Pathogenicity, antigenicity, and detection of turkey astroviruses.

Astroviruses are etiologic agents of acute gastroenteritis in human beings and in a variety of animal species. Turkey astrovirus is an important pathogen of poult enteritis and mortality syndrome (PEMS), which has caused significant losses to turkey industry in the USA. Unlike the situation of human astroviruses, turkey astroviruses have not been well characterized in terms of pathogenicity, and antigenicity. In the first part of this study, a pathogenicity investigation was carried out in specific pathogen free (SPF) turkey embryos and commercial poults. Turkey astrovirus 1987 (TAstV1987) isolate and turkey astrovirus 2001 (TAstV2001) isolate were used in this investigation. Both virus isolates caused gastroenteritis and growth depression in both SPF turkey embryos and commercial poults. The histopathologic lesions on thymus and bursa induced by TAstV2001 in embryos and poults were not remarkable. Similarly, no abnormal morphologic changes were noted on thymus of embryos and poults inoculated with TAstV1987. There was no statistically significant difference in bursa weight / body weight ratios (P>0.05). However, TAstV1987 infection resulted in microscopic lesions of infected bursas of embryos and poults. In the second part of this study, The antigenicity of TAstV1987 and TAstV2001 were compared with each other, and the antigenic relatedness were determined by crossneutralization tests in turkey embryos as well as by enzyme-linked immunosorbent assay ii (ELISA). The antigenic relatedness values (R) were calculated using the Archetti and Horsfall formula. The R value as measured by cross-neutralization tests was 0.56%, indicating the TAstV1987 and TAstV2001 belong to different serogroups; whereas the R value measured by ELISA was 70.7%, suggesting these two viruses share common antigen(s). So far, the diagnosis of astroviruses was mainly dependent on electron microscopy (EM) or immune electron microscopy (IEM) techniques. To develop other diagnostic assays, an antigen capture enzyme-linked immunosorbent assay (AC-ELISA) was developed using polyclonal hyperimmune antisera against TAstV1987 and TAstV2001 isolates, and monoclonal antibody (MAb) for TAstV2001. Monoplex and multiplex reverse transcription /polymerase chain reactions (RT-PCR) were developed as well using non-degenerate primer sets specific to the capsid region, and degenerate primer pairs covering the polymerase area of turkey astrovirus genome. One ssRNA internal control (IC) template reagent was produced, and applied to the RT-PCR to reduce the false negative percentage of the test. Direct EM was used also in this study and the results obtained from both EM and RT-PCR were compared. The results showed that the polyclonal AC-ELISA had higher sensitivity and a wider detection spectrum than that of the monoclonal AC-ELISA with group-specific MAb, but with higher background, whereas the monoclonal AC-ELISA had very high specificity but lower sensitivity (0.06 μg of viral proteins). Fecal samples or gut content samples from naturally infected or experimentally infected poults with PEMS were examined by EM and RT-PCR. The EM results indicated that the positive percentage of small round viruses (SRVs) that could include astroviruses as well as enteroviruses was 33.4%. The iii monoplex RT-PCR results amplified with primers SRV-1-3 and SRV-1-5 revealed that the positive percentage of astroviruses was 45.3%, which is 10.9% higher than that of direct EM even if other SRVs were not excluded. Multiplex RT-PCR with both SRV-1-3 and SRV-1-5 and AFCP-F1 and AFCP-R1 and the monoplex RT-PCR with degenerate primers showed good specificity and wider detection spectrum than that of earlier published data and the monoplex RT-PCR with the positive percentage of 59.4%, 25% higher than that of EM. With the internal control, an overall test inhibition rate of 12.5% was found for the astrovirus RT-PCR assay, which can be used to decrease the falsenegative percentage of the detection.