Detection of reticuloendotheliosis virus as a contaminant of fowl pox vaccines

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1 Detection of reticuloendotheliosis virus as a contaminant of fowl pox vaccines A. M. Awad,* 1 H. S. Abd El-Hamid, A. A. Abou Rawash,* and H. H. Ibrahim * Department of Avian and Aquatic Animal Medicine, Faculty of Veterinary Medicine, Alexandria University, Behera, Egypt 22758; Department of Avian and Aquatic Animal Medicine, Faculty of Veterinary Medicine, Alexandria University (Damanhour Branch), Behera, Egypt 58012; and Center Laboratory for Evaluation of Veterinary Biologics, Abbasia, Cairo, Egypt ABSTRACT This study was designed to detect reticuloendotheliosis virus (REV) as a contaminant in fowl pox vaccines. A total of 30 fowl pox vaccine samples were examined for the presence of REV using both in vitro and in vivo methods. In in vitro testing, the fowl pox vaccine samples were inoculated into chicken embryo fibroblast cultures prepared from specific-pathogen-free embryonated chicken eggs, and the cultures were examined using PCR to detect REV. In in vivo testing, each fowl pox vaccine sample was inoculated into 5-dold specific-pathogen-free chicks, which were kept under observation for up to 12 wk postinoculation; serum samples were collected at 15, 30, and 45 d postinoculation for the detection of REV-specific antibodies using ELISA. Tissue samples were collected at 8 and 12 wk postinoculation for histopathological examination. Of the tested vaccines, only one imported vaccine sample tested positive for REV using PCR. Serum samples collected from chicks infected with the PCR-positive vaccine batch also tested positive for REV-specific antibodies using ELISA. Histopathological examination of the liver, spleen, and bursa of Fabricius demonstrated the presence of tumor cells in these organs, confirming the results obtained using PCR and ELISA, and indicating that the sample was contaminated with REV. These data clearly indicate that the screening of all commercial poultry vaccines for viruses is an important factor in assuring the biosafety of animal vaccines. Key words: poultry, reticuloendotheliosis virus, fowl pox vaccine, polymerase chain reaction, ethylenediaminetetraacetate INTRODUCTION 2010 Poultry Science Association Inc. Received May 17, Accepted July 28, Corresponding author: ashrafawad_1100@yahoo.com 2010 Poultry Science 89 : doi: /ps Poultry pathogens constitute one of the major problems facing the poultry industry today and cause severe economic losses. For this reason, vaccinations are a major component of programs that are used to control these economically important diseases. Therefore, poultry vaccinations must be safe, potent, and free from any biological contaminants, such as bacterial, fungal, or viral pathogens. Vaccines are either locally produced or imported. In Egypt, approximately 90% of the vaccines required for the poultry industry are imported from different producers in Europe and the United States. Over the last few years, reticuloendotheliosis virus (REV) has been considered one of the most important vaccine contaminants (Fadly et al., 1996). Reticuloendotheliosis virus is an oncogenic avian retrovirus that is antigenically and structurally unrelated to viruses of the leucosis-sarcoma group. Reticuloendotheliosis virus is associated with immunosuppression, runting, and neoplasia in domestic poultry as well as other avian species (Dren et al., 1988; Witter, 1991; Witter and Fadly, 2003). Furthermore, Nicholas and Thornton (1983) suggested that REV is a potential hazard in the use of chicken embryos and cells for preparation of vaccines, and REV infection can persist at the same production site over a period of several years (Bagust, 1993). Reticuloendotheliosis virus has been isolated from Marek s disease vaccines (Yuasa et al., 1976). Jackson et al. (1977) reported a high mortality rate, neurological symptoms, and feathering abnormalities (nakanuke) in chickens vaccinated with a contaminated Marek s disease vaccine. These cases were attributed to REV, which was detected as a vaccine contaminant in one commercial vaccine batch. Bagust and Dennett (1977) isolated REV from a commercial Marek s disease vaccine (herpesvirus of turkeys) by serial passage of the REV-contaminated vaccine on chicken embryo fibroblast (CEF) and detected REV antigen using a fluorescence antibody test. In the Middle East and Africa, REV-associated lymphoma, not related to a contaminated vaccine, has been reported (Meroz, 1992; Okoye et al., 1993). On the other hand, in the United States, Fadly et al. (1996) reported an outbreak of lymphoma 2389

