Unit of Avian Health, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA1
Author for correspondence: K. Schat. Fax +1 607 253 3384. e-mail kas24{at}cornell.edu
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Abstract |
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Introduction |
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CAV was first isolated in 1979 in Japan (Yuasa et al., 1979 ). Since its first description, it has been detected in chickens all over the world and is considered to be ubiquitous (Von Bülow & Schat, 1997
). All characterized isolates of CAV belong to a single serotype (Von Bülow & Schat, 1997
). However, there are differences between isolates that affect virus cell tropism in vitro (Renshaw et al., 1996
). All known strains cause a similar disease syndrome, chicken infectious anaemia (CIA).
Clinical disease, CIA, occurs only in chicks infected before 3 weeks of age or in older immunosuppressed chickens. Susceptible chicks develop a severe anaemia (Goryo et al., 1985 ; Taniguchi et al., 1983
; Yuasa et al., 1979
) and immunosuppression that may lead to secondary infections (Goryo et al., 1987
; Vielitz & Landgraf, 1988
; Von Bülow & Schat, 1997
). Chicks experimentally infected at 1 day of age develop lesions 8 days post-inoculation (p.i.) and anaemia 12 to 16 days p.i. (Von Bülow & Schat, 1997
). Histologically, there is complete atrophy of lymphoid cells in a wide variety of tissues. In the bone marrow, cells of all haematopoietic lineages are depleted during active infection (Goryo et al., 1989
; Pope, 1991
; Smyth et al., 1993
). Most chicks completely recover by 32 to 36 days p.i., when neutralizing antibodies are present (Von Bülow & Schat, 1997
). Virus can be isolated from most tissues except serum up to 28 days p.i. and can be detected in rectal contents up to 49 days p.i.
Hoop (1993) reported that experimentally infected males transmitted CAV through their semen until they developed antibodies to CAV. Similarly, experimentally infected hens also transmitted CAV to their offspring until the hens developed CAV antibodies between 8 to 14 days p.i. (Hoop, 1992
). In both cases the percentage of offspring infected was less than 10%. In natural outbreaks, vertical transmission may occur from 3 to 9 weeks after exposure, but vertical transmission has not been reported after development of immunity (Chettle et al., 1989
; Engström & Luthman, 1984
; Vielitz & Landgraf, 1988
). The tissues or cells involved in the vertical transmission of CAV have not yet been identified.
The Department of Microbiology and Immunology at our Institute maintains three genetic strains of specific-pathogen-free (SPF) chickens in two filtered-air, positive-pressure (FAPP) houses. These SPF flocks were considered to be free of CAV infection based on periodic serologic testing of randomly selected birds (B. Lucio & K. A. Schat, unpublished data) and chick susceptibility tests (Lucio et al., 1990 ). However, these flocks became accidentally infected with CAV (SH-1 strain), most likely in July 1996 based on the development of antibodies to CAV. Interestingly, the seroconversion was associated with the development of sexual maturity and genetic background of the chicken strain. Thus, not all chickens became antibody positive, even while the three strains were maintained in the same rooms (