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 µg 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)-1β and IL-2 mRNA levels were not significantly affected by CAV infection at 7 or 14 days p.i. Similar assays for interferon-γ (IFN-γ) transcripts demonstrated a 10-fold increase in IFN-γ 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.

Introduction

Chicken infectious anaemia virus (CAV) is a small, icosahedral, non-enveloped virus belonging to the Circoviridae family, and was recently placed into the Gyrovirus genus.¹ The virus has a 2·3-kb genome consisting of single-stranded, negative sense, covalently closed circular DNA that encodes for three partially overlapping reading frames (ORF)1, 2, and 3. One polycistronic mRNA is transcribed from which the three corresponding viral proteins, VP1, 2, and 3 are translated.^2,3 VP1 is the only capsid protein and VP2 functions as a scaffold protein so that VP1 is properly folded.^4,5 CAV-neutralizing antibodies are specific for a conformational epitope formed by the interaction of VP1 and VP2. VP3, also known as apoptin⁶ induces apoptosis of thymocytes in vivo and of cultured transformed avian cells in vitro.⁷

CAV is the causative agent of chicken infectious anaemia, which is characterized by anaemia, immunosuppression, and secondary bacterial infections when chickens are infected before 2 weeks of age.⁸ Virus transmission can occur horizontally as well as vertically in the case that seronegative hens become infected during egg production.^9,10 Chickens positive for CAV-specific maternal antibodies are protected from clinical disease.¹¹ Infection of chickens with CAV at or after 3 weeks of age results in the formation of virus-neutralizing antibodies before clinical symptoms appear, although subclinical effects may occur.^12,13 Recently, it was reported that latent CAV was present in the gonads of adult chickens in a specific-pathogen-free (SPF), closed flock that had become positive for CAV antibodies. CAV or CAV DNA could be transmitted vertically from CAV antibody-positive and -negative hens to their offspring without causing clinical disease in the newly hatched chicks. These chickens often did not develop CAV antibodies until the onset of sexual maturity or afterwards (14,15 and M. M. Miller, K. A. Ealy, W. B. Oswald and K. A. Schat, manuscript submitted).

Numerous studies have shown that CAV is an important cofactor for a number of avian diseases. Experimental coinfection of CAV with Marek's disease virus (MDV) caused an increase in mortality^16–19 although this depended on the challenge dose¹⁸ or virulence¹⁹ of the MDV strains. Likewise, dual infection of CAV with reticuloendotheliosis virus (REV),¹⁶ infectious bursal disease virus (IBDV),¹⁶ reovirus,²⁰ or adenovirus²⁰ resulted in increased morbidity and mortality. CAV infection has also been associated with poor vaccine-induced protection to Marek's disease (MD),^21–24 Newcastle disease^25,26 and infectious laryngotracheitis.²⁶ The enhancement of these diseases by concomitant CAV infection is most likely the consequence of immunosuppression, yet the mechanism of CAV-induced immunosuppression has not been completely characterized. Infection of maternal antibody-negative, susceptible chicks caused the destruction of CD4⁺ CD8⁺ thymocytes and CD8⁺ splenocytes.^27,28 Infection of susceptible 1-day-old chicks also resulted in decreased mitogen responsiveness of spleen cells and production of T-cell growth factors (TCGF, presumably interleukin (IL)-2) at 8 and 15 days post infection (days p.i.). Interferon (IFN) production was increased at 8 days p.i., but decreased between 15 and 29 days p.i..²⁹ Interestingly, infection in 3-week-old chickens caused immunosuppression in the absence of clinical disease. McConnell et al.^12,13 found a decrease in IL-1, IL-2, and IFN-γ between 14 and 28 days p.i. using bioassays. Suppression of macrophage functions was also reported when 3-week-old chickens were naturally exposed to CAV.

After the onset of the CAV infection in the SPF flocks at Cornell University in the summer of 1996^14,15 chromium release assays (CRA) to study cytotoxic T lymphocyte (CTL) responses to MDV and REV no longer worked. These assays, which were routinely performed using splenocytes obtained from approximately 5–6-week-old chickens infected with MDV or REV, failed to yield any positive results, which was unexpected because CRA to detect REV-specific CTL have been routinely performed since 1987.^30–33 It was speculated that these negative results could be caused by the presence of CAV in the SPF flocks. However, specific information on the importance of CAV for the generation of specific antiviral immune responses is lacking, although preliminary data have suggested that CTL but not natural killer cells are impaired.³⁴ As a consequence, the effect of natural or experimental infection with CAV on the generation of antigen-specific CTL and transcription of selected cytokines was examined. The studies presented in this demonstrate the impairment of pathogen-specific CTL development after natural and experimental infection with CAV in CAV antibody-negative chickens. Taqman™ real-time quantitative polymerase chain reaction (PCR) and reverse transcriptase (RT)–PCR assays³⁵ were used in some of the experiments to quantitate CAV-specific DNA and transcript levels in CAV antibody-positive and -negative chickens inoculated with CAV.

