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(A) (B)
10 10
15 15
6 6 10 10
mRNA log 2 copy number mRNA log 2 copy number 5 5 8 8 10 10 5 5 7 7 6 6
10
10

5 5 5 5 5 5 5
3 3
2 2

9 9 10 1110 11 12
B B B B 3 3 3 3 4 4 4 4 5 5 5 5 6 6 6 6 7 7 7 7 8 8 8 8 9 9 9 9 B B BB 1 1 11 2 2 22 3 3 33 4 4 44 5 5 55 6 6 66 7 7 77 8 8 88 9 10 11 12 129 10 11 12
Embryonation day
Embr
on
Embr
ti
on
y
on
on
on
da
on
Embryonation day
da
y
Embr y y y on a a a ti ti ti on da y Embr y y y on a a a ti on da y y y
Embr
Embr
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on
Figure 9.10 Determination of CAV transcripts in embryos using real-time quantitative RT-PCR. Total RNA was extracted from 10 fertile
Figure 10.
blastoderm (B) samples and 10 embryos/sampling day at embryonation day (ED) 1 and 3–9 in Expt. 1 (A), and from 10 blastoderm (B)
samples and 10 embryos/sampling day at ED 1–12 in Expt. 2 (B). RNA copy numbers were normalized for equal loading using the cellular
housekeeping gene, GAPDH. The results are reported as log 2 of the average copy number and error bars indicate the standard error of
the mean. Numbers above the bars indicate the number positive/10 samples. Sampling days without a number had no positive samples.
Unpublished data from M.M. Miller, K.A. Stucker and K.A. Schat, with permission from Miller and Stucker. From Schat 2009, CTMI 331, pp.
151–184, with permission from Springer-Verlag.
rate to sentinel birds kept in the same isolator as infected chick- Transmission of CAV in conventional chickens
ens (Tan and Tannock, 2005). Miller et al. (2001) reared 90 There are several ways that CAV can be transmitted, and the
1-day-old SPF chickens from a commercial SPF producer in manner of transmission has important consequences for the
individual cages up to 20 weeks of age. These chicks were the development of disease: (1) horizontal transmission, (2) vertical
offspring of an antibody-negative parent flock. At 4 weeks of transmission when antibody negative hens become infected by
age one bird was antibody positive and culled. Antibody tests natural exposure or through virus-positive semen, (3) the use of
for all remaining chickens were negative at 6, 8 and 12 weeks of contaminated vaccines.
age. At 16 and 20 weeks of age, two birds were antibody posi-
tive and both birds were immediately culled. The three positive Horizontal infection
birds were also positive in a nested PCR assay. In addition, one Virus is spread into the environment through faeces, feathers
hen was culled for a toe problem and tested positive in the and perhaps tears. Horizontal infection occurs by the oral and
PCR assay but was antibody negative. All remaining birds were or ocular route. Virus shedding into the environment through
negative at the termination of the experiment. These results faeces was shown by Hoop (1992) and Dren et al. (2000) by
certainly suggest that CAV does not spread rapidly when the virus isolation after experimental infection. The source of the
birds are kept in low density cages. virus in the faeces is most likely coming from infected lympho-
Based on these data it was proposed by Miller and Schat cytes in the intestinal tract and caecal tonsils (Smyth et al., 1993;
(2004) that CAV can cause a latent infection with control of van Santen et al., 2004a). CAV-positive cells were consistently
transcription tightly regulated by a balance between negative found in lymphoid aggregates in the ascending duodenum with
regulators (COUP-TF1 and δEF1; Miller et al., 2008) and posi- occasionally positive intra-epithelial lymphocytes between 7
tive regulators (oestrogen; Miller et al., 2005). The concept of and 13 days pi of 1-day-old SPF chicks (Smyth et al., 1993).
latency was previously raised by McNulty (1991) and Dren et al. Van Santen et al. (2004a) found CAV DNA in caecal tonsils
(2000). It is not clear how latent virus is maintained in the host. and Harderian glands with peak concentrations at 10 and 14
Schat (unpublished data) transfected MSB1 cells with mutated days, respectively, post im inoculation of 1-day-old chicks. These
double-stranded circular CAV DNA lacking the start-codon for peak concentrations occurred a few days later following oral
VP3, so that virus could not replicate. After eight passages of the challenge. The presence of rather high concentrations of viral
MSB1 cells viral DNA could still be recovered suggesting that cir- genomes in the Harderian gland suggests that tears could be
cular dsDNA was replicating as a minichromosome or that it may a source of infectious virus. Feathers are probably the second
have integrated in MSB1 DNA. The latter is a distinct possibility major source of virus contaminating the environment. Smyth et
because integration of circular ssDNA viruses into host genomes al. (1993) noticed a few CAV-antigen positive cells in the feather
can occur, but the mechanism(s) involved in integration remain pulp, which are likely to be infected lymphocytes. Davidson et
obscure (Krupovic and Forterre, 2015). al. (2008) used feather extracts from CAV -ositive chickens to