2 2390 in 2 broiler breeder flocks after the use of a REV-contaminated fowl pox vaccine. The insertion of the REV sequence into the fowl pox virus genome, in both field isolates and vaccine strains, has increased recently (Diallo et al., 1998; García et al., 2003) and fowl pox virus infection can cause a lymphoproliferative response in chickens. Also, the REV provirus is present in the majority of the genomes from field fowl pox virus isolates (Singh et al., 2003, 2005). Moreover, current strains of fowl pox virus that carry REV sequences are becoming more pathogenic to poultry (Tadese et al., 2008). In this study, we aimed to screen locally produced and imported fowl pox vaccines used in poultry production for the presence of REV. We isolated REV from fowl pox vaccine-inoculated CEF and used PCR to detect REV in specific-pathogen-free (SPF) chickens. Reticuloendotheliosis virus was also detected using an ELISA to detect REV-specific antibodies from infected SPF chickens and histopathological examination. MATERIALS AND METHODS Vaccine Samples Different batches of fowl pox vaccines, either imported or locally produced, were examined for REV contamination. A total of 30 fowl pox vaccine batches (4 batches were locally produced and 26 batches were imported) were examined. Each vaccine batch was aliquoted into 6 vials: 2 were pooled, propagated on CEF, and examined by PCR; 2 were pooled and inoculated in SPF chicks; and 2 vials were kept at 4 C for future studies. SPF Fertile Eggs and Chickens A total of 250 SPF fertile chicken eggs were obtained from a SPF farm in Egypt. Twenty chicken embryos, 9 to 11 d old, were used for the preparation of CEF culture, whereas the remainder were kept in the incubator until hatching. A total of 200, five-day-old SPF chickens were used for the experimental infection. Tissue Culture Media Dehydrated Eagle s minimum essential medium (MEM; Sigma, St. Louis, MO) with Eagle s salts and l-glutamine but without sodium bicarbonate was reconstituted in double-distilled water and used for tissue culture. Heat-inactivated fetal calf serum was added at a concentration of 5% for growth medium and 2% in maintenance medium. The medium was supplemented with antibiotics (1,000 IU of penicillin and 500 µg of streptomycin) before use. Testing of Vaccines for REV Contamination by Tissue Culture Inoculation Awad et al. Preparation of the Inoculum. The preparation of the fowl pox vaccine samples for detection of REV contamination using tissue culture was carried out according to Fadly et al. (1996). The fowl pox vaccine samples were reconstituted in 10 ml of MEM, and 10-fold serial dilutions were made until the vaccines reached a dilution that contained 10 bird doses/ml. Inoculation of the Prepared Inocula. The culture medium was aspirated from CEF cultures in 6-well tissue culture plates, and the prepared vaccines were inoculated onto the CEF. The cultures were incubated for 30 min at 37 C to allow adsorption. One uninoculated well was included as a negative control. Maintenance medium (with 2% calf serum) was added to all wells, and the plates were incubated at 37 C in a 5% CO 2 incubator for 4 d with daily observation. Harvesting the Inoculated Cultures. Four days postinoculation, the plates were subjected to 3 freethaw cycles. The cell lysates were harvested in small vials and kept at 70 C for the detection of REV using PCR. PCR for REV Detection Fowl Pox Virus Genome Extraction. Deoxyribonucleic acid was extracted according to the manufacturer (Biobasic, Amherst, NY). Briefly, 0.8 ml of tributyl phosphate buffer and 3 μl of proteinase K (0.1%) were added to an aliquot (0.5 ml) of each thawed sample. The mixture was vortexed and allowed to digest at 55 C for 30 min. The mixture was applied to an EZ-10 column (Bio Basic, Markham, Ontario, Canada), which was centrifuged (2,290 g) for 1 min. The flow-through was discarded to remove any residual amount of wash solution. Deoxyribonucleic acid was eluted from the column in 30 to 50 µl of elution buffer by centrifugation (3,575 g) for 1 min. PCR Amplification. The detection of REV using PCR was performed as recommend by the manufacturer (Fermentas, Glen Burnie, MD). Each PCR reaction consisted of 50 μl of nuclease-free water, 25 μl of 2 PCR master mix, 0.1 μm of forward primer (5 -CATACTG- GAGCCAATGGTGTAAAGGGCAGA-3 ), 0.1 μm of