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`Dot blot hybridization assay for chicken anemia
`agent using a cloned DNA probe.
`D Todd, J L Creelan and M S McNulty
`1991, 29(5):933.
`J. Clin. Microbiol. 
`  
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`JOURNAL OF CLINICAL MICROBIOLOGY, May 1991, p. 933-939
`0095-1137/91/050933-07$02.00/0
`Copyright © 1991, American Society for Microbiology
`
`Vol. 29, No. 5
`
`Dot Blot Hybridization Assay for Chicken Anemia Agent
`Using a Cloned DNA Probe
`DANIEL TODD,* JULIE L. CREELAN, AND M. STEWART McNULTY
`Veterinary Research Laboratories, Stoney Road, Stormont, Belfast BT4 3SD, United Kingdom
`
`Received 17 August 1990/Accepted 7 February 1991
`
`A dot blot hybridization assay capable of detecting chicken anemia agent (CAA)-specffic DNA in tissues from
`infected birds has been developed. The assay uses a 32P-labeled DNA probe prepared from cloned CAA-specific
`fragments representing the entire virus genome and has a sensitivity limit of between and 1 and 10 pg. DNAs
`from CAA isolates originating in the Federal Republic of Germany, Japan, the United States, the United
`Kingdom, and Australia were detected. Investigation of specimens from experimentally infected chicks
`indicated that virus-specffic DNA was detected in the tissues of birds from 5 through 42 days after infection and
`that greater amounts were usually detected in the thymus than in the spleen, liver, feces, or blood. Tissues from
`specific-pathogen-free and broiler chicks which had become infected at an older age through contact with
`experimentally infected anemic chicks also contained CAA-specific DNA detectable by the assay. Thymuses
`from 1- to 2-week-old chicks from eight commercial broiler flocks which had been showing clinical signs
`characteristic of anemia-dermatitis syndrome were found positive by the hybridization technique, but thymuses
`from chicks obtained from broiler flocks which did not show such signs were found negative. Of the 35 positive
`samples (from 46 samples tested), 19 (54%) contained virus-specific DNA in sufficiently great amounts to
`permit 4-h autoradiography exposures and sample throughput times of 2 days. When compared with virus
`isolation, the CAA dot blot hybridization assay is time- and labor-saving.
`
`Chicken anemia agent (CAA), which was first recognized
`in Japan in 1979 (21), is now known to be a nonenveloped,
`icosahedral virus which measures 23.5 nm in diameter and
`which contains a circular, single-stranded DNA genome
`(17). As such, it does not belong to any previously described
`family of animal viruses. Serological evidence indicates that
`infection of chickens with CAA is widespread worldwide (6).
`Experimental infection of 1-day-old susceptible chicks
`with the virus results in aplastic anemia, atrophy of the
`lymphoid organs, liver changes, and increased mortality
`(21). A similar syndrome, known as anemia-dermatitis syn-
`drome (19) or blue wing disease (1), occurs in the field after
`primary infection of in-lay breeder flocks. No clinical signs
`are seen in breeders, but CAA is vertically transmitted to the
`progeny chicks, which typically show the disease at 2 to 4
`weeks of age. In addition, subclinical CAA infections can
`adversely affect the economic performance of broiler flocks
`(11).
`As a method of diagnosing infection, virus isolation is
`labor-intensive and time-consuming. In this paper, we de-
`scribe the detection of CAA-specific DNA in tissues from
`infected birds with a dot blot DNA hybridization assay
`which uses a cloned DNA probe.
`
`MATERIALS AND METHODS
`Viruses. The Cux-1 and Gifu-1 isolates of CAA were
`obtained from V. von Bulow (Free University, Berlin,
`Federal Republic of Germany) and N. Yuasa (National
`Institute of Animal Health, Tokyo, Japan), respectively.
`CAA viruses which we have designated EF88/78/276, 87/10/
`44, and 89/3713 were isolated in our laboratories from
`material originating in the United States (9), the United
`
`* Corresponding author.
`
`Kingdom (8), and Australia, respectively. The avian adeno-
`viruses used in this investigation were isolate 127 of egg drop
`syndrome 1976 (EDS) virus (18) and isolate CELO-Phelps of
`fowl adenovirus type 1 (FAV-1). An adeno-associated virus
`is known to be present in stocks of this FAV-1 isolate. The
`Sinkovic strain of infectious laryngotracheitis (ILT) virus
`and the Marexine vaccine strain of turkey herpesvirus
`(THV) were the avian herpesviruses used.
