Studies on the Pathogenicity and Vaccines of Avian Reticuloendotheliosis Viruses and Subgroup J Avian Leukosis Viruses

Abstract: Both reticuloendotheliosis viruses(REV) and avian leucosis viruses(ALV) belong to Retroviridae, and they could cause different syndromes and lesions, from subclinical infections to grow retardation, immtmosuppression and tumors. To further understand the negative impacts of REV and ALV infections on poultry industry, the pathogenicity of REV and subgroup J ALV (ALV-J) was studied. It was demonstrated and proved that immunosuppressive levels of HI antibody titers to NDV and AIV after vaccination in chickens could successively be used as the objective and quantitative criteria to compare pathogenicity of different strains of REV and ALV-J. The results indicated that the early REV infection could dramatically and persistently inhibit antibody reactions after vaccination, and co-infection of REV and ALV-J will significantly enhance such immunosuppression. Then, it would severely interfere with the protective efficacy of vaccinations against NDV and H5-AIV in chickens and ducks. Epidemiological studies indicated that REV and ALV-J infection/co-infection is very common in chicken flocks in China. The results in this study also demonstrated that vaccination of breeders before laying with live REV vaccine could effectively prevent their progenies from REV infection and REV-induced immunosuppression.1 Pathogenicity of REV on young chickens and ducks was influenced by several facors1.1 Comparison of 6 REV strains with different origins for their pathogenicity in 1-day-old SPF chickens.The pathogenicity of 6 REV strains isolated from chickens or ducks was very similar when they were inoculated into 1-day-old SPF chickens. It was demonstrated as followings: growth retardation, atrophy of thymuses and the Bursa at different levels, suppressed immune reactions to vaccination against NDV, H5-, H9-AIV. When SPF chickens were inoculated respectively with 6 different REV strains with 3000 TCID_(50) at 1 day, HI antibody titers to NDV, H5-AIV and H9-AIV 3-5 w after vaccination were significantly lower in infected chickens than that in control chickens(p<0.01). However, the difference among chickens inoculated with 6 strains was not significant statistically (p>0.05). The results indicated that all REV strains tested from different origins had the similar immunosuppressive effects.1.2 Relationship between pathogenicity and doses of REV.When SPF chickens were inoculated at 1 day with 3000 or 100 TCID_(50) of REV-C99 strain, both groups caused growth retardation and decreased antibody reactions to vaccination. However, the higher the dose used, the more severe the immunosuppression. Three weeks after second vaccination against H5-AIV, for example, HI antibody titers to H5 in chickens infected at 1 day with 3000 or 200 TCID50 were 2.2±3.0(n=10) and 6.5±4.3(n=11) Log2 compared to 1.0.0±1.4(n=8) in control chickens.1.3 Relationship between pathogenicity of REV and chicken ages for infection.When SPF chickens were infected with REV-C99 strain at days 1, 8 and 15 respectively, HI antibody titers against NDV, H5-AIV or H9-AIV after vaccination were decreased at different levels compared to the control, but immunosuppression was much more severe in chickens infected at day 1. Especially, HI antibody titers were still significantly lower than the control even after 2 and 3 repeated vaccinations if chickens were infected with REV at day 1. In contrast, immunosuppression induced by REV infection 8-day-old or older chickens was transient, the difference in HI antibody titers between REV-infected and control chickens became less and less after 2 or 3 vaccinations. The results demonstrated that the pathogenicity of REV to chickens is strictly age-dependent.1.4 REV-infection could severely interfere with protective efficacy of vaccine against H5-AIV.REV infection in 1-day-old chickens could significantly depress the HI antibody titers to H5-AIV after vaccination. And the challenge with high pathogenic H5-AIV in BSL3 lab indicated that the early REV infection did severely interfere with the protective efficacy of vaccine against H5-AIV. Also, such immunosuppressive effects induced by REV could last for at least 4 months. When chickens were vaccinated against H5-AIV 3 times at 2w, 7w and 12w of ages, actually, HI antibody titers in birds infected with REV at 1 day of age always significantly lower than the control. When challenged with HPAI-H5N1 5w after the third vaccination, the protective rates in control chickens was 11/12(91.7%), but it was low as 14/26(53.8%) in REV-infected chickens.1.5 Immunosuppression and tumorgenesis of different REV strains in ducksWhen inoculated in ducks at·1 day of age, pathogenicity of both REV-C99 of duck origin and HA9901 of chicken origin was more strong in ducks than in chickens. Besides REV infection could significantly depress HI antibody titers to vaccine against H5-AIV, REV could induce acute tumors and death at a high percentage in 4-8 w after inoculation with different REV strains in 1-day-old ducklings. In contrast, tumors were possibly detected only a very low percentage of chickens 2-3 months after infection at 1 day of age.2 Comparisons of different ALV-J strains for their pathogenicity to chickensDifferent strains of ALV-J isolated from white meat-type chickens or Chinese local “yellow” chickens were compared for their pathogenicity to SPF chickens. All isolated could induce growth retardation and immunosuppdression on HI antibody titers to NDV, H5-AIV or H9-AIV after vaccination, but at different levels. With the age, such inhibitory effects of ALV-J on chickens became less and less. Relative to REV, pathogenicity of ALV-J was milder in SPF chickens. Also, there is big difference in immunosuppression among different ALV-J strains.3 Synergism of REV and ALV-J infections in pathogenicitySingle infections or co-infection of REV and/or ALV-J were compared for their pathogenicity in