reverse primer (5 -AATGTTGTAGCGAAGTACT-3 ), and 10 pg to 1 µg of template DNA. The thermocycling parameters were 94 C for 2 min; 39 cycles at 94 C for 1 min, 55 C for 2 min, and 72 C for 1 min; and 72 C for 6 min (Aly et al., 1993). Testing of Fowl Pox Vaccines for REV Contamination by Chicken Inoculation Preparation of the Inoculum. The fowl pox vaccine sample was reconstituted in MEM. It was reconstituted at a concentration of 100 doses/ml (Fadly and Witter, 1997). Inoculation of Chickens and Blood Collection. The diluted vaccine samples were subcutaneously inoculated into five 5-d-old SPF chicks (0.2 ml of the inoculum per chick). The chicks were kept in an isolator for 45 d postinoculation. A negative control group of

3 DETECTION OF RETICULOENDOTHELIOSIS VIRUS 2391 Figure 1. Detection of proviral reticuloendotheliosis virus sequences in DNA of different fowl pox vaccine samples. A 291-bp fragment was detected in lane 3. Lane 1 = negative control cells; lane 2 = sample 21; lane 3 = sample 22; lane 4 = sample 23; lane 5 = sample 24; lane 6 = sample 25; lane 7 = sample 26; lane 8 = molecular size marker. Color version available in the online PDF. chicks was also inoculated subcutaneously with sterile saline and kept parallel to the inoculated group. After 30 and 45 d postinoculation, blood samples were collected from surviving birds. The serum was separated, inactivated by heating at 56 C for 30 min, and kept at 20 C until testing. Commercial ELISA kits (IDEXX Laboratories, Westbrook, ME) were used for detection of REV-specific antibodies in these samples, as recommended by the manufacturer. In Vivo REV Detection. Fowl pox vaccine 22, which produced positive results in the PCR and ELISA assays, was retested in vivo. Before the inoculation of the chicks, the vaccine sample was reconstituted in MEM at a concentration of 100 doses/ml. Fifteen 5-d-old SPF chicks were used in this experiment: 10 chicks were inoculated subcutaneously with 0.2 ml/bird (100 doses of fowl pox vaccine), and 5 chicks were inoculated with saline as a negative control. All chicks were kept for 12 wk postinoculation. Individual serum samples were obtained at 15, 30, and 45 d postinoculation and tested for REV-specific antibodies as described previously. Histopathological Examination. At 8 and 12 wk postinoculation, 5 birds from fowlpox vaccine 22-inoculated and negative control groups were killed and tissue specimens were collected from the liver, spleen, and bursa of Fabricius for histopathological examination. The tissue specimens were immediately fixed in 10% neutral-buffered formalin solution for at least 24 h. The fixed specimens were washed in tap water, processed in ascending concentrations of ethyl alcohol for dehydration, and cleared in xylol and paraffin-embedded. Hematoxylin and eosin staining was carried out on 4- to 5-µm-thick sections, and stained sections were examined with a light microscope (Banchroft and Stevens, 1996). RESULTS Testing of Vaccine for REV Contamination In Vitro Assay. The inoculated SPF cultures were observed daily for 4 d, and then the cultures were subjected to 3 freeze-thaw cycles. Polymerase chain reaction was used for the detection of REV in the CEF cultures inoculated with the vaccines. As shown in Figure 1, a product of 291 bp was obtained from vaccine sample 22, whereas REV was not detected in the other vaccine samples. In Vivo Assay. The inoculated birds were kept under daily observation for 7 wk postinoculation, and blood samples were collected at both 30 and 45 d postinoculation. As seen in Tables 1, 2, and 3, one group of chickens (vaccine 22) was positive for REV-specific antibodies at both 30 and 45 d postinoculation, whereas the remaining groups were negative for REV-specific antibodies at both 30 and 45 d postinoculation. Table 1. Results of ELISA test for detection of reticuloendotheliosis virus (REV) antibodies in sera of specific-pathogen-free chicks 45 d postinoculation with fowl pox vaccine samples from no. 1 to 10 1 Chick group (n = 5) Mean of optical density S:P ratio 2 No. positive/ no. examined Interpretation /5 Negative /5 Negative /5 Negative /5 Negative /5 Negative /5 Negative /5 Negative /5 Negative /5 Negative /5 Negative Uninoculated chicks /5 Negative 1 Negative control mean = 0.052; positive control mean = 0.53; corrected positive control = S:P ratio = sample mean/correct positive mean (positive control mean negative control mean). S:P ratio more than 0.5 was considered positive for REV antibodies.