`Virus growth. Virus isolates were grown in MDCC-MSB1
`cells, kindly provided by V. von Bulow (Free University), as
`described previously (7). The Cux-1 isolate of CAA was
`semipurified by differential centrifugation and discontinuous
`sucrose gradient centrifugation as described previously (10).
`EDS virus was grown in duck eggs and purified by ultracen-
`trifugation as described previously (18). FAV-1 and ILT
`virus were grown in chicken embryo liver (CEL) cells
`harvested at 24 h postinfection. Cultures of chicken embryo
`fibroblasts (CEF) cells that had been infected with THV
`were harvested at 6 days postinfection. The growth of THV,
`FAV-1, and ILT virus was checked by indirect immunoflu-
`orescence performed with acetone-fixed coverslip cultures.
`DNA extraction. DNA was extracted from uninfected
`MDCC-MSB1 cells or MDCC-MSB1 cells infected sepa-
`rately with the Cux-1, Gifu-1, EF88/78/276, 87/10/44, and
`89/3713 isolates of CAA and harvested at 48 to 72 h postin-
`fection by treatment of suspensions of approximately 107
`cells contained in 200 ,ul of 0.001 M EDTA-0.01 M Tris-HCl
`(pH 8.0) (TE buffer) with sodium dodecyl sulfate (SDS) (1%)
`and proteinase K (1 mg/ml) for 2 h at 37°C; extraction with
`phenol, phenol-chloroform-isoamyl alcohol (25:24:1), and
`chloroform-isoamyl alcohol (24:1), and three diethyl ether
`washes. Virus DNA was recovered from semipurified Cux-1
`virus by the same extraction procedure.
`DNA was extracted from specimens (0.05 to 0.1 g) of
`thymus, spleen, liver, feces, clotted blood, or unclotted
`
`933
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`934
`
`TODD ET AL.
`
`J. CLIN. MICROBIOL.
`
`blood present in hematocrit tubes by treatment of samples
`diluted in 200 ,ul of TE buffer with SDS (1%) and proteinase
`K (1 mg/ml) for 17 h at 37°C, extraction with phenol-
`chloroform-isoamyl alcohol (25:24:1) and chloroform-iso-
`amyl alcohol (24:1), and one diethyl ether wash.
`For cloning purposes, DNA was recovered from MDCC-
`MSB1 cells (109) infected with the Cux-1 isolate of CAA and
`harvested at 48 h postinfection by a modification of the Hirt
`(3) procedure described by Molitor et al. (12).
`CEL and CEF cells infected with FAV-1, THV, or ILT
`virus were scraped from 25-cm2 culture flasks and recovered
`by centrifugation at 2,000 x g for 10 min. Cell pellets were
`resuspended in 200 p.l of TE buffer, and the DNA was
`extracted as described for CAA-infected MDCC-MSB1
`cells. DNA was isolated from purified EDS virus as de-
`scribed previously (18).
`Cloning of CAA-specific DNA. DNA (1 to 5 p,g) extracted
`by the Hirt procedure was treated with Si nuclease (Amer-
`sham) in accordance with the manufacturer's instructions
`and fractionated by electrophoresis in 1% agarose gels in the
`presence of ethidium bromide as described previously (17).
`A faint band of CAA-specific, double-stranded, replicative-
`form (RF) DNA was visible at a position which corre-
`sponded to a molecular size of 2.3 kbp relative to the
`X-HindIII DNA fragments used as markers. The gel contain-
`ing this band was excised, and the DNA was recovered by
`electroelution; extracted with phenol (twice), phenol-chloro-
`(once), and chloroform-
`form-isoamyl alcohol (25:24:1)
`isoamyl alcohol (24:1) (once); washed with diethyl ether
`(three times); and precipitated with ethanol.
`The fragments generated by digestion of the CAA-specific
`RF DNA with Hindlll were ligated to a pGEM-1 transcrip-
`tion vector that had been cut with HindlIl and treated with
`alkaline phosphatase. Escherichia coli ED8767 transformed
`by recombinant plasmids was identified by the colony hy-
`bridization technique with a virus DNA probe which had
`been labeled with [32P]dCTP by the oligo priming method of
`Feinberg and Vogelstein (2) (Multiprime DNA labeling sys-
`tem; Amersham). The DNA used for the preparation of this
`probe had been extracted from semipurified virus (Cux-1
`isolate) and recovered as a band in low-melting-point agar-
`ose (GIBCO-BRL) after electrophoresis. Following Hindlll
`treatment, electrophoretic analysis indicated that the recom-
`binant plasmids contained CAA-specific inserts of 1.3 and
`1.0 kbp. One plasmid, designated pCAA-1, contained both
`inserts and was selected for probe production.