Materials and methods

Chickens

All experimental chickens were obtained from departmental SPF N2a (MHC: B²¹B²¹) and P2a (MHC: B¹⁹B¹⁹) flocks.³⁰ These closed flocks are maintained in a filtered air, positive pressure house. These flocks had become exposed to CAV in 1996 and remained positive since then.^14,15 Production flocks are monitored at 16, 24, 32 and 60 weeks of age to determine their serological status. Most hens of these flocks seroconvert between 16 and 32 weeks of age and chicks are maternal antibody-positive, but some chicks are hatched from hens that have not seroconverted (M. M. Miller and K. A. Schat, unpublished data). All experimental procedures were conducted in compliance with all applicable federal and institutional animal use guidelines.

Cell cultures and cell lines

Chick kidney cell (CKC) and chicken embryo fibroblast (CEF) cultures were prepared from 2-week-old SPF chicks or 10-day-old chicken embryos, respectively, as previously described.³⁶ The MDV-transformed chicken cell line MDCC-CU147³⁷ and the REV-transformed chicken cell lines RECC-CU91 and CU205³⁸ were propagated in LM medium containing 10% fetal bovine serum (FBS) in 5% CO₂ at 41°. The REV cell line RECC-CU371 expressing the MDV glycoprotein gB has been described.³³

Viruses and bird inoculations

For CTL assays, chickens in MDV-infected groups were inoculated intra-abdominally with 1000–2000 focus-forming-units of the serotype 1 strain JM-16/passage (p)19. JM-16 was propagated in CKC as previously described.³⁹ REV-infected chickens were inoculated intra-abdominally with 10^4·3 tissue culture infective doses 50% (TCID₅₀) of the nontransforming REV-CS strain^31,40 that was originally obtained from R. L. Witter (Avian Disease and Oncology Laboratory, East Lansing, MI). REV was propagated in CEF as previously described.³¹ MDV and REV stocks were free of CAV as determined by PCR assays. For experimental infection with CAV, chickens were inoculated intramuscularly with 10^4·2 (Exp. II) or10^5·7 (Exp. III-V) TCID₅₀ of the CIA-1 strain⁴¹ which was propagated in MDCC-CU147 as described.³⁷

Serology

The presence of CAV antibodies was analysed by enzyme-linked immunoabsorbant assays (ELISA) using the Flokchek CAV Antibody Test kit (Idexx Laboratories, Westbrook, ME). Sera were diluted 1 : 10 according to the manufacturer's instructions. Absorbances (Abs.) were read at 650 nm in a Microplate EL310 Autoreader (Bio-Tek Instruments, Inc., Winooski, VT).

DNA and RNA extractions

DNA and RNA were extracted from spleen cell suspensions that were prepared as described for effector cell preparation in chromium release assays (see below). DNA extractions were performed by a standard phenol : chloroform extraction method⁴² followed by ethanol precipitation and a 70% ethanol wash. RNA extractions were performed using RNAzolB (Teltest, Inc., Friendswood, TX) according to the manufacturer's instructions. Aliquots of RNA were further treated with DNase (Ambion, Inc., Austin, TX) before use in RT–PCR assays.

CAV-specific PCR

One µg of genomic DNA was subjected to PCR analysis with the following CAV-specific primers (located within ORF3): O3F (5′-CAAGTAATTTCAAATGAACG-3′) and O3R (5′-TTGCCATCTTACAGTCTTAT-3′)¹⁵ using the following cycling parameters: 94° for 5 min, followed by 35 cycles of denaturation at 94° for 1 min, annealing at 45° for 2 min, and extension at 72° for 1 min, ending with a final extension at 72° for 10 min. All PCR reactions were performed in a PE2400 thermal cycler (PE Biosystems, Foster City, CA) in a total volume of 50 µl. PCR products were resolved on 2% agarose gels, stained with ethidium bromide, and visualized in an Eagle Eye detection system (Stratagene, LaJolla, CA).

Southern blotting

Following visualization of PCR products, gels were denatured and DNA transferred to nylon membranes by standard methods.⁴² Hybridization was performed overnight at 65° using a digoxigenin (DIG)–horseradish peroxidase (HRP) conjugate-labelled (Boeringher-Mannheim, Germany) probe derived from a plasmid containing the complete CIA-1 strain genome.⁴³ After addition of substrate, blots were exposed to an autoradiograph (Kodak, Rochester, NY) for 30 min to several hours.

Qualitative RT–PCR assays

Assays for transcript detection of IFN-γ, IL-1β, IL-2, and β-actin were performed by RT–PCR using a ‘touchdown’ cycling procedure as previously described⁴⁴ with the exception that all cDNA PCR reactions were performed in 50 µl volumes in a PE9700 thermal cycler (PE Biosystems).

Taqman™ real-time quantitative PCR and RT–PCR

Primers and probes.

Taqman real-time PCR and RT–PCR were used to determine the levels of CAV DNA replication and to estimate the relative levels of transcripts for CAV ORF1, IFN-γ, IL-2, and IL-1β, respectively. All PCR and RT–PCR reagents, primers, and Taqman probes were purchased from PE Biosystems and used in conjunction with the ABI Prism 7700 thermal cycler. All primers and probes were designed using Primer Express v.1.0 software and are listed in Table 1. Individual reactions contained 25 pmol of each primer and 10 pmol of the corresponding probe in a total volume of 25 or 50 µl.

Table 1.

