| Avian reoviruses (ARV) are members of the Orthoreovirus genus, one of the 12 genera of the Reoviridae family. Avian and mammalian reoviruses are the two principal groups of the Orthoreovirus genus. Avian reoviruses contain 10 double-stranded RNA (dsRNA) genome segments, which are divided into 3 size classes, designated L (large), M (medium) and S (small), on the basis of their electrophoretic mobility.ARVs are important pathogens of birds that can cause considerable economic losses in the poultry industry. These viruses were initially discovered as pathogenic agents that induced tenosynovitis in young chickens, and were subsequently found to be ubiquitous among poultry flocks, although most avian reoviruses cause asymptomatic infections in poultry. Avian reoviruses have been associated with a variety of disease conditions in poultry including enteric and respiratory diseases, myocarditis, hepatitis and the so-called stunting/malabsorption syndrome, but a direct link between the presence of the virus and disease has only been conclusively demonstrated for viral arthritis syndrome (also known as tenosynovitis), which is characterized by swelling of the hock joints and by lesions in the gastrocnemius tendons. There were at least 11 avian reovirus serotypes.In spite of their economic significance as pathogens of poultry, there is very little systematic surveillance of pathogenesis and the antigenic relatedness on domestic avian reovirus isolates until very recently. In order to conduct the effect of field avian reovirus isolates on our poultry industry, 10 field avian reovirus strains were isolated from chickens with different diseases conditions and compared their serologic relatedness, sigma C sequences and pathogenicity on SPF white leghorn chickens and broiler chickens.1. The isolation, identification and purification of 10 field avian reovirusesIn order to study the relationships between the pathogenicity , the antigenicity and theσC gene sequence of the prevalence strains of avian reovirus in our country, 10 field avian reovirus isolates were separated from chickens with viral arthritis and lower growth in Shandong, Xinjiang, Jilin, Jiangsu and other different provinces. They were determined by indirect fluorescence immune antibody and RT-PCR. 7 isolates from the chickens with arthritis are DL, DF, LC, CHX, NT, LY, and XJ strains; Two from the broiler chicken with lower growth are ZZ and CC strains; The YT isolate was from chickens with both sarthritis and lower growth syndrome. These isolates were cultivated successfully in primary CEF cell culture and purified 3 times by the infinite continuous dilution method.2 Sigma C Gene sequence comparison and antigenic relatedness of different avian reovirus isolatesProteinσc is encoded by the 3'-proximal cistron of S1 gene induce specific neutralizing antibodies. Reverse transcription polymerase china reaction (RT-PCR) was used to amplify the 3rd open reading frame (ORF) of S1 segment encoding sigma C protein of 5 isolates. These viruses were: CHX, LY, CC, DL, and LC strains. Sequence analysis showed that the homology of these fragments encoding sigma C protein is above 99%, and sigma C gene homology of CC, CHX and LY strains are 100%. It is suggested that these isolation has the common evolved history.The serological relatedness of seven avian reovirus isolates (ZZ, CC, CHX, LY, NT, XJ, S1133 ) was determined using a virus-neutralization (VN ) test. Seven groups of 8 SPF leghorn chickens each were twice infected with one of the seven isolates per group. Serum was reacted against each isolate in a beta VN test in CEF. Relatedness (R) values, determined by cross VN, revealed that R values among all isolates were 25%~74%. The data showed these six isolates had been antigenically distantly related to each other.It is indicated from ARVσc gene sequence and virus neutralization test that the ARV antigenicity by no means merely concerns correlated completely withσc .It has the possibility to have other structure proteins correlating with ARV antigenicity, and need further study.3. Comparative study of pathogenicity of avian reovirus isolates on SPF white leghorn chickens3.1 Influence of growth-supress on chicken of ARV infection Pathogenicity and immunosuppression on specific pathogen free (SPF)chickens of 11 field avian reovirus isolates were investigated. One-day-old SPF chickens were inoculated subcutaneous with above purified