`Transfection was performed with
`the
`Transfection.
`DEAE-dextran method of Sompayrac and Danna (13). This
`method involved incubating 0.1 to 0.5 jig of DNA contained
`in 10 ,ul of TE buffer with approximately 105 MDCC-MSB1
`cells (350 Pd) in the presence of DEAE-dextran (200 p.g/ml)
`for 4 to 6 h before resuspending the transfected cells in fresh
`medium. After 2 to 3 days, the cells were subcultured into
`fresh cells and passaged at 2- to 3-day intervals as described
`previously (7). Virus infection was detected by indirect
`immunofluorescence with acetone-fixed cells dried on mul-
`tispot slides (7).
`The CAA-specific DNA used for transfection was pre-
`pared as follows. Recombinant plasmid pCAA-1 was purified
`by equilibrium density gradient centrifugation in CsCl con-
`taining ethidium bromide (4). Purified plasmid DNA was
`partially digested by treating 5- to 10-jig amounts for 10 min
`at 37°C with 16 to 20 U of HindIII. Following agarose gel
`electrophoresis, the 2.3-kbp fragment from which the 1.3-
`and 1.0-kbp fragments were derived was recovered from the
`gel by electroelution and purified by extraction with phenol-
`
`chloroform as described for CAA-specific RF DNA. In some
`experiments, the 2.3-kbp fragment was treated with T4 ligase
`under conditions stipulated by the manufacturer (Amer-
`sham) and reextracted with phenol-chloroform prior to
`transfection.
`Southern blot hybridization. The CAA-specific 1.3- and
`1.0-kbp fragments generated by treatment of pCAA-1 with
`HindlIl were fractionated by electrophoresis in 1% agarose,
`and the DNA was Southern blotted (14) onto nylon mem-
`branes (Hybond-N; Amersham). Blots were hybridized with
`32P-labeled DNA probes prepared by the oligo priming
`method from either the 1.3- or the 1.0-kbp fragment present
`in slices of low-melting-point agarose after electrophoretic
`separation.
`Preparation of the cloned DNA probe. The CAA-specific
`1.3- and 1.0-kbp fragments generated by HindlIl treatment
`of pCAA-1 were separated from the vector fragment by
`electrophoresis in 1% agarose gels and purified by electroe-
`lution and phenol-chloroform extraction as described above.
`The pooled, cloned CAA-specific DNA fragments (approxi-
`mately 25 ng) were labeled by the Multiprime DNA labeling
`system with [32P]dCTP (approximately 800 Ci/mmol; code
`10385; Amersham).
`Dot blot hybridization assay. Each DNA sample (50 ,uI)
`was treated with an equal volume of 0.7 N NaOH for 30 min
`on ice prior to the addition of 100 Rl of 2 M ammonium
`acetate. Duplicate 100-pl aliquots were dotted onto Hy-
`bond-N nylon membranes with a 96-well manifold apparatus
`(GIBCO-BRL) coupled to a vacuum pump. Sample dots
`were washed twice with 200 RI of 2x SSC (1 x SSC is 0.15 M
`NaCl plus 0.015 M sodium citrate). After being dried, the
`membranes were exposed to UV light for 10 min. The
`membranes were prehybridized for 6 h at 68°C and hybrid-
`ized for 17 h at 68°C. The same solution, which contained 6x
`SSC, 5x Denhardt's solution, 0.15% SDS, and salmon
`sperm DNA (500 ,ug/ml), was used for both the prehybrid-
`ization and the hybridization steps, except that the hybrid-
`ization solution contained the 32P-labeled cloned DNA probe
`(105 dpm/ml). The membranes were washed successively
`with 2x SSC for 15 min (twice), 2x SSC containing 0.1%
`SDS for 30 min (once), and 0.1 x SSC for 10 min (once) at
`68°C. Autoradiography was performed with Hyperfilm-MP
`(Amersham), and the hybridized blots were exposed for
`periods of 4 h to 7 days. Each assay was done with serial
`10-fold dilutions of DNA from uninfected and Cux-1-infected
`MDCC-MSB1 cells. The starting dilution contained approx-
`imately 1 ,ug of DNA. DNAs from the avian viruses ILT
`virus, FAV-1, THV, and EDS virus were assayed by this
`hybridization method. In these experiments, samples of
`infected CEL or CEF cell DNA (1 to 5 ,ug) or purified EDS
`virus DNA (0.1 jig) were used. Tissue DNA samples, which
`represented about 25% of the tissue extracts, were undiluted
`and contained between 1 and 10 jig of DNA. To gain more
`information from each dot blot hybridization assay, we
`semiquantitatively assessed test DNA samples by visually
`comparing their hybridization signals with those of the
`dilutions of infected-cell DNA. Thus, test samples producing
`signals equal to or greater than that of the 1:1,000 dilution
`but less than that of the 1:100 dilution were scored 1+, those
`producing signals equal to or greater than that of the 1:100
`dilution but less than that of the 1:10 dilution were scored
`2+, and those producing signals equal to or greater than that
`of the 1:10 dilution were scored 3+. Samples producing
`signals less than the signal produced by the 1:1,000 dilution
`of infected-cell DNA but still above the background were
`considered weakly positive (w+).