Primers and probes for Taqman™ real-time PCR and RT–PCR

Gene	Oligo	Sequence	Length	Position	Exon(s)	Accession no.*
CAV ORF1	Q5′	5′-GCCCCGGTACGTATAGTGTGAG-3′	22-mer	989–1010	N/A†	L14767
	Probe	5′-(6FAM)-CTGCCGAACCCCCAATCT-ACTATGACTATCC-(TAMRA)-3′	31-mer	1012–1042
	Q3′	5′-CCGTGAGAAATATGATTCCTTGG-3′	23-mer	1047–1069
IFN-γ	Q5′	5′-AAACAACCTTCCTGATGGCGT-3′	21-mer	408–428	3/4	U27465
	Probe	5′-(6FAM)-TGAAAGATATCATGGACC-TGGCCAAGCTC-(TAMRA)-3′	29-mer	437–465
	Q3′	5′-CTGGATTCTCAAGTCGTTCATCG-3′	23-mer	467–489
IL-1β	Q5′	5′-GCTCTACATGTCGTGTGTGATGAG-3′	24-mer	572–595	5/6	Y15006
	Probe	5′-(6FAM)-CCACACTGCAGCTGGA-GGAAGCC-(TAMRA)-3′	23-mer	607–629
	Q3′	5′-TGTCGATGTCCCGCATGA-3′	18-mer	634–651
IL-2	Q5′	5′-GATTCATCTCGAGCTCTACACACC-3′	24-mer	191–214	2/3	AF017645
	Probe	5′-(6FAM)-CTGAGACCCAGGAGTG- CACCCAGC-(TAMRA)-3′	24-mer	217–240
	Q3′	5′-ACCACTTCTCCCAGGTAACACTG-3′	23-mer	249–271
GAPDH	Q5′	5′-TGACGTGCAGCAGGAACACT-3′	20-mer	26–45	1/2/3	K01458
	Probe	5′-(TET)-AAGGCGAGATGGTGAAAG-TCGGAGTCAA-(TAMRA)-3′	28-mer	49–76
	Q3′	5′-GTGACCAGGCGGCCAATAC-3′	19-mer	88–106
28S	Q5′	5′-GGCGAAGCCAGAGGAAACT-3′	19-mer	4703–4721	N/A	X59733
	Probe	5′-(VIC)-AGGACCGCTACGGACC- TCCACCA-(TAMRA)-3′	23-mer	4723–4745
	Q3′	5′-GACGACCGATTTGCACGTC-3′	19-mer	4746–4764

^*GenBank accession number.

^†N/A = Not Applicable.

Cycling parameters.

The cycling parameters for the detection of CAV DNA and mRNA have recently been established.³⁵ Briefly, for the detection of DNA all samples were incubated at 50° for 2 min, then at 95° for 10 min to activate the AmpliTaq Gold polymerase (PE Biosystems), followed by 40 cycles consisting of denaturation at 95° for 15 s, and annealing/extension at 60° for 1 min. The RT–PCR parameters began with a hold at 48° for 30 min for reverse transcription, followed by a hold at 95° for 10 min, followed by 40 cycles consisting of 95° for 15 s and 60° for 1 min.

Standards.

To determine viral load, 10-fold dilutions ranging from 1 to 10⁶ copy numbers of the plasmid pBP-CIAΔ3 (7·8 kb) was used as previously described to generate a standard curve.³⁵ Standards for all CAV and cytokine RT–PCR assays were generated using a twofold dilution series of known positive total RNA samples for a given transcript. For the CAV standard, RNA from CIA-1 strain-infected CU147 cells was used. For the IFN-γ standard, CU205 RNA was used, since CU205 constitutively expresses IFN-γ.^45,46 For IL-2 and IL-1β, selected splenic RNA samples previously testing positive by qualitative RT–PCR were used. Transcript levels were all expressed in relative terms since the exact number of transcripts in each standard was not known.

Analysis.

The results for PCR and RT–PCR experiments were analysed using Sequence Detection Systems v.1.6.3 software. The standard curve generated for each primer set in an assay was used to extrapolate the amounts in the unknown samples.

To ensure comparison of equivalent amounts of DNA in determination of viral load, real-time PCR for the inducible nitric oxide synthetase (iNOS) gene was also performed on all DNA samples. The plasmid pBS–iNOS was used to generate a standard curve from which the relative amounts of DNA were extrapolated. Only samples exhibiting equivalent amounts of iNOS DNA within a factor of 10 were analysed for CAV load. Viral and cytokine transcript levels were normalized using the values for the housekeeping gene glyceraldehyde phosphate dehydrogenase (GAPDH) in Exp. III–V. A standard curve was generated for the GAPDH using twofold dilutions of splenic RNA. The viral or cytokine transcript level for each sample was then divided by the corresponding value of the GAPDH transcript level.