field avian reovirus isolates respectively, 105TCID50/chicken. At 7-day-old ages, avian influenza virus (AIV) and new castle-disease virus(NDV) inactivated vaccine were injected, influence of the avian reovirus isolates infection on chickens body weight and on the bursa of fabric us were investigated. Results showed that, after the infection of the virus isolates CHX, NT, LY, YT and XJ at one-day old SPF chicken, within 49 days post infection (PI), there was no remarkable difference (P>0.05) in the body weight between ARV isolates infection groups and control group, although some groups have temporary higher or lower than control group. It is showed the isolates from arthritis do not have the obvious influence on the body weight of SPF chickens. The YT strain, from chickens with both arthritis and lower growth signs, decreased the growth of SPF chickens within 42 days PI. At 28,35, and 42 days PI . The mean body weight of YT infected group and control group were 219±28.8g vs. 249±21.6 g,301±29.3g vs. 343±47.8 g,371±39.7g vs. 424±45.2 g separately. The differences are significant (P<0.05). CC and ZZ strains, from poor growth broiler chickens, infected 1-day old SPF chickens, significant reduction in the growth rates were found compared with the control group at 45 days of age (P<0.05). The mean weights of CC, ZZ strain infected groups and control were 481g±66.7g, 488±55.1g, and 533±62.6g respectively.3.2 ARV infection and viral arthritisDuring all the experiment process, up to 8 weeks of age, no leg problem caused on chickens by each of 10 ARV isolates appeared.3.3 Influences of ARV infection on lymphoid organ of chickensThe results showed ARV infection at one-day-old chickens could induce damage on lymphoid cells of bursa. In this study both ZZ and CC isolates from poor growth chickens and LY isolates from chickens with arthritis could induce lymphoid cells depletion. This study also suggested that most of the strains in the experiments had no significant influence on lymphoid organ weight to body weight rates, for example DF, DL, LC, LY, and ZZ isolates. When chickens infected with CC strains, at 30 days PI, the bursa to body weight rate(BBWR) and the thymus to body weight rate(TBWR) were 0.44±0.11 and 0.42±0.06,obviously lower than the control group of 0.55±0.14,0.54±0.0(9P<0.05).The group inoculated with ZZ strain, its TBWR was lower than the control group slightly, and difference was not significant (P>0.05). 3.4 Influence of the ARV isolates infection on antibody titers against AIV and NDVInfluence of the avian reovirus isolates infection on chickens antibody titers against NDV and AIV were also investigated. The results showed that some of the isolates depressed the hemagglutination-inhibition (HI) response of chickens to NDV and AIV transitory on different degree, for example the antibody titer to NDV of DF strain 6W post-vaccination (PV) and to AIV 4W PV, CC strain against both AIV and NDV 3W PV. Some avian reovirus strains have the mild influence on AIV HI-antibody response, for example antibody titers to AIV of XJ strain infection group 6W PV and NT strain infection group 4W and 6W PV. Some of these isolates infection inhibit moderately the response of chicken to NDV, for example antibody titers to NDV of LC strain group 6W PV, LY strain 3W PV and CHX strain 4W PV. ZZ isolate demonstrated the ability to interfere significantly with NDV HI responses, The antibody titers to NDV of ZZ infection group and control group 3W, 4W and 6W postvaccination were 4.3±2.22 vs. 6.2±3.55 Log2;4.2±1.12 vs. 5.8±1.97Log2;4.7±0.95 vs. 5.9±1.17Log2(P<0.05)respectively.4. Pathogenicity on broiler chickens of ARV4.1 Influence of ARV infection on growth of broiler chickensIn order to study the pathogenicity and the immunosuppression in broilers, purified CC, ZZ, LY and XJ strains were inoculated separately one-day-old broilers by pad + oral or oral route and vaccinated with NDV inactivated vaccine at 7-day old. The chicken weights and the HI antibody titers to NDV were collected in different times. The result showed that the mean body weights of chickens infected with ZZ, CC isolate were 591±73.7g, 584±46.0g respectively and significantly lower than that of the control group of 682±74.3g (P<0.01) at 19-day-old of chickens. At 26, 56 days of age the infected birds growth still decreased significantly than control group. In another trail, the mean body weights of chickens infected with LY, XJ strain respectively were 655±67.7g, 643±72.7g, significantly lower than control group of 714±47.8g at 21 days of age(P<0.01). These results indicated that these isolates (ZZ, CC, LY, and XJ strains) decreased the growth rates of broiler chickens.4.2 Influence of the ARV on antibody titers against NDVWhen chickens infected with ZZ, CC, LY, XJ strains respectively at one-day old and vaccinated with NDV inactivated vaccination at 7 days of age, ZZ strain showed the ability to interfere significantly with NDV-HI responses 3W post vaccination (P<0.01), while CC, LY, XJ strains have no significant effect on the NDV-HI response. Pathogenicity study of ARV isolates on SPF chickens and commercial broilers showed that broiler chickens were more susceptible to ARV than SPF white leghorn chickens.5. Influence of LY strain infection on pathogenicity of v-IBDV and immune-reactions in SPF chickensThe influences of LY strain avian reovirus infection at 1 day of age on bursa development, antibody responses after vaccinations to AIV, NDV and infectious bursal disease virus (IBDV), and pathogenicity of virulent IBDV (v-IBDV) were studied in chickens of SPF-origin. The results indicated that LY strain ARV infection at 1-day-old chickens caused atrophy of the Bursa and decreased lymphocyte numbers in the bursa, but it gave no significant negative effects on growth rates and antibody titers to AIV and NDV after vaccination. LY strain ARV infection decreased antibody titers to IBDV in B87 vaccinated birds but all vaccinated birds infected with LY strain were still fully protected from death or clinical syndromes after v-IBDV challenge. Although all B87-vaccinated birds were fully protected from death after v-IBDV challenges, the antibody titers to AIV or NDV after vaccinations with inactivated vaccines were significantly lower in v-IBDV challenged birds than controls. Surprisingly, LY isolate infection at ages of 1-7 days could compromise the immune suppression induced by v-IBDV in B87-vaccinated birds as HI antibody titers to AIV and NDV in ARV-infected groups were significantly higher than chickens with no ARV infection.SPF chickens infected with LY strain at one day old, and attacked with virulent IBDV at 30 days of ages respectively, the mortality rate was 5/24, remarkably lower than ARV negative chickens challenged with IBDV group (14/24). But at 52 days old chicken, LY isolate infection at 1 day old did not show significant influence on chickens'mortality. It may be showed that LY strain infection induces chickens'bursa changes and interferes v-IBDV growth in target tissues. A discussion was made on the interactions between ARV infection and vaccine IBDV or v-IBDV to explain such sophisticated phenomena in the bird experiments.6. Detection of Avian reovirus in Chicken Tissues and Feather Tips by Dot Blot with Dig-lableled DNA ProbeThe Avian reovirusσc gene DNA fragment was labeled with Digoxigenin as cDNA probe for detection of ARV in chicken tissue and feather tips in Dot blot hybridization. As little as 1.6 pg of ARV RNA could be detected with the DIG-labeled probe. Chickens were infected with ARV by inoculating at one-day old and in-contact infection. ARV could be detected in most tissues of inoculated chickens 24 h post infection (PI) and spread to un-inoculated birds by contact rapidly. Positive rates in feather tips were correlated with other tissues. The result demonstrated that the dot blot with DIG probe to detect ARV in feather tips is a useful method for determination of ARV infection and spread in chickens, and it can be helpful when a large number of samples should be tested.7. InclusionIn brief, this research has carried on the systematic comparison ofσc sequences, pathogenicity and the antigenicity of ARV isolates from different districts and from different clinical signs of chickens the first time. The results showed that these ARV isolates from our country did not demonstrate the ability to cause the obvious clinical lesion on birds when birds were inoculated with each of these isolates alone. The sequence encodingσc protein are relatively conserved, and the pathogenicity and antigenicity of these isolates had changed greatly respectively.Simultaneously this study demonstrated that the dot blot with DIG probe to detect ARV in feather tips is a useful method for determination of ARV infection and spread in chickens... |
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