`
`CEV Exhibit 1027_003
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`

`VOL. 29, 1991
`
`DOT BLOT HYBRIDIZATION ASSAY FOR CHICKEN ANEMIA AGENT
`
`935
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`Q
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`CS
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`*1
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`2 3
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`FIG. 2. Dot blot hybridization of CAA-specific DNA. Samples
`containing gel-purified, cloned CAA-specific 1.3- and 1.0-kbp DNA
`fragments (row 1), DNA from uninfected MDCC-MSB1 cells (row
`2), and DNA from MDCC-MSB1 cells infected with the Cux-1
`isolate (row 3) were serially (10-fold) diluted. An autoradiography
`exposure period of 6 days was used.
`
`fragments of 1.3 and 1.0 kbp (Fig. 1). Southern blot hybrid-
`ization with probes prepared from DNA extracted from
`semipurified virus showed that both inserts were CAA
`specific. Additional hybridization experiments showed that
`DNA probes prepared with each of these fragments did not
`cross-hybridize (data not shown). Partial digestion of
`pCAA-1 with HindIII resulted in the generation of a 2.3-kbp
`fragment, the precursor of the 1.3- and 1.0-kbp fragments
`(Fig. 1). The 2.3-kbp fragment was separated by agarose gel
`electrophoresis and recovered by electroelution. Transfec-
`tion experiments showed that this fragment, in either the
`ligated or the unligated form, produced infectious virus in
`MDCC-MSB1 cells following four to seven passages. Trans-
`fection attempts with undigested pCAA-1 were unsuccess-
`ful.
`Sensitivity and specificity of the cloned DNA probe in dot
`blot hybridization reactions. The DNA probe used for dot
`blot hybridization was prepared from the 1.3- and 1.0-kbp
`HindIII fragments of the pCAA-1 2.3-kbp fragment, which
`
`(i)
`S
`
`L
`*0 .O
`
`T
`
`L
`
`(i i)
`S
`
`0
`
`2 3 4 5 6
`
`_
`
`FIG. 3. Dot blot hybridization of CAA-specific DNA in tissue
`extracts from experimentally infected chicks. Duplicate samples of
`liver (L), spleen (S), and thymus (T) tissue extracts were assayed for
`each of the two birds (i and ii) examined on each experimental
`occasion (Table 1); shown are SPF chicks on day 19 (row 1) and day
`23 (row 2) and broiler chicks on day 23 (row 3) and day 44 (row 4)
`that had been in contact with infected chicks. Serial (10-fold)
`dilutions of uninfected-cell (row 5) and infected-cell (row 6) DNAs
`were included for control purposes. An autoradiography exposure
`period of 6 days was used.
`
`9
`
`Kbp
`
`1.3
`
`1.0
`
`bp
`
`4361
`
`2322
`2027
`
`564
`
`FIG. 1. Agarose gel electrophoresis of cloned CAA-specific
`DNA. Ethidium bromide-stained DNA was visualized with UV
`light. Fragments of X phage DNA digested with HindIll were
`included for size reference (lane 3). The products of complete (lane
`1) and partial (lane 2) HindIll digestions of pCAA-1 are shown. The
`cloned CAA-specific fragments of 1.3 and 1.0 kbp are labeled, and
`the partial digestion product of 2.3 kbp is indicated by an arrow.
