Detection of Chicken Anemia Virus DNA in Embryonal Tissues and Eggshell Membranes

Abstract

Chicken infectious anemia virus (CIAV) is a ubiquitous and highly resistant virus of chickens that causes anemia and death in chicks less than 3 wk of age and immunosuppression in chickens older than 3 wk of age. The production of specific-pathogen-free eggs free of CIAV is essential for research and vaccine production. Currently, flocks are screened for CIAV by antibody tests to ensure freedom from CIAV infection. Recent evidence, however, indicates that chickens may carry and vertically transmit CIAV DNA independently of their antibody status. In this study, we tested embryos and eggshell membrane residues by nested polymerase chain reaction (PCR) as a sensitive method of detecting CIAV DNA. CIAV DNA could be detected in the blastodisks and semen obtained from antibody-positive and -negative chickens. Examination of different tissues between 18 and 20 days of incubation indicated that many but not all organs of individual embryos were positive. The lymphoid organs and gonads had the highest incidence of CIAV DNA, which was significantly different (P < 0.05) from the incidence in the liver. Eggshell membrane samples from embryos or newly hatched chicks were an excellent noninvasive source for the detection of CIAV DNA, identifying significantly more positive embryos than did pooled lymphoid organs. The use of dexamethasone injections as a method to improve the detection of carrier birds did not result in an increase of vertical transmission or cause seroconversion in the treated hens. A combination of testing eggshell membrane residues at hatch and periodic testing of blood DNA by nested PCR can be used to identify chickens carrying CIAV DNA and may be used to eradicate carrier birds.

Um protocolo de "nested-PCR" para detecção do vírus da anemia das galinhas

“…A alta sensibilidade da "nested-PCR" também é uma característica importante nas investigações que visam determinar a transmissão vertical do vírus ou a provável latência do mesmo (Todd et al 1992, Miller et al 2003. Ao realizar a "nested-PCR" com soro de matrizes com anticorpos para CAV, constatou-se que o DNA do agente pode persistir mesmo depois de ocorrida a soroconversão, como igualmente observado por outros autores (Cardona et al 2000).…”

Negative modulation of the chicken infectious anemia virus promoter by COUP-TF1 and an E box-like element at the transcription start site binding δEF1

“…CAV infects T cell precursors in the thymic cortex and CD8 + splenic lymphocytes and haemocytoblasts in the bone marrow (Adair, 2000;Jeurissen et al, 1992;Noteborn et al, 1994a). There is also evidence that CAV can be present in the reproductive system (Cardona et al, 2000a, b) and be vertically transmitted in antibody-positive or -negative hens (Miller et al, 2003).Sequences in the 59 non-transcribed region of the CAV genome are believed to be the sole promoter enhancer for CAV (Noteborn et al, 1994b;Phenix et al, 1994). This region has four or five direct repeat (DR) regions of 21 bases, with a 12 base insert between the first two (or three) and the last two DR.…”

“…The most wellcharacterized is chicken ovalbumin upstream promoter transcription factor 1 (COUP-TF1) (Cooney et al, 1993;Park et al, 2003). Together, these nuclear receptors can bind a wide range of ERE-like motifs and often compete for binding to the same DNA sequence.Due to the common occurrence of seroconversion at the time of sexual maturation in specific-pathogen-free flocks (Miller et al, 2001) and the presence of viral DNA in male and female gonads (Cardona et al, 2000b) and in freshly laid fertilized eggs (Miller et al, 2003), we hypothesized that CAV gene regulation is influenced by the regulation of Miller et al, 2005), and that the ERE-like sequences from the CAV promoter could compete for binding to unidentified proteins recognizing a consensus ERE. This suggested that members of the nuclear receptor superfamily provide a mechanism to regulate CAV activity when low viral genome copy numbers are present (Miller et al, 2005).…”

“…CAV infects T cell precursors in the thymic cortex and CD8 + splenic lymphocytes and haemocytoblasts in the bone marrow (Adair, 2000;Jeurissen et al, 1992;Noteborn et al, 1994a). There is also evidence that CAV can be present in the reproductive system (Cardona et al, 2000a, b) and be vertically transmitted in antibody-positive or -negative hens (Miller et al, 2003).…”

“…Due to the common occurrence of seroconversion at the time of sexual maturation in specific-pathogen-free flocks (Miller et al, 2001) and the presence of viral DNA in male and female gonads (Cardona et al, 2000b) and in freshly laid fertilized eggs (Miller et al, 2003), we hypothesized that CAV gene regulation is influenced by the regulation of the reproductive system. Previous experiments found a significant increase in CAV promoter-driven expression in the ER enhanced cell line, LMH/2A, when treated with oestrogen (Miller et al, 2005), and that the ERE-like sequences from the CAV promoter could compete for binding to unidentified proteins recognizing a consensus ERE.…”