Chromium release assay

Effector cells were prepared from spleens at 7 days p.i. as described.⁴⁷ Briefly, spleens were decapsulated and pushed through a 0·60-µm pore-size nylon mesh (Tetko, Inc., Kansas City, MO) to create single cell suspensions. Cells were resuspended in phosphate-buffered saline (PBS) and centrifuged over Ficoll-Paque (Amersham Pharmacia Biotech) at 500 g for 20 min to remove red blood cells. Lymphocytes were harvested from the interface, washed once with PBS, and resuspended to a final concentration of 5 × 10⁷ cells/ml in LM20 (RPMI-1640 + 20% FBS; Gibco Life Technologies, Inc., Gaithersburg, MD). The target cell lines CU91, CU205, and/or CU371 were labeled with 150–200 µCi of Na⁵¹CrO₄ (New England Nuclear, Boston, MA) as previously described.⁴⁷ CRA were performed in triplicate at an effector:target cell ratio of 100 : 1 in 96-well round-bottomed plates (Costar, Corning, Inc., Corning, NY) using 5 × 10⁴ target cells per well as previously described^33,47 except that radioactivity was counted in a Trilux 1450 Microbeta (EG & G Wallac, Turku, Finland) scintillation counter. Following a 4-hr incubation at 38°, the plates were centrifuged at 500 g for 10 min. Half of the supernatant (100 µl) from each well was harvested, mixed with an equal volume of SuperMix scintillation fluid (EG & G Wallac) in Microbeta 96-well flexible plates and counts per minute (c.p.m.) were determined for each well based on 5 min counts. The pellets were resuspended in the remaining supernatant, and half (50 µl) from each well was transferred to a new Microbeta plate containing 50 µl of 2% Triton-X and lysed overnight. SuperMix (100 µl) was then added to the lysate in each well and mixed by shaking the plates gently on a Vortex Genie 2 (Fisher Scientific, Pittsburgh, PA) for 5–10 min, and the c.p.m. were determined. The percent specific release (% SR) was calculated using the following formula: % SR = [(c.p.m. supernatant of sample − c.p.m. supernatant avg. control)]/[(c.p.m. pellet + supernatant − c.p.m. supernatant avg. control)] × 100.

Statistical analysis

Student's t-tests⁴⁸ were performed to determine statistical significance between different treatment groups for viral and cytokine transcript levels. For CRA analysis, the percent releases (% release = c.p.m. supernatant/total c.p.m.) between control and infected groups were also compared using the Student's t-test.

Experimental design

Three types of experiments were designed. Exp. I, consisting of two trials which are representative of a series of trials, was used to determine if the generation of antigen-specific CTL was influenced by natural exposure to CAV in the presence or absence of maternal antibodies. Because this experimental approach depended on natural infection with CAV, which is difficult to control, chickens in Exp. II–V were experimentally infected with the CIA-1 strain. Exp. II was a pilot experiment designed to examine if experimental infection of CAV by intramuscular injection influenced transcriptional activity of selected cytokines. The results of this experiment were used to design Exp. III-V to study the effects of experimental infection with CAV by intramuscular injection in CAV-antibody positive and negative chicks on the generation of CTL, cytokine expression and CAV replication. The design of each experiment is detailed below.

Experiment I.

Chicks were hatched in the CAV-contaminated SPF facility and transferred at 9 or 30 days to the experimental units. To generate CTL, groups of P2a chickens (n = 6) were inoculated at 9 or 30 days of age with REV-CS in Trial 1 and with MDV strain JM-16/p 19 in Trial 2 or left as uninoculated controls. The infected and control groups were housed in separate experimental units. At 7 days p.i. all birds were killed and spleens and sera were collected. Spleen cells were prepared and tested in CRA against CU91 and CU205 (Trial 1) and CU371 (Trial 2). DNA was extracted from leftover spleen cells from selected samples and used to determine the presence of CAV DNA and β-actin (DNA control) by PCR analysis. Sera were analysed for CAV antibodies by ELISA.

Experiment II.

N2a chickens from the SPF flocks were hatched in CAV-free experimental units. All chicks were tested for CAV maternal antibodies at 10 days of age by ELISA and positive chicks were randomly divided into 6 groups (n = 6). At 14, 21, and 28 days of age, groups 2, 4, and 6, respectively, were inoculated with CIA-1, while groups 1, 3, and 5 served as uninoculated controls. Details on numbers of chicks/treatment/group are presented in Table 2. All groups were housed in separate positive-pressurized isolator units for the duration of the experiment. Two or three birds from each group were killed at 4 and 7 days p.i. and spleens and sera were collected. DNA and RNA were extracted from each spleen sample and used in qualititative PCR and RT–PCR assays for CAV and various cytokine transcripts.

Table 2.

Cytokine expression in CAV maternal antibody-positive chickens experimentally inoculated with CAV at 14 and 21, and 28 days of age (Exp. II)

	Treatment			CAV antibodies at		No pos/total in RT–PCR for
Group	CAV	Age of infection (days)	Examination at days p.i.	10 days of age	Day of examination	IL-2	IL-1β	IFN-γ	β-actin
1	–	14	4	3/3	3/3	3/3	3/3	3/3	3/3
			7	3/3	2/3	3/3	3/3	3/3	3/3
2	+		4	3/3	3/3	3/3	3/3	3/3	3/3
			7	3/3	3/3	3/3	3/3	2/3	3/3
3	–	21	7	2/2	NT	2/2	2/2	0/2	2/2
4	+		7	3/3	NT	3/3	2/3	3/3	3/3
5	–	28	4	3/3	1/3	2/3	3/3	1/3	3/3
			7	2/2	1/2	1/2	2/2	1/2	2/2
6	+		4	3/3	1/3	0/3	3/3	1/3	3/3
			7	3/3	1/3	3/3	1/3	3/3	3/3

Experiments III–V.