`
`Experimental infections. One-day-old specific-pathogen-
`free (SPF) chicks that were free of maternal antibody to
`CAA were infected by intramuscular inoculation (0.1 ml) of
`CAA isolates (105 to 106 50% tissue culture infective doses
`per ml). Alternatively, infection of chicks was achieved by
`yolk sac inoculation (0.1 ml) of 7-day-old chicken embryos
`with the CAA Cux-1 isolate (105 to 106 50% tissue culture
`infective doses per ml). In one experiment involving "in-
`contact" infection, chicks hatched from virus-inoculated
`embryos were reared from the age of 1 day with chicks
`hatched from uninfected SPF eggs and broiler chicks
`hatched from eggs collected from a commercial broiler-
`breeder flock which was found to be serologically positive
`for CAA by indirect immunofluorescence (7). Birds were
`removed on different days postinfection (posthatching), and
`their thymuses, spleens, and livers were removed for inves-
`tigation. In some experiments, feces, clotted blood, or
`unclotted blood samples used for hematocrit measurements
`were also collected for investigation.
`Field material. Thymus specimens were taken from 46 1-
`to 2-week-old broiler birds submitted to our laboratories for
`postmortem examination. These birds originated from eight
`flocks containing chicks showing clinical signs and lesions
`characteristic of anemia-dermatitis syndrome. Thymuses
`taken from 42 1- to 3-week-old broiler birds collected from
`seven "normal" flocks and 24 1- to 3-week-old broiler birds
`submitted to our laboratories for the investigation of another
`disease condition, runting-stunting syndrome, were also
`examined.
`
`RESULTS
`Characterization of pCAA-1. Treatment of pCAA-1 with
`HindIlI and subsequent electrophoresis in 1% agarose gels
`indicated that this recombinant plasmid contained 2 HindIII
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`936
`
`TODD ET AL.
`
`J. CLIN. MICROBIOL.
`
`Downloaded from
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`
`results obtained for the dilutions of infected-cell DNA with
`those obtained for the dilutions of purified, cloned 1.3- and
`1.0-kbp fragments indicated that the first dilution (1:10) of
`the infected-cell DNA contained between 100 and 1,000 pg of
`CAA-specific DNA (Fig. 2). In some experiments, particu-
`larly those with long exposure periods, weak signals were
`obtained with the 1:10 dilution of uninfected-cell DNA. In
`such cases, however, these signals were always weaker than
`that of the 1:1,000 dilution of infected-cell DNA. An example
`of the autoradiography results for a hybridization assay
`performed with extracts of tissues from experimentally
`infected chicks and the semiquantitative assessments (w+,
`1+, 2+, 3+) of the hybridization signals are given in Fig. 3
`and Table 1, respectively.
`Experimental infections. Infection by intramuscular inocu-
`lation of 1-day-old SPF birds resulted in anemia with asso-
`ciated changes in the thymus, spleen, and liver (19). CAA-
`specific DNA could be detected by the dot blot hybridization
`assay in extracts of each of these three tissues (Table 2). The
`patterns of DNA detection obtained after infection with
`three different isolates of CAA were similar. Thus, at 5 days
`postinfection, CAA-specific DNA was readily detected in
`the thymus and, with increasing time, became detectable in
`the spleen and liver. Although more CAA-specific virus was
`detected in the thymus at earlier times (5 and 7 days)
`postinfection, even at later times this tissue usually con-
`tained more virus DNA than did the spleen and liver.
`Subsequent experimental infection by intramuscular inoc-
`ulation with the Cux-1 isolate of CAA indicated that virus
`DNA was not detectable in the thymus, spleen, or liver at 3
`days postinfection but was detectable at 27, 34, and 42 days
`postinfection. Samples of feces, clotted blood, or unclotted
`blood taken from birds infected by this method at different
`times postinfection were rarely positive (data not shown).
`Birds were experimentally infected by yolk sac inocula-
`tion to simulate more closely the route of vertical transmis-
`sion observed in the field. When inoculated with undiluted
`virus (105 to 106 50% tissue culture infective doses per ml),
`chickens became anemic 1 to 2 weeks posthatching and often
`died. Investigation of these birds indicated that the thymus
`was again the best source of detectable CAA-specific DNA
`(Table 3). Additional embryo inoculation experiments with
`serial 10-fold dilutions of virus showed that virus-specific
`DNA was detected in tissues from birds hatched from eggs
`inoculated with a 1:1,000 dilution of the original virus
`inoculum. This inoculum was sufficient to produce clinical
`anemia in the birds at 16 days posthatching. Embryo inocu-
`lation with the next (1:10,000) dilution of virus failed to
`produce anemia or virus-specific DNA detectable by dot blot
`hybridization (data not shown).
