Paul G. , Hess M. . 2010 . Specific-pathogen-free chickens vaccinated with a live FAdV-4 vaccine are fully protected against a severe challenge even in the absence of neutralizing antibodies . Avian Dis . 54 : 905 – 910 . Google Scholar CrossRef Search ADS PubMed Shivachandra S. B. , Sah R. L. , Singh S. D. , Kataria J. M. , Manimaran K. . 2003 . Immunosuppression in broiler chicks fed aflatoxin and inoculated with fowl adenovirus serotype-4 (FAV-4) associated with hydropericardium syndrome . Vet. Res. Commun. 27 : 39 – 51 . Google Scholar CrossRef Search ADS PubMed Singh A. , Oberoi M. S. , Grewal G. S. , Hafez H. M. , Hess M. . 2002 . The use of PCR combined with restriction enzyme analysis to characterize fowl adenovirus field isolates from northern India . Vet. Res. Commun. 26 : 577 – 585 . Google Scholar CrossRef Search ADS PubMed Toro H. , Gonzalez C. , Cerda L. , Hess M. , Reyes E. , Geisse C. . 2000 . Chicken anemia virus and fowl adenoviruses: association to induce the inclusion body hepatitis/hydropericardium syndrome . Avian Dis. 44 : 51 – 58 . Google Scholar CrossRef Search ADS PubMed Toro H. , González C. , Cerda L. , Morales M. A. , Dooner P. , Salamero M. . 2002 . Prevention of inclusion body hepatitis/hydropericardium syndrome in progeny chickens by vaccination of breeders with fowl adenovirus and chicken anemia virus . Avian Dis. 46 : 547 – 554 . Google Scholar CrossRef Search ADS PubMed Toro H. , Prusas C. , Raue R. , Cerda L. , Geisse C. , González C. , Hess M. . 1999 . Characterization of fowl adenoviruses from outbreaks of inclusion body hepatitis/hydropericardium syndrome in Chile . Avian Dis. 43 : 262 – 270 . Google Scholar CrossRef Search ADS PubMed Ye J. , Liang G. , Zhang J. , Wang W. , Song N. , Wang P. , Zheng W. , Xie Q. , Shao H. , Wan Z. , Wang C. , Chen H. , Gao W. , Qin A. . 2016 . Outbreaks of serotype 4 fowl adenovirus with novel genotype, China , Emerg Microbes Infect 5 : e50 – e50 . Google Scholar CrossRef Search ADS PubMed Zhao J. , Zhong Q. , Zhao Y. , Hu Y. X. , Zhang G. Z. . 2015 . Pathogenicity and complete genome characterization of fowl adenoviruses isolated from chickens associated with inclusion body hepatitis and hydropericardium syndrome in China . PLoS One 10 : e0133073 . Google Scholar CrossRef Search ADS PubMed © 2018 Poultry Science Association Inc. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

Poultry ScienceOxford University Press

Published: Jul 11, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off