Eighty-seven N2a chicks from the SPF flock were hatched in CAV-free experimental units and randomly assigned to three different experiments (Exp. III–V). At 14–17 days of age the maternal antibody status for CAV was determined for all birds. Because the breeding flock had not completely seroconverted for CAV, a number of chicks were negative for CAV antibodies at 14 days of age. Antibody-positive and -negative chicks were divided over the different treatment groups in each experiment (see Tables 3–5 for details). Chicks were inoculated with REV-CS, CIA-1, CIA-1 + REV-CS, or left as uninoculated controls at 15 (Exp. III), 30 (Exp. IV) or 45 (Exp. V) days of age as outlined in Tables 3–5. Based on the data obtained in Exp. I, additional groups (n = 18) of 9- or 10-day-old N2a birds were added to Exp. IV and V, respectively, as a control for the CTL assays. These chickens were inoculated at the same time as the 30- and 45-day-old chickens in Exp. IV and V, respectively, with REV-CS, CIA-1 + REV-CS, or left as uninoculated controls (n = 6/treatment). Birds were euthanized at 7 or 14 days p.i. as outlined in Tables 3–5. Spleens were harvested for CRA assays at 7 days p.i., and for analysis of CAV replication and cytokine transcription at 7 and 14 days p.i.. Sera were also collected at the time of death for all chickens and at the time of experimental infection of the 30- and 45-day-old (Exp. IV and V, respectively) chickens to evaluate CAV antibody status.

Table 3.

Experimental infection of 9– to 15–day-old, CAV maternal antibody-negative chickens with CAV results in increased viral load and viral transcript levels and abrogation of REV-specific CTL responses (Exp. III–V)

		Treatment
			Age in days (d) at		Virus challenge		CAV Antibodies at			CAV replication
Exp	Group	No. birds	harvest	CAV	REV	14 days	Challenge	Harvest	Viral DNA load	Rel mRNA level*	% Specific release ± SEM for REV CTL†
III	1	4	15	22	–	–	+	ND	–	0	1	ND
	2	4	15	22	+	–	+	ND	–	2·4 ± 2·5 × 101	1	ND
	3	4	15	22	+	–	–	ND	–	3·3 ± 2·4 × 106	5·6 ± 3·5 × 104	ND
IV	1	6	9	16	–	–	NA	ND	+	ND	1	0
	2	6	9	16	–	+	NA	ND	+	ND	1	14·4 ± 7·5
	3	3	9	16	+	+	NA	ND	+	ND	1	21·6 ± 7·7
	4	3	9	16	+	+	NA	ND	–	ND	1·5 ± 1·4 × 106	0
V	1	6	10	17	–	–	NA	ND	+	ND	1	ND
	2	6	10	17	–	+	NA	ND	+	ND	1	13·8 ± 9·3
	3	6	10	17	+	+	NA	ND	+	ND	1·3 ± 0·6 × 101	12·5 ± 8·9

^*Relative to average transcript level of age-matched control group (defined value of 1).

^†% Specific release using syngeneic CU205 target cells.

Table 5.

Experimental infection of 45-day-old, CAV maternal antibody-negative chickens with CAV results in increased viral load and viral transcript levels and abrogation of REV-specific CTL responses (Exp. V)

		Treatment
		Age in days at		Virus challenge		CAV Antibodies at			CAV replication
Group	No. birds	infection	harvest	CAV	REV	14 days	45 days	Harvest	Viral DNA load	Rel mRNA level*	% Specific release ± SEM for REV CTL†
4	6	45	52	−	−	+	−	−	0	1	ND
5	6	45	52	−	+	+	−	−	0	1	10·0 ± 7·2
6	6	45	52	+	−	+	−	−	1·2 ± 1·9 × 106	2·7 ± 3·7 × 103	0
7	5	45	52	+	+	+	−	−	2·6 ± 2·2 × 107	1·0 ± 0·9 × 105	4·6 ± 2·6
8	4	45	59	−	−	+	−	−	0	1	ND
9	4	45	59	−	+	+	−	−	0	1	ND
10	6	45	59	+	−	+	−	+	3·0 ± 3·3 × 107	2·9 ± 4·6 × 102	ND
11	5	45	59	+	+	+	−	+	3·2 ± 1·8 × 107	3·4 ± 3·5 × 102	ND

^*Relative to average transcript level of age-matched control group (defined value of 1).

^†% Specific release using syngeneic CU205 target cells.

Results

Development of CTL responses in CAV maternal antibody-positive chickens infected with CAV despite viral DNA presence in all groups (Exp. I)

Because the role of neutralizing antibodies is critical in conferring protection from CAV-induced immunosuppression, CTL responses in CAV-antibody positive chickens were compared with responses in CAV antibody-negative chickens. REV-specific (Trial 1) and MDV-specific (Trial 2) CTL were generated in antibody-positive chicks infected at 10 days but not in antibody-negative chickens infected at 30 days of age (Fig. 1). As expected, no specific release was detected against the allogeneic REV target cell line (CU205) for either age group in Trial 1 (data not shown).