`Chicks that had been infected as embryos with sufficient
`virus to produce anemia were reared in the presence of
`uninfected SPF hatchmates found negative for CAA anti-
`
`5
`
`9
`
`13
`
`16
`
`19
`
`23
`
`1
`2
`
`1
`2
`
`1
`2
`
`1
`2
`
`1
`2
`
`1
`2
`
`-
`-
`
`-
`-
`
`-
`-
`
`1+
`-
`
`w+
`w+
`
`-
`
`w+
`
`-
`-
`
`-
`
`-
`-
`
`1+
`-
`
`w+
`w+
`
`-
`
`w+
`
`-
`-
`
`-
`
`-
`1+
`
`3+
`3+
`
`3+
`3+
`
`-
`
`1+
`
`44
`
`NA
`NA
`NA
`1
`NA
`NA
`NA
`2
`a Two birds were investigated at each age.
`b -, No virus DNA was detected.
`c NA, Not assayed.
`
`NAC
`NA
`
`NA
`NA
`
`NA
`NA
`
`_
`
`-
`-
`
`-
`-
`
`NA
`NA
`
`-
`-
`
`-
`-
`
`_
`
`-
`-
`
`-
`-
`
`NA
`NA
`
`-
`w+
`
`-
`-
`
`_
`
`-
`-
`
`-
`-
`
`NA
`NA
`
`-
`2+
`
`-
`-
`
`had been separated by agarose gel electrophoresis and
`recovered by electroelution. 32P-labeled probes were pro-
`duced by the oligo priming method and had specific activities
`of 108 to 109 dpm/,ug. Dot blot hybridization experiments
`showed that between 1 and 10 pg of the purified cloned DNA
`fragments could be detected (Fig. 2). Additional experiments
`indicated that the DNAs extracted from MDCC-MSB1 cells
`infected with the Cux-1, Gifu-1, EF88/78/276, 87/10/44, and
`89/3713 isolates of CAA produced positive hybridization
`signals of similar intensity, whereas the DNAs extracted
`from uninfected MDCC-MSB1 cells or from tissues obtained
`from SPF chickens did not. Similarly, DNAs extracted from
`purified EDS virus or from cell cultures infected with
`FAV-1, THV, or ILT virus failed to produce positive hy-
`bridization signals with the CAA probe under the conditions
`of the assay (data not shown).
`For control purposes, serial 10-fold dilutions of the DNAs
`extracted from uninfected and CAA (Cux-1)-infected
`MDCC-MSB1 cells were included in each hybridization
`assay (Fig. 2). The autoradiography exposure period was
`considered sufficient and experiments were judged satisfac-
`tory if the 1: 1,000 dilution of the infected-cell DNA produced
`a signal stronger than the background. Comparison of the
`
`TABLE 2. Detection of CAA-specific DNA in chicks infected by intramuscular inoculation with three different isolates
`Scorea for the indicated tissue from chicks infected with:
`
`(dAys)
`
`Liver
`
`Cux-1 isolate
`Spleen
`
`5
`-
`-
`7
`2+
`1+
`15
`w+
`1+
`a -, No virus DNA was detected.
`
`Thymus
`
`2+
`3+
`2+
`
`Liver
`
`-
`-
`w+
`
`EF88/78/276 isolate
`Spleen
`
`w+
`1+
`1+
`
`Thymus
`
`3+
`3+
`2+
`
`Liver
`
`-
`-
`1+
`
`87/10/44 isolate
`Spleen
`
`-
`1+
`w+
`
`Thymus
`
`2+
`3+
`w+
`
`TABLE 1. Detection of CAA-specific DNA in SPF and broiler
`chicks reared in contact with CAA-infected birds
`Scoreb for the indicated tissue from the following chicks:
`SPF
`Spleen
`
`Broiler
`
`Thymus
`
`Liver
`
`Spleen
`
`Thymus
`
`(days)
`
`Bird'
`
`Liver
`
`CEV Exhibit 1027_005
`
`

`

`VOL. 29, 1991
`
`DOT BLOT HYBRIDIZATION ASSAY FOR CHICKEN ANEMIA AGENT
`
`937
`
`Downloaded from
`
`http://jcm.asm.org/
`
` on September 24, 2014 by KANSAS STATE UNIV
`
`0
`
`e
`
`f
`
`c
`
`0*
`
`0"
`
`a
`1 00
`
`b
`
`2 3
`
`FIG. 4. Dot blot hybridization of CAA-specific DNA in thymus
`extracts from broiler chicks showing signs characteristic of anemia-
`dermatitis syndrome. Six samples (a to f) were assayed in duplicate
`in each of rows 1 and 2. Serial dilutions of uninfected-cell (row 3)
`and infected-cell (row 4) DNAs were included for control purposes.
