Department of Biotechnology and Environmental Biology, RMIT University, PO Box 71, Bundoora, Victoria 3083, Australia
Correspondence
Gregory A. Tannock
gtan{at}rmit.edu.au
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ABSTRACT |
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The growth of CAV in chicken embryos was first described by von Bülow & Witt (1986). Most studies of CAV pathogenesis have involved inoculation of chickens by the intramuscular (i.m.) route, which has been shown to be the most sensitive for inducing clinical signs in susceptible day-old chickens (Rosenberger & Cloud, 1989
; Yuasa, 1989
). The size of the CAV inoculum influences the severity of anaemia and other clinical signs and the proportion of chickens infected (Yuasa et al., 1980a
; Rosenberger & Cloud, 1989
; McNulty et al., 1990
). In the present study, the growth of strain 3711, an Australian vaccine virus prepared from infected chickens (Connor et al., 1991
), was examined in various tissues of specific-pathogen-free (SPF) chicken embryos by using a microplate DNA-hybridization assay for estimation of the CAV DNA copy number. These studies were extended to SPF chickens, where its growth was compared with that of other preparations of strain 3711 after passage in cells of the MDCC-MSB1 line. The relationship between the occurrence of clinical signs and viral load was also measured.
CAV vaccine strain 3711 was supplied by Dr Gordon Firth, Intervet (Australia) Pty Ltd, as a liver homogenate in PBS after three passages in SPF chickens and was shown to be free from other avian pathogens. Before use, the virus preparation was clarified, sterilized by filtration and diluted in PBS to contain 40 50 % chicken infectious doses (CID50). Strain 3711 was also adapted to grow to high titre (>106·0 TCID50 in 0·1 ml) in cells of the MDCC-MSB1 line (Tannock et al., 2003). Viruses used in the present study were passaged 65 and 129 times.
CAV DNA extracted from tissue samples by the method of Todd et al. (1992) was amplified with the forward primer 5'-CAGTGAATCGGCGCTTAGC-3' and the reverse primer 5'-GCTCGTCTTGCCATCTTACAG-3'. Standard PCR mixtures (50 µl) contained 2·0 µM each dNTP, 2·0 mM MgCl2, 0·2 µM forward and reverse primers, 1·0 U AmpliTaq DNA polymerase (Roche Molecular Systems) and 5 µl template DNA. Each reaction was allowed to take place for 35 cycles, each consisting of 30 s at 95 °C, 30 s at 55 °C and 1 min at 72 °C.
A 266 bp probe within the 452 bp segment was amplified by PCR using primers 5'-GACCATCAACGGTGTTCAG-3' and 5'-CTCGCTTACCCTGTACTCG-3'. The PCR product was purified and then labelled with Photoprobe biotin (Vector Laboratories), according to procedures described by Forster et al. (1985) and Hibma et al. (1994)
.
The microplate DNA-hybridization assay was based on the method described by Inouye & Hondo (1990). Briefly, the amplified DNA segment (452 bp) was purified (Todd et al., 1992
) and coated onto a microplate well at 37 °C for 2 h. After washing three times, 5 ng heateddenatured biotin probe (266 bp) and 4·5 µg salmon-sperm DNA were added per well and incubated at 42 °C overnight. After a further three washes, 100 µl horseradish peroxidase-conjugated streptavidin (1 : 500) was added to each well and the plate was held at room temperature for 1 h. The wells were again washed and 100 µl TMB substrate (3,3',5,5'-tetramethylbenzidine; KPL) was added per well and incubated for 30 min. The reaction was then stopped by addition of 50 µl 2 M H2SO4 per well and A450 was read. The DNA-hybridization assay could detect 0·0050·025 ng of the 452 bp CAV segment; bands containing 510 ng of the 452 bp segment could be detected by a standard PCR (Tan, 2002
). Novak & Ragland (2001)
also demonstrated that a competitive DNA-hybridization assay in microtitre plates for the detection of CAV was more sensitive than in situ hybridization, dot-blot and ELISA assays.
The Mr of the 452 bp segment was estimated to be 2·9832x105 and its concentration was determined by A260 measurement. The copy number of the 452 bp segment was calculated according to the following formula (Mackay, 1999):
DNA copy number=Avogadro's number (6·02x1023)xmass (g)/Mr.
For internal standardization, serial 10-fold dilutions of the purified 452 bp segment were prepared in parallel. A 50 µl aliquot of each dilution, containing 1011010 copies of the 452 bp segment, was amplified by the standard PCR and the products were quantified by microplate DNA hybridization using the biotin probe. A good linear correlation between A450 and copy number for the 452 bp segment was observed (y=0·1751x0·0561). As each 452 bp segment represented a single CAV genome, the number of CAV DNA templates in samples could be estimated by using the standard PCR to amplify the 452 bp segment, followed by DNA hybridization with the biotin probe and extrapolation from the standard curve. For the assay of tissue extracts, standard preparations containing 104107 copies of the 452 bp segment were run in parallel. Assays were considered valid if the mean A450 of each standard preparation fell within ±1SD of the expected values.
Strain 3711, prepared from infected chickens, was inoculated to 25 5-day-old SPF chicken embryos via the yolk sac. Fifteen eggs were inoculated with PBS as controls and all were examined at days 1620. Deaths occurred in 12 infected embryos (48 %) and in one from the control group (6 %). Of the 12, four appeared normal, four were stunted and four were stunted with unilateral abnormalities of the eye. The organs from six individual surviving embryos at day 20 were harvested and extracts were prepared for testing for CAV viral load by the microplate DNA-hybridization assay. The CAV copy number for the various organs was 3·68·6 log10 (g tissue)1. CAV DNA could be detected in all thymus and pancreas samples [mean viral loads, 8·60±1·08 and 5·60±0·81 log10 copies (g tissue)1, respectively]. For the brain, bursa, kidney, liver and lung samples, CAV DNA was detected in one or two of three samples tested [4·85±0·75 or 7·43±0·62 log10 copies (g tissue)1]. Only one of three duodenum samples tested was positive and the viral copy number [4·60 log10 copies (g tissue)1] was lower than that in other organs.
A further batch of 25 embryos was infected similarly with another batch of CAV vaccine and 15 chickens (60 %) hatched. Three surviving chickens from infected and control groups were euthanized at days 5, 10, 15, 20 and 25 after hatch and individual organ samples were collected and tested for virus load. Hatched chickens inoculated in ovo showed clinical signs, characterized by moderate to severe depression, anorexia, anaemia and dehydration at days 5 and 10 after hatch. One bird from the infected group died at day 15. Fig. 1 shows that the mean body weight of the infected group was lower than that of the controls at day 10 (Student's t-test; P<0·05), but the greatest differences were seen at days 1525 (P<0·01). The thymus : and bursa : body-weight ratios of inoculated chickens were significantly lower than those of the control group at days 515 (P<0·01). However, for the spleen : body-weight ratio, significant differences were noted only between inoculated and control birds at day 25 (P<0·05).
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Extracted DNA was tested by the microplate DNA-hybridization assay. The results (Table 1) indicate that all tested tissues were positive, but highest viral loads were present in the thymus [8·6011·60 log10 copies (g tissue)1]. An unexpected observation was the very high load in the pancreas [7·6010·85 log10 copies (g tissue)1]. Organs with moderate loads were the liver [7·859·53 log10 copies (g tissue)1], spleen [7·609·25 log10 copies (g tissue)1], bursa [5·608·10 log10 copies (g tissue)1], kidney [6·607·85 log10 copies (g tissue)1] and lung [4·608·10 log10 copies (g tissue)1]. Lower loads were present in the brain and duodenum [3·355·60 and 3·105·60 log10 copies (g tissue)1]. Viral loads in the organs of chickens were significantly higher than in those of embryos. The very high load in clotted blood is partly attributable to viraemia and partly due to the growth of CAV in the bone marrow. The presence of CAV DNA in most organs is probably a consequence of viraemia and not an indication that virus replication had taken place. The high CAV load in the pancreas suggests that it, like the thymus, is a primary target organ. Smyth et al. (1993)
described the presence of CAV antigen in the pancreas of infected birds and similar findings have been reported for coxsackievirus B4 virus, which produces infections of the pancreas and a focal myositis of mice (Pallansch & Roos, 2001
).
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All birds infected with CAV by i.m. inoculation developed characteristic anaemia by days 1012 post-infection (p.i.), with peak signs being present at days 1518. Affected birds appeared anorexic and depressed, with pallor of the comb and wattles and ruffled feathers. Only one bird in the group died, at day 20. Birds in groups inoculated by the oral route developed the same clinical signs by day 15 and no deaths occurred during the experimental period, confirming earlier findings (Rosenberger & Cloud, 1989; Yuasa, 1989
) that the i.m. route is most efficient for inducing clinical disease. Neither clinical signs nor deaths occurred in the contact group.