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.

To determine if the absence of CTL responses correlated with the presence of CAV antibodies or DNA, sera were analysed for CAV antibody status and spleen cell DNA was subjected to qualitative CAV PCR analysis, followed by hybridization to a CAV-specific probe. Only antibody status consistently correlated with CTL responses (Fig. 1), although the presence of viral DNA did appear to correspond to some degree with the magnitude of specific release from individual birds (data not shown).

Influence of CAV infection on cytokine transcription as maternal antibodies wane (Exp. II)

The effects of experimental infection with CAV on IFN-γ, IL-1β, and IL-2 transcript levels were examined in maternal antibody-positive chickens inoculated at 14, 21, or 28 days of age. All birds were negative for CAV mRNA in the RT–PCR assays, while the β-actin samples were positive suggesting that the maternal antibodies prevented or reduced viral replication. The results of qualitative RT–PCR assays for cytokines using RNA from infected spleen material harvested at 4 and 7 days p.i. are summarized in Table 2. There were no differences between control and infected chickens infected at 14 days of age (groups 1 and 2). Infection at 21 days of age (group 4) resulted in the activation of IFN-γ compared to the controls at 7 days p.i.. IFN-γ transcripts were also detected at 7 days p.i. in all chickens infected at 28 days of age (group 6) and in 1 of 2 chickens from the age-matched controls (group 5). These data suggest that CAV infection may activate the transcription of IFN-γ. Infection of 28-day-old chickens resulted also in the absence of IL-2 transcripts at 4 days p.i. and the absence of IL-1β transcripts in 2/3 chickens at 7 days p.i.. These two birds were negative for CAV antibodies at 7 days p.i..

Impaired CTL responses in chickens experimentally infected with CAV in the absence of CAV maternal antibodies (Exp. III–V)

The relationship between antigen-specific CTL response, maternal antibody status, CAV DNA load, and CAV transcript levels was examined in different age groups of chickens infected with CAV by intramuscular inoculation (Exp. III–V).

CAV replication and REV-specific CTL in chickens infected between 9 and 15 days of age.

CAV maternal antibody-positive 9- and 10-day-old birds inoculated with CIA-1 or REV + CIA-1 (Table 3: Exp. III, group 2, Exp. IV and V, groups 3) had negligible CAV DNA load and/or transcription levels. In contrast, the CAV antibody-negative hatchmates inoculated with CIA-1 or REV + CIA-1 (Table 3: Exp. III, group 1, Exp. IV, group 4) had high levels of CAV DNA, and/or CAV transcript levels relative to uninfected controls (Table 3). Significant REV-specific CTL activity was detected in all groups that were inoculated with REV alone compared to uninoculated control groups (P < 0·05) (Table 3). CTL were also present at 7 days p.i. when maternal antibody-positive chickens were inoculated with CIA-1 + REV at 9 and 10 days of age (Table 3: Exp. IV, group 3; Exp. V, group 3). CTL did not develop when maternal antibody-negative chickens were infected with CIA-1 + REV (Table 3: Exp. IV, group 4).

CAV replication and REV-specific CTL in chickens infected at 30 and 45 days of age.

Similar results were obtained when chickens were infected at 30 days of age with CIA-1 or REV + CIA-1 and examined at 7 days p.i. (Table 4: Exp. IV, groups 5–10). The maternal antibody status at 14 days of age determined if CAV replication occurred even while CAV antibodies could not be demonstrated at the time of experimental infection. CAV transcription was sharply reduced at 14 days p.i. (Table 4: group 12) compared to 7 days p.i. (Table 4: groups 8 and 10), but the CAV DNA load remained high. CAV antibodies could be demonstrated in the virus-infected group at 14 days p.i. (Table 4: group 12). REV-specific CTL responses at 7 days p.i. in dually infected birds that were positive for CAV maternal antibodies at 14 days of age were comparable to the responses in chickens only inoculated with REV (Table 4: group 9 and 6, respectively). In contrast, CTL were not detected in dually infected chickens that were negative for CAV maternal antibodies at 14 days of age (Table 4: group 10). The two groups infected with CIA-1 alone (Table 4: groups 7 and 8) were negative for REV-specific CTL as expected and served as negative controls for the CTL assays.

Table 4.

Experimental infection of 30-day-old, CAV maternal antibody-negative chickens with CAV results in increased viral load and viral transcript levels and abrogation of REV-specific CTL responses (Exp. IV)

	Treatment
		Age in days at		Virus challenge		CAV Antibodies at			CAV replication
Group	No. birds	infection	harvest	CAV	REV	14 days	30 days	Harvest	Viral DNA load	Rel mRNA level*	% Specific release ± SEM for REV CTL†
5	4	30	37	−	−	+	−	−	0	1	ND
6	4	30	37	−	+	+	−(+)‡	−	0	1	11·7 ± 8·4
7	5	30	37	+	−	+	−	−	8·5 ± 6·3 × 101	1	0
8	3	30	37	+	−	−	−	−	2·3 ± 1·1 × 106	1·2 ± 0·5 × 104	0
9	4	30	37	+	+	+	−	−	5·8 ± 4·1 × 101	1	8·2 ± 6·3
10	4	30	37	+	+	−	−	−	4·0 ± 0·6 × 107	4·7 ± 3·4 × 105	0
11	4	30	44	−	−	+	−	−	0	1	ND
12	4	30	44	+	−	+	−	−(+)§	1·1 ± 1·4 × 106	1·9 ± 2·6 × 102	ND

^*Relative to average transcript level of age-matched control group (defined value of 1).