4 2392 Awad et al. Table 2. Results of ELISA test for detection of reticuloendotheliosis virus (REV) antibodies in sera of specific-pathogen-free chicks 45 d postinoculation with fowl pox vaccine samples from no. 11 to 20 1 Chick group (n = 5) Mean of optical No. positive/ density S:P ratio 2 no. examined Interpretation /5 Negative /5 Negative /5 Negative /5 Negative /5 Negative /5 Negative /5 Negative /5 Negative /5 Negative /5 Negative Uninoculated chicks /5 Negative 1 Negative control mean = 0.039; positive control mean = 0.42; corrected positive control = S:P ratio = sample mean/correct positive mean (positive control mean negative control mean). S:P ratio more than 0.5 was considered positive for REV antibodies. Histopathological Examination Liver. Histopathological examination of the liver revealed marked atrophy of hepatocytes as a result of pronounced congestion of the hepatic sinusoids after 8 and 12 wk postinoculation. Small foci of lymphoblast infiltration were detected 12 wk postinoculation (Figure 2B and 2C). Spleen. Histopathologic examination of the spleen at 8 wk postinoculation revealed ill-defined multiple focal proliferations of lymphocytic-histocytic cells with hyperchromatic nuclei and moderate to abundant, slightly basophilic cytoplasm. The cells were located close to arterioles (Figure 3B). After 12 wk, the spleen had diffuse focal proliferation of lymphocytic-histocytic cells. The cells were pleomorphic, ranging from small and rounded ovoid to large lymphoblast cells with a vesicular nucleus. Some elongated and degenerated cells were also observed. Neoplastic cells with a large round-tooval vesicular nucleus and prominent chromatin clumps adjacent to the inner surface of the nuclear envelope were detected (Figure 3B and 3C). Bursa of Fabricius. Eight weeks postinoculation, the bursa of Fabricius showed mild, ill-defined aggregations of lymphoid cells, which were difficult to distinguish from normal bursal lymphoid cells. The cells were inter- and intrafollicularly distributed (Figure 4B). At 12 wk postinoculation, the bursa showed marked atrophy with moderate inter- and intrafollicular proliferation of lymphoid cells (lymphoreticular cells; Figure 4C). The cells had the same morphology as described in the spleen, and many of the cells showed degeneration. DISCUSSION In the present study, 30 batches of fowl pox virus vaccines were collected from different sources (locally and imported) and were screened for REV contamination. Vaccines were tested by 2 assays: in vitro using CEF cultures and PCR for the detection of REV and in vivo with inoculation of fowl pox vaccines into 5-d-old SPF chicks and detection of REV-specific antibodies using ELISA and histopathological examination of the internal organs. Table 3. Results of ELISA test for detection of reticuloendotheliosis virus (REV) antibodies in sera of specific-pathogen-free chicks 45 d postinoculation with fowl pox vaccine samples from no. 21 to 30 1 Chick group (n = 5) Mean of optical No. positive/ density S:P ratio 2 no. examined Interpretation /5 Negative /5 Positive /5 Negative /5 Negative /5 Negative /5 Negative /5 Negative /5 Negative /5 Negative /5 Negative Uninoculated chicks /5 Negative 1 Negative control mean = 0.048; positive control mean = 0.51; corrected positive control = S:P ratio = sample mean/correct positive mean (positive control mean negative control mean). S:P ratio more than 0.5 was considered positive for REV antibodies.