`An autoradiography exposure period of 4 h was used.
`
`because, despite the availability of a method for purifying
`the virus from infected cells (17), low yields of virus prevent
`sufficient DNA of the required purity from being obtained.
`On the basis of its circular, single-stranded DNA genome,
`CAA cannot be assigned to any known animal virus family.
`However, results obtained with other single-stranded DNA
`viruses, including the similarly unclassified porcine circovi-
`rus (16), led us to select a cloning strategy based on the
`isolation and subsequent ligation of the putative CAA-
`specific, double-stranded, RF DNA. This was extracted by a
`modification of the Hirt (3) procedure described for porcine
`parvovirus RF DNA (12). On the basis of our findings that
`CAA-specific Hindlll fragments of 1.3 and 1.0 kbp were
`cloned either separately or, in the case of pCAA-1, together
`and that no cross-hybridization was detected between these
`fragments, we suggest that the 2.3-kbp CAA-specific RF
`DNA from which the fragments were cloned was open
`circular and possessed two HindIII sites. We also suggest
`that the generation of the recombinant plasmid pCAA-1
`resulted from the ligation of a partially digested RF DNA in
`which only one of the two HindIII sites was cleaved. The
`ability of the cloned 2.3-kbp fragment recovered by electro-
`elution after partial HindIII digestion to transfect MDCC-
`MSB1 cells coupled with the knowledge that the circular,
`single-stranded virus DNA genome is 2.2 to 2.3 kb in size
`(17) indicates that the fragment present in pCAA-1 contains
`the entire sequence of the CAA genome. For detection
`purposes, hybridization probes based on the whole genome
`are likely to be more sensitive than are those based on a
`fragment representing a small proportion of the genome.
`The hybridization probe prepared with the 1.3- and 1.0-
`kbp HindIII fragments of pCAA-1 by the oligo priming
`technique proved to be sensitive and specific. The limit of
`sensitivity, 1 to 10 pg, was similar to that achieved in other
`DNA-detecting dot blot hybridization assays (15), while
`evidence for the specificity of the probe was provided by our
`failure to detect hybridization with DNAs extracted from
`MDCC-MSB1 cells and tissues from SPF birds and with
`DNAs from avian viruses, including FAV-1, EDS virus,
`THV, and ILT virus. We have been unable to test avian
`parvovirus DNA for cross-hybridization, as an isolate of this
`virus is unavailable to us. However, the negative result
`obtained with DNA extracted from cells infected with
`FAV-1 and the knowledge that an adeno-associated virus is
`present in stocks of this FAV-1 isolate suggest that CAA-
`specific DNA does not cross-hybridize with DNA from this
`type of avian parvovirus. In addition, the absence of hybrid-
`
`TABLE 3. Detection of CAA-specific DNA in chicks infected
`by embryo inoculation
`
`Days
`posthatching
`
`Birda
`B
`
`Scoreb for the following tissue or specimen from
`chicks infected with the Cux-1 isolate:
`Clotted blood
`Spleen
`Thymus
`Feces
`
`Liver
`
`7
`
`10
`
`1
`2
`
`1
`2
`
`-
`-
`
`-
`1+
`
`w+
`2+
`
`1+
`2+
`
`12
`
`1
`w+
`-
`2
`w+
`-
`a Two birds were investigated at each age.
`b -, No virus DNA was detected.
`
`2+
`-
`w+
`3+ -w+
`
`2+
`2+
`
`3+
`3+
`
`-
`1+
`
`-
`-
`
`-
`w+
`
`-
`-
`
`body by indirect immunofluorescence and broiler chicks
`hatched at the same time from eggs laid by hens which
`possessed CAA antibody. For the antibody-negative SPF
`hatchmates, virus-specific DNA was detected in the thy-
`muses from birds 13, 16, 19, and 23 days old (Table 1). In
`contrast, CAA-specific DNA was not detected in the anti-
`body-positive broiler birds at 13 or 16 days of age but was
`detected in one bird at 23 days posthatching (Fig. 3).