^†% Specific release using syngeneic CU205 target cells.

^‡Two of the chickens in this group were positive for CAV maternal antibodies.

^§Two of the chickens in this group were positive for CAV antibodies.

The CAV maternal antibody status at 14 days did not influence viral replication when chickens were inoculated at 45 days of age (Exp. V, Table 5).

All chickens inoculated with CIA-1 or CIA-1 + REV had high levels of CAV DNA and transcripts at 7 days p.i. (Table 5, groups 6 and 7). CAV DNA levels remained high at 14 days p.i., but the level of viral transcripts was sharply reduced and CAV antibodies were present (Table 5: groups 10 and 11). One chicken in group 7 of Exp. V was positive for antibodies at 7 days p.i.. It had a viral titre load of 10³ and was not included in the analysis. All chickens that were not inoculated with CIA-1 (groups that were not inoculated or inoculated with REV alone) remained negative for CAV viral DNA and transcripts indicating that latent virus was not transferred from the hens to their offspring or that it remained latent in the offspring. Infection of 45-day-old chickens with CIA-1 + REV significantly impaired CTL responses but did not completely abrogate the response. The chicken that was antibody positive at 7 days p.i. had high CTL activity with 24·6% SR.

The combined data on CTL activity in the different age groups clearly demonstrate that antigen-specific CTL are not generated when CAV is actively replicating at the same time.

Influence of infection with REV and/or CAV on IFN-γ, IL-1β, and IL-2 transcript levels

To determine if the increased CAV DNA and viral transcription can cause an alteration of cytokine levels important for the generation of CTL, transcript levels of IFN-γ, IL-1β, and IL-2 were examined by real-time RT–PCR for all birds. No significant changes in the levels of IL-1β and IL-2 transcripts were observed between treatment groups at either 7 or 14 days p.i. (Figs 2 and 3, respectively).

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.

The analysis of the impact of infection with REV, CAV, or REV + CIA-1 on IFN-γ transcription was complicated by the finding that REV or REV + CIA-1 induced significant increases in IFN-γ mRNA levels at 7 days p.i. in all three experiments (Fig. 4a–c) compared to age-matched controls. Inoculation of antibody-positive chickens with CIA-1 alone also increased the level of IFN-γ transcription significantly compared to age-matched controls (P < 0·05) in CAV maternal antibody positive chickens at 7 days p.i. (Exp. III and Exp. IV). Even in CAV maternal antibody-negative birds infected with CIA-1 at 30 days of age a significant increase was noted. The differences in IFN-γ transcription levels at 7 days p.i. between REV or REV + CIA-1 vs. CIA-1 in antibody-positive and -negative chickens infected at 30 days of age (Fig. 4b) are significant at P < 0·02. At 14 days p.i. no significant differences were detected.

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.

Discussion

This report provides for the first time functional evidence that the immunosuppressive effects associated with subclinical CAV infection are, at least in part, associated with the impairment or absence of pathogen-specific CTL. The absence of REV-specific CTL at 7 days p.i. with CAV + REV in chickens that lacked CAV-specific maternal antibodies contrasts with the presence of the CTL after infection of chickens possessing CAV-specific maternal antibodies. The results also provide quantitative data confirming that the presence of maternal antibodies is critical in protection from viral replication. The fact that only 76% of the experimental birds tested at 2 weeks of age in Exp. III–V were positive for maternal antibodies was in accordance with the seroconversion status (73%) of the parental flock (M. M. Miller and K. A. Schat, unpublished data). This became an experimental advantage to further demonstrate the importance of maternal antibodies to CAV in protection from immunosuppressive effects and viral replication in age-matched chickens. It is of interest that the protective effects of CAV maternal antibodies were still present, when chickens challenged at 30 days of age were not longer CAV antibody-positive as determined by ELISA.

These results have important consequences for the poultry industry because CAV is likely to be a much more important pathogen than was previously believed. Protective immunity induced by vaccination or natural infection with coccidia or viruses requires a strong cell-mediated component for protection or recovery from infection as has been documented for coccidiosis,⁴⁹ MDV,⁵⁰ and infectious bronchitis (IB) virus.⁵¹ Increased incidence of IB in broilers has been linked with an increased incidence of CAV in thymus tissues of affected birds (F. Hoerr, University of Alabama, Auburn, AL, personal communication). This suggests that CAV can have an impact on IB incidence, probably when vaccine-induced antibodies to IB virus provide suboptimal protection as a consequence of mutations in the S1 gene of IBV.