5 DETECTION OF RETICULOENDOTHELIOSIS VIRUS 2393 The in vitro testing revealed that only 1 of the 30 (3.33%) fowl pox virus vaccines was positive for REV using PCR. A 291-bp fragment was obtained, similar to previous reports (Witter and Fadly, 2003). They detected proviral DNA using PCR that amplified a 291- bp product of the REV long terminal repeat (LTR), and this PCR has been shown to be a sensitive and specific method for the detection of various strains of REV in CEF cultures, as well as in blood and tumors of infected chickens. The percentage of positive vaccines (3.33%) is relatively low when compared with that obtained by García et al. (2003), who examined field isolates. This study used primers specific for the 5 LTR or the env gene (specific gene fragments as LTR) for the detection of REV using PCR. They found that all field isolates produced positive results with both primer sets, whereas 50% of the examined vaccine strains produced positive results with the 5 LTR primer set but not with the env primers. These results are similar to studies by Diallo et al. (1998), Singh et al. (2000), and Kim and Tripathy (2001), who found that none of the examined vaccine strains contained the env sequence; however, 12 of 17 vaccines tested were positive for REV using PCR and the 5 LTR primers. The different percentage of positive vaccines in these reports and our study may be due to the fact that the fowl pox vaccines tested were produced by different manufacturers and were imported from many countries; therefore, the vaccines may not have the exact same strain in each batch. Additionally, we used only 1 primer pair (5 LTR), whereas other studies used 2 primer pairs (the 5 LTR and env). However, Hertig et al. (1997) reported that PCR amplification using env-specific primers was observed for 1 of 4 tested vaccine strains. Antibodies against REV were detected in the group of chickens inoculated with fowl pox vaccine sample 22 using ELISA. The REV-specific antibodies were first detected at 15 d postinoculation, which is in agreement with Motha (1984), who detected REV-specific antibodies in inoculated chickens as early as 16 d postinoculation. However, Aly et al. (1993) detected REVspecific antibodies starting at 3 wk postinoculation. In contrast to these findings, Fadly and Witter (1997) failed to detect REV-specific antibodies using immunofluorescence 3 or 4 wk postinoculation after inoculation of 5-d-old SPF chickens with a REV-contaminated fowl pox vaccine. This discrepancy may be due to the fact that Fadly and Witter (1997) used a contaminated fowl pox vaccine as an inoculum, as compared with Motha (1984) and Aly et al. (1993), who used a pure REV preparation. These results suggest that the dose of REV in the inoculum may play a role in the response of the chickens and that the presence of other agents (fowl pox virus in our study) may interfere with the ability of inoculated chickens to produce antibodies against REV. These results indicate that ELISA-based detection is a more sensitive and specific method for the detection of REV-specific antibodies (Smith and Witter, 1983). Figure 2. Photomicrograph of the liver stained with hematoxylin and eosin. A) The histoarchitecture of the liver is intact in controls (400 ). B) Eight weeks postinfection, the liver histology showing a large area of extensive infiltration and aggregation of hyperplastic mononuclear cells (arrow) between the severely degenerated and necrotic hepatocytes (400 ). C) Twelve weeks postinfection, the liver histology showing marked atrophy of hepatic acinai as well as pronounced congestion of hepatic sinusoids (200 ). Color version available in the online PDF.

6 2394 Awad et al. Figure 3. Photomicrograph of the spleen stained with hematoxylin and eosin. A) The histoarchitecture of the spleen is intact in controls (400 ). B) Spleen histology 8 wk postinfection showing an excess of the proliferated mononuclear cells with reticuloendothelial elements (arrow) and one large hypertrophied lymph follicle (F) (400 ). C) Spleen histology 12 wk postinfection showing widespread focal proliferation of lymphocytic-histocytic cells (50 ). Color version available in the online PDF. Figure 4. Photomicrograph of the bursa of Fabricius stained with hematoxylin and eosin. A) The histoarchitecture of the bursa of Fabricius is intact in controls (400 ). B) Bursa of Fabricius histology 8 wk postinfection showing diffuse hyperplastic changes in most of the lymph follicles (arrows) (250 ). Note the vesicular nuclei and chromatin margination (inset; 200 ). C) Bursa of Fabricius histology 12 wk postinfection showing inter- and intrafollicular aggregation of lymphocytic-histocytic cells (notice atrophy of the follicles) (200 ). Color version available in the online PDF.

7 DETECTION OF RETICULOENDOTHELIOSIS VIRUS 2395 In this study, specific REV neoplastic lesions were observed in the liver, spleen, and bursa 8 and 12 wk postinoculation, similar to what has been reported in prairie chickens inoculated with REV 8 wk postinoculation (Bohls et al., 2006). Finally, we can conclude that vaccine contamination, partial or complete insertion of the REV genome into the vaccine, and development of new control methods represent important challenges that must be addressed to develop effective strategies for controlling REV infection in poultry and for obtaining maximum vaccination results. Hence, the vaccine manufacturers, diagnostic laboratories, and World Organisation for Animal Health should be more involved and diligent about vaccine control. REFERENCES Aly, M. M., E. J. Smith, and A. M. Fadly Detection of reticulo-endotheliosis virus infection using the polymerase chain reaction. Avian Pathol. 22: Bagust, T. J Reticuloendotheliosis virus. 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