`Field infections. Thymuses taken from 46 chicks removed
`from broiler flocks showing clinical signs and lesions char-
`acteristic of vertically transmitted anemia-dermatitis syn-
`drome were investigated for the presence of CAA-specific
`DNA by the dot blot hybridization assay (Table 4). Samples
`taken from each of the eight flocks were positive, although
`the numbers and strengths of the positive samples varied
`from flock to flock. Nineteen of the 35 positive samples
`(54%) were assessed as 2+ or 3+. With such samples,
`autoradiography exposure periods of about 4 h were suffi-
`cient to permit detection (Fig. 4).
`No positive reactions were obtained when thymus sam-
`ples taken from 42 normal 1- to 3-week-old broilers (six birds
`per flock) or from 24 broilers submitted from a flock showing
`runting-stunting syndrome were investigated.
`
`DISCUSSION
`In this paper, we report the detection of CAA-specific
`DNA in tissues from experimentally and naturally infected
`chickens by a dot blot hybridization assay based on the use
`of a cloned DNA probe. On the grounds of purity, it is
`preferable to use cloned virus-specific DNA in hybridization
`assays rather than DNA extracted from purified virus prep-
`arations. This is of particular significance in the case of CAA
`
`TABLE 4. Detection of CAA-specific DNA in thymuses from
`broiler chicks showing signs characteristic of
`anemia-dermatitis syndrome
`No. of birds with CAA-specific DNA in flockb:
`
`A
`
`B
`
`C
`
`D
`
`E
`
`F
`
`G
`
`Dot
`blot
`score'
`
`3
`1
`2
`-
`1
`1
`1
`w+
`2
`2
`0
`1+
`0
`1
`2+
`1
`0
`1
`2
`3+
`a -, No virus DNA was detected.
`b Six birds were taken from each flock, except for flocks G and H.
`
`2
`0
`2
`1
`1
`
`0
`0
`1
`4
`1
`
`2
`3
`1
`0
`0
`
`1
`0
`1
`2
`1
`
`H
`
`0
`0
`1
`3
`1
`
`CEV Exhibit 1027_006
`
`

`

`Downloaded from
`
`http://jcm.asm.org/
`
` on September 24, 2014 by KANSAS STATE UNIV
`
`938
`
`TODD ET AL.
`
`J. CLIN. MICROBIOL.
`
`ization for thymus samples taken from normal broiler birds
`and from those submitted to our laboratories for the inves-
`tigation of another disease syndrome indicate that the probe
`does not cross-hybridize with nucleic acid sequences present
`in this tissue as a result of infection with other commonly
`occurring agents. The ability of the hybridization probe
`derived from the cloned DNA genome of the Cux-1 isolate of
`CAA to detect DNAs extracted from cells infected with
`CAA isolates from the United Kingdom, United States,
`Australia, and Japan with similar sensitivities probably re-
`flects the high base sequence homology that exists among
`these isolates, which are serotypically indistinguishable and
`which produce identical pathological changes upon experi-
`mental infection of susceptible chickens (5).
`Experimental infections achieved by either intramuscular
`inoculation of 1-day-old birds or yolk sac inoculation of
`chicken embryos resulted in characteristic clinical signs and
`pathological lesions. On the basis of the observed patholog-
`ical changes, we selected the thymus, spleen, and liver for
`investigation by the DNA dot blot hybridization assay.
`Semiquantitative assessments of the amounts of CAA-spe-
`cific DNA determined by comparing the hybridization sig-
`nals of the test samples with those of dilutions of positive
`control DNA indicated that CAA-specific DNA, with very
`few exceptions, was usually present in greater amounts in
`the thymus than in the other tissues. This result was partic-
`ularly evident at early times (5 days) postinfection, when the
`spleen and liver samples were seldom positive. Feces or
`blood specimens proved unreliable sources of CAA-specific
`DNA. Our finding that CAA-specific DNA could be detected
`for up to 42 days postinfection supported the results of
`Yuasa et al. (20), who found that the virus could be isolated
`from most organs at 28 days postinfection and from the brain
`and feces at 49 days postinfection.
`In addition to investigating tissues from directly infected
`birds, most of which showed clinical signs of anemia, we
`examined specimens from birds infected indirectly at an
`older age through contact with infected anemic chicks. It has
`been established that birds infected by contact with inocu-
`lated chicks do not usually show clinical signs of anemia (8,
`21). We found that virus DNA was detected in thymuses
`from 13-day-old birds infected through contact and that
`similarly infected birds, at 16 and 19 days of age, contained
`as much DNA in their thymuses as did younger, anemic
`birds. The ability to replicate in the thymus may therefore
`bear little relation to the development o