SPF chickens and commercial broilers. The results indicated that co-infection of REV and ALV-J caused more severe immunosuppression compared to each single infections. Six weeks after vaccination at 1 w of age in SPF chickens were infected with REV, ALV-J or both at 1 day of age, for example, 6.88±2.56(n=24) and 6.0±3.13(n=11) of HI titers to NDV in ALV-J or REV infected chickens were significantly lower than 7.43±1.5 (n=28) in control chickens (p<0.05). However, HI titer of 4.29±3.38(n=14) in chickens co-infected with both REV and ALV-J were further significantly lower than that in chickens infected with each single viruses(p<0.01). Obviously, there was a synergism between both REV and ALV-J infections in immunosuppression. Also, co-infection of REV and ALV-J dramatically increased conditional becterial infections and death rates in commercial broilers.4 Epidemiological studies on REV and ALV-J infections in Chinese local “yellow chicken” flocks.4.1 Vertical infection and co-infection of REV and ALV-J are common in chickens in China.The epidemiological studies demonstrated that REV and ALV-J were isolated not only from chickens with immunosuppression and/or tumor lesions but also from embryos at certain percentages. Among 4 batches of eggs from parent breeder flocks of “yellow” chickens, REV were isolated from 2/14, 5/17, 5/12 and 5/29 embryos, ALV-J were isolated from 2/14, 1/17, 2/17 and 3/29 embryos. Meanwhile, the co-infection of REV and ALV-J was confirmed 3of 57 embryos.4.2 Phelogenie analysis of env genes of REV strains isolated from Chinese local “yellow” chickens.Among 8 REV isolates obtained from “yellow” chickens in Guangdong province, env genes of 2 strains GD051029 and GD06LG1 were amplified and sequenced, their sequences were compared to other 14 strains isolated in China or other countries. Results indicated that GD051029 and GD06LG1 had identity of 99.8%-99.9%to reference strain SNV of duck origin, but only 93.7%-93.8%of identity to another Chinese REV strain HA9901 isolated from chickens in 1999. The env gene fragments of 401 bp of another 6 REV strains isolated in Guandong in 2005-2006 were also more homologous to SNV than HA9901. It seems like that there is no relationship betwe
en the env gene identity and host origins of REV.4.3 Epidemiological studies of ALV-J in Chinese local “yellow” chickens.Studies indicated that ALV-J infection was very common in Chinese local “yellow” chickens. Pathological studies demonstrated that ALV-J specific myelocytomatosis was detected not only in livers, spleens and kidneys as in white meat-type chickens, but also in thymuses and the Bursa, which was not reported before. The gp85 and gp37 genes of 6 ALV-J strains isolated from “yellow” chickens had very high homology to ALV-J which was isolated from white meat-type chickens. Especially, there were high as 95.1% and 99.5% identity for gp85 and gp37 respectively between GD0512 isolated from “yellow” chicken in Guangdong in 2005 and NN0101 isolated from white meat-type chickens in Henan province in 2001. It is indicating that the ALV-J in “yellow” chickens may come from white meat-type chickens.5 Comparative studies on gross lesions and histopathology of tumors induced by MDV, REV and ALV-J.It was always difficult to make a differential diagnosis on the natures of tumors from field cases. To understand if it is possible to make differential diagnosis according to pathological observations, tumor samples from chickens inoculated with reference MDV, REV or ALV-J respectively were compared by both gross lesion and histo-sections. Results demonstrated that all 3 viruses along could induce white proliferating tumor nodules with irregular shapes in livers. MDV could induce small number of large tumor masses with clear boundary in round shape or large number of small tumor nodules in the size of green beans. REV induced mainly tumors of irregular shapes of different sizes in livers, it was difficult to differentiate it from the later form of MD tumors. ALV-J induced many very small white tumor nodules (less than 1 mm in diameter) distributed in the whole enlarged livers, which was easy to be distinguished from MDV and REV tumors. But sometimes, ALV-J also induced many tumor nodules in the size of green beans, which was also difficult to be distinguished from MDV tumors. In histo-sections, there were many acidophil particles in the cytoplasm of ALV-J induced myelocytoma cells, which is characteristic of ALV-J induced tumor cells and easy to be differentiated from MDV or REV tumors. In the chickens inoculated with known MDV or REV, the most tumor cells belonged to lymphocytes or mononuclear cells, it make it difficult to differentiate from each other.6 Development of REV live vaccine and its protective efficacySo far, there is no vaccine against REV infection in the world. To prevent and control REV infection in chickens, some studies were made to develop vaccine against REV infection. After breeders were inoculated with cell-adapted REV-C99-p30 vaccine virus 4 w before laying, anti-REV antibody was demonstrated in 2w, and neither negative influence on the egg production nor vertical transmission of the virus through eggs was detected in vaccinated chickens. Maternal antibody was proved in all progenies from vaccinated breeders. Bird experiments also indicated that maternal antibody to REV not only prevent viremia after REV inoculation at 1 day of age, but also protected broilers from REV-induced growth retardation and immunosuppression. Four weeks after vaccinations against NDV, H5-AIV and H9-AIV at 1w of age, HI antibody titers (Log2) to NDV, H5-AIV and H9-AIV in broilers with or without maternal antibody to REV were: 3.36±2.04 vs 1.58±1.69 (p<0.01), 6.27±3.87 vs 0.71±1.6 (p<0.01), and 6.72±3.92 vs 0.54±1.44 (p<0.01)…
Key words: reticuloendotheliosis virus; subgroup J avian leucosis virus; co-infevtion; epidemiology; synergism; immunosuppression; HI antibody; vaccine

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