The finding that CAV impairs the development of pathogen-specific CTL may have important implications for understanding the pathogenesis of other circo(-like) viruses that have recently been isolated from different animal species including humans. For example, columbid circovirus and canary circovirus are often isolated from pigeons and canaries with immunosuppressive symptoms^52,53 but the mechanism of the immunosuppression is poorly understood. The porcine circovirus serotype 2 has also been linked to immunosuppressive disorders and Darwich et al.⁵⁴ described an actual decrease in CD8⁺ cells in experimentally infected piglets. In addition, the human virus group known as TT virus resembles CAV in some aspects, especially the promoter/enhancer and genomic organization. Infections with TT virus lead to chronic viraemia. Although these infections have not been linked to a specific disease,⁵⁵ the possibility can not be excluded that immunosuppressive disorders are linked to TT virus infection.

It is known that CAV-neutralizing antibodies are specific for a conformational epitope formed by the interaction of VP1 and VP2, the major capsid and scaffolding proteins, respectively. These antibodies obtained either through active immunity or passive transfer from the laying hen confer protection from the disease^4,5 and were previously believed to clear the virus. However, recent studies concluded that despite seroconversion of the flock viral DNA could be detected in gonadal tissues and to a lesser degree in spleens,^14,15 suggesting a more complex pathogenesis of the virus. Thus CAV may continue to exist in a latent state in the offspring to be reactivated after sexual maturity. This may result in a subclinical infection with immunosuppressive consequences including impaired capacity for CTL generation. As a consequence, it can be argued that the chickens used in experiments II–V may have been infected vertically in addition to the inoculation with CIA-1. However, the absence of viral transcripts or high copy numbers of viral DNA in the uninfected and REV-infected, maternal antibody-negative chickens indicates that virus was not vertically transmitted or that it remained latent. It certainly did not cause problems for the negative controls.

Whether the impairment of CTL development is a direct result of CAV infection, i.e. by destruction of lymphoid precursors, or an indirect result, such as alteration of relevant cytokine levels, remains unclear. Because commercial ELISA assays are not available for the detection of chicken cytokines, relative transcript levels were used to evaluate the effects of CAV infection on IFN-γ and IL-2, which are associated with antigen-specific CTL responses, and IL-1β. CAV did not significantly influence the transcription levels of IL-2 or IL-1β at 7 and 14 days p.i.. Infection with CAV in antibody-positive and -negative chickens resulted in a low but significant increase in IFN-γ transcription at 7 days p.i.. Interestingly, this does not seem to be related to virus replication, because maternal antibodies protected against virus replication. These results are in contrast with the work by Adair et al.²⁹ who reported suppression of IL-2 activity at 8 and 15 days p.i. as well as significant reductions in IFN-γ titres at 15, 22, and 29 days p.i. following CAV infection. Similarly, McConnell et al.¹³ found that natural exposure of 3-week-old chickens to CAV resulted in impaired functional activity of IFN-γ, IL-1β, and IL-2 at 14, 21, and 28 days p.i., and also at 42 days p.i. for IFN-γ and IL-1β. The differences between this study and their work could be because different techniques were used to evaluate the effects of CAV infection on cytokine production. Adair et al.²⁹ and McConnell et al.¹³ used supernatant fluids from spleen cell cultures treated with concanavalin A or adherent splenocytes stimulated with Staphylococcus aureus lysates in bioassays which could have measured a mixture of cytokines. Unfortunately, ELISA assays for avian cytokines were not commercially available at the time of the study. For this reason, the studies presented in this paper used RT–PCR assays to specifically measure transcript levels. An alternate explanation for the differences may be that CAV infection interferes with post-transcriptional regulation of cytokine production.

The impaired cytokine production reported by McConnell et al.¹³ started at 2 weeks after CAV exposure, while the impairment of CTL responses was found at 7 days p.i. It seems therefore unlikely that the impairment of the CTL responses is related to the immunosuppressive effects caused by altered cytokine responses starting at 2 weeks post exposure.¹³ However, impaired cytokine responses after 2 weeks may contribute to an overall state of immunosuppression that could affect CTL development when exposure to a secondary pathogen occurs one to two weeks post-CAV exposure. A more likely scenario for the effects of CAV infection on CTL development is that CTL precursor populations are reduced as a consequence of CAV infection. CAV infects mature spleen cells²⁷ and subsequently causes apoptosis of these cells.⁷ It has also been previously demonstrated that clinical CAV infection results in lymphoid destruction between 14 and 21 days p.i. of both CD4⁺ and CD8⁺ T-cell subsets.²⁸ It is certainly plausible that apoptosis of CTL precursors occurs to a lesser extent during subclinical infection, which subsequently may lead to decreased cytokine production and increased susceptibility to secondary infections.

Infection with REV alone resulted in a 10-fold increase in IFN-γ mRNA levels in 9- to 10- or 30-day-old birds. The increase in IFN-γ levels was augmented or maintained by dual infection with REV and CAV in birds possessing maternal antibodies and lacking maternal antibodies. It is not clear why REV is such a strong inducer of IFN-γ. REV-transformed cell lines are strong producers of IFN-γ,^45,46 but these cell lines are expressing v-rel, an oncogene belonging to the nuclear factor-κβ rel family of transcription factors, which are capable of up-regulating genes.^56,57 However, the CS strain of REV lacks the v-rel gene and another mechanism must be responsible for the strong induction of IFN-γ transcription.