Infection with chicken anaemia virus impairs the generation of pathogen-specific cytotoxic T lymphocytes

Abstract

Infection with chicken anaemia virus (CAV), a circovirus, can result in immunosuppression and subsequent increased susceptibility to secondary infections. This is the first report of impairment of pathogen-specific cytotoxic T lymphocytes (CTL) after natural and experimental infection of chickens with CAV and Marek's disease virus (MDV) or reticuloendotheliosis virus (REV). MDV- and REV-specific CTL were generated at 7 days post infection by 9-30-day-old-chickens that were positive for maternal antibodies to CAV at 9-17 days of age. Replication of CAV could not be demonstrated in these chickens using quantitative real-time polymerase chain reaction (PCR) and reverse transcriptase (RT)-PCR assays. In contrast, REV-specific CTL failed to develop when chickens negative for maternal antibodies at 9-17 days of age were infected. Infection with CAV at 45 days of age after CAV maternal antibodies had waned also caused a decreased REV-specific CTL response. In these chickens increased levels of CAV DNA of up to 107 copy numbers per micro g DNA and increased relative transcript levels of CAV by up to a factor of 106 were detected by quantitative real-time PCR and RT-PCR. Interleukin (IL)-1beta and IL-2 mRNA levels were not significantly affected by CAV infection at 7 or 14 days p.i. Similar assays for interferon-gamma (IFN-gamma) transcripts demonstrated a 10-fold increase in IFN-gamma mRNA levels at 7 days post infection following REV or REV + CAV infection, while CAV alone caused a two- to fourfold increase. These results show a strong link between CAV antibody status, CAV replication, and the ability to generate REV-specific CTL. It is likely that the immunosuppressive effects of subclinical infection have previously been underestimated.

Figure 1

REV- and MDV-specific CTL responses in 9- vs. 30-day-old birds (Exp. I). P2a chickens (six per treatment group) were inoculated intra-abdominally at 9 or 30 days of age with 10^4·3 TCID₅₀ REV-CS in Trial 1 and with 1000 FFU JM-16/p 19 in Trial 2. Uninfected hatch-mate control groups were housed separately and included in each of the trials. At 7 days p.i. all birds were killed for spleen and blood collection. Splenocytes were prepared and tested in triplicate in CRAs with an effector : target ratio of 100 : 1 against CU91 (syngeneic REV target cell line) in Trial 1 and CU371 (syngeneic MDV target cell line) in Trial 2 as described in Materials and Methods. DNA extracted from spleen cells was subjected to CAV-specific PCR. Amplicons were visualized on 2% agarose gels, transferred to nylon membranes and hybridized to a DIG-labelled probe containing the entire CIA-1 DNA sequence. Sera were tested in an ELISA to determine the maternal antibody status for CAV in individual birds. *, statistically significant (P < 0·01) compared to the 30-day control group in each respective trial.

Figure 2

Relative IL-1β mRNA levels in CAV- and/or REV-infected chickens (Exp. III–V). (a) 9- to 15- (b) 30-, and (c) 45-day-old CAV maternal antibody-positive and -negative N2a chickens were infected with 10^5·7 TCID₅₀ CIA-1 and/or 10^4·3 TCID₅₀ REV-CS as indicated in the key, or used as uninfected controls. At 7 days p.i. (a, b, c) and 14 days p.i. (b, c) three to six birds per group were killed for spleen and sera collection. RNA was extracted from spleens and subjected to real-time RT–PCR analysis. Average transcript levels are expressed relative to the average age-matched controls, defined as 1. The absence of a column in a given panel indicates that this group was not tested in that trial. Differences between treatment groups were not significantly different.

Figure 3

Relative IL-2 mRNA levels in CAV- and/or REV-infected chickens (Exp. III–V). (a) 9- to 15- (b) 30-, and (c) 45-day-old CAV maternal antibody-positive and –negative N2a chickens were infected with 10^5·7 TCID₅₀ CIA-1 and/or 10^4·3 TCID₅₀ REV-CS as indicated in the key, or used as uninfected controls. At 7 days p.i. (a, b, c) and 14 days p.i. (b, c) three to six birds per group were killed for spleen and sera collection. RNA was extracted from spleens and subjected to real-time RT–PCR analysis. Average transcript levels are expressed relative to the average age-matched controls, defined as 1. The absence of a column in a given panel indicates that this group was not tested in that trial. Differences between treatment groups were not significantly different.

Figure 4

Relative IFN-γ mRNA levels in CAV- and/or REV-infected chickens (Exp. III–V). (a) 9- to 15- (b) 30-, and (c) 45-day-old CAV maternal antibody-positive and -negative N2a chickens were infected with 10^5·7 TCID₅₀ CIA-1 and/or 10^4·3 TCID₅₀ REV-CS as indicated in the key, or used as uninfected controls. At 7 days p.i. (a, b, c) and 14 days p.i. (b, c) three to six birds per group were killed for spleen and sera collection. RNA was extracted from spleens and subjected to real-time RT–PCR analysis. Average transcript levels are expressed relative to the average age-matched controls, defined as 1. The absence of a column in a given panel indicates that this group was not tested in that trial. *, P < 0·01; **, P < 0·03; ***, P < 0·05.