Birds inoculated by the i.m. route showed a significant decline in weight gain after i.m. inoculation at days 10 and 25, compared with the controls (Fig. 2a; P<0·05), and greater differences were seen at days 15 and 20 (P<0·01). Similar results were obtained in the groups inoculated orally at days 15, 20 and 25 (P<0·01), but there was no decline in body weight for the contact group. Only thymic atrophy was observed in i.m.- and orally infected chickens and the thymus : body-weight ratios were greater than for the control group at days 1520 (P<0·01) and 25 (P<0·05). Lower HVs were observed in groups infected by i.m. inoculation. HVs of <27 % were observed at days 1020 but, in 53 % of cases, increases to near-normal values had occurred by day 25 (Fig. 2a
). For groups inoculated orally, HVs of <27 % were seen up to day 20. Most birds were still anaemic by day 25 and recovery to normal values occurred in one-third of birds. There were no changes in the mean HV of the contact group. Overall, no differences in pathogenesis could be detected between the groups inoculated with chicken- or cell culture-grown viruses.
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In the groups inoculated by the i.m. route, CAV DNA could be detected in all organs tested at days 525 p.i., with highest copy numbers being present in samples obtained from clotted blood, pancreas and thymus (Fig. 2b). Peak levels [10·1011·60 log10 copies (g tissue)1] were obtained at days 1020. Intermediate levels [3·359·76 log10 copies (g tissue)1] were present in samples from the brain, bursa, kidney, liver, lung and spleen and lowest levels [3·105·60 log10 copies (g tissue)1] from the duodenum. There were no significant differences in load between chicken- or cell culture-passaged viruses (Fig. 2b
). Peak loads in the thymus, pancreas and blood of chickens inoculated by the i.m. route occurred at day 15 and by the oral route by day 20, although the extent of each was similar. CAV was present in thymus and clotted blood at day 5 p.i. and other organs of chickens inoculated by the oral route by day 10. Highest loads were present in the thymus, clotted blood and pancreas. In the contact group, CAV DNA could not be detected in any organ before day 10. Fewer birds were positive and the viral loads [3·107·76 log10 copies (g tissue)1] in individual organs were significantly lower than those in birds inoculated by the i.m. [3·1011·60 log10 copies (g tissue)1] and oral [3·1010·60 log10 copies (g tissue)1] routes.
Apart from route of inoculation, the ability to produce anaemia after experimental infection is related directly to the virus dose (Yuasa et al., 1979; Yuasa, 1989
; McNulty et al., 1990
). The present study demonstrates, for the first time, a relationship between viral load and the extent of clinical disease. All chickens in groups inoculated by the i.m. and oral routes or by inoculation of embryos showed typical clinical signs. Chickens infected by contact transmission did not show signs and exhibited similar increases in total weight and in the weights of immune organs to those of the controls. Viral loads in all organs of the chickens infected by contact were significantly lower than those infected by the i.m., oral or in ovo routes (Fig. 2b
).
There were no differences in pathogenicity between preparations of strain 3711 that were propagated in chickens or passaged to different levels in the MDCC-MSB1 cell line (Fig. 2a). By contrast, Todd et al. (1995
, 1998)
found that the Cux-1 strain became substantially less pathogenic after 173 and 320 passages in MDCC-MSB1 cells and that the pathogenicity of attenuated viruses could be restored after 10 passages in young chickens. The reasons for these differences are unclear, but could be related to undefined genetic differences between strain 3711 and the Cux-1 strain. There were also no significant differences with respect to viral load, the appearance of clinical signs, reductions in weight gain, declines in HV or levels of antibody induced between chickens inoculated in ovo at day 5 of embryogenesis and others inoculated at day 1 after hatch (Table 1
; Figs 12
). However, mortality rates following egg inoculation were high (48 and 40 %). In support of this, decreases in the bursa : and spleen : body-weight ratios occurred in hatched chickens inoculated in ovo, but not in chickens inoculated at day 1 (Tan, 2002
).
There have been several recent reports on the use of real-time PCR for measuring viral loads of CAV in infected chickens (Markowski-Grimsrud et al., 2002) and for the measurement of viral DNA copy number in isolated lymphocytes (Markowski-Grimsrud & Schat, 2003
). This test has also been adapted to measurement of CAV-neutralizing antibody (van Santen et al., 2004a
). An advantage of the test used in the present study is its low cost and suitability for the simultaneous processing of large numbers of samples without the need for an expensive, dedicated instrument.
Overall, these findings confirm that the bone marrow and thymus are the most important target organs in the pathogenesis of CAV infection. The destruction of haemocytoblasts in bone marrow results in a severe depletion of erythroid and myeloid cells and produces anaemia; the destruction of T-lymphocyte progenitor cells in the thymus results in depletion of mature cytotoxic and helper T cells and induces severe atrophy (Adair, 2000). Virus is spread to other organs by viraemia and lymphoid foci develop in the liver, kidney and lung. However, the reason for higher viral loads in the pancreas is unclear. N.B. A paper on the measurement of peak CAV loads by real-time PCR in chickens infected by the i.m. and oral routes appeared at the time that our paper was submitted, and showed similar results (van Santen et al., 2004b
).
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Received 10 September 2004;
accepted 25 January 2005.
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