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Table 9.2. Susceptibility of Marek’s disease virus-derived cell lines developed at Cornell University to infection with the Cux-1 strain of
chicken anaemia virus
Phenotype of the cell lines No. of lines positive a
CD4 CD8 TCR No. of lines 3–4 dpi 5–7 dpi
+ – 2 6 0 5
+ – 3 2 0 1
– + 2 5 2 5
– + 3 4 3 4
– – 2 5 1 5
– – 3 2 2 2
a Determined by indirect immunofluorescence using 50,000 cells/cell line at days post infection.

Figure 9.6 Fluorescent antibody staining of MSB1 cells infected with chicken anaemia virus. Note the positive intranuclear staining. Source:
From Shivaprasad and Lucio, 1994, with permission from the American Association of Avian Pathologists.

the Cux-1 isolate. Stimulation with concanavalin-A (Con-A) of inoculated with the CL-1 isolate at ED5 by the same route. Fifty
spleen and bone marrow cells increased the titre. Thymocytes per cent of these embryos died between ED 16 and 20. The
required the addition of Con-A or 10% ‘conditioned’ medium dead embryos were smaller than controls, and had oedema and
to become susceptible to infection (McNeilly et al., 1994). haemorrhages (Lamichhane et al., 1991). Davidson et al. (2007)
Manoharan et al. (2012) used peripheral blood mononuclear inoculated embryos at ED7 by the yolk-sac route with liver
cells to isolate and propagate CAV from PCR-positive field extracts from 12 flocks, which were CAV-positive by PCR assays.
cases. Con-A stimulation increased the titre and sensitivity of Two of the isolates caused significant growth retardation of the
detection. Although both groups used SPF chickens to obtain embryos. Although a few embryos had lesions such as haemor-
the cells, careful analysis of control versus infected cultures will rhages and pale livers, there was no clear association between
be essential because CAV DNA can be present in SPF chickens lesion development and specific isolates. If embryonated chicken
even in the absence of CAV-specific antibodies (Cardona et al., eggs are used for primary virus isolation, a definitive diagnosis
2000b). needs to be made using other assays such as qPCR.
Chicken embryos 1-Day-old chicks
Embryonated chicken eggs can be used for virus isolation and The first isolates of CAV were obtained by intramuscular inocula-
propagation. Von Bülow and Witt (1986) inoculated 5-day-old tion of 1-day-old SPF chicks. Fourteen to 16 days pi haematocrit
SPF embryos by the yolk-sac route. At embryonation day (ED) values were established to determine if anaemia was present
19, whole embryos without the head and livers were titrated. (Yuasa et al., 1979; Goryo et al., 1985). The use of chicks embryo-
Titres were similar for livers and embryos and showed a 1000-fold nally bursectomized at 18 days of embryonation may increase the
increase over the inoculum. There were no lesions detected in sensitivity due to lack of antibody responses to curtail virus rep-
the inoculated embryos. This finding contrasts with embryos lication. This approach was used by Lucio et al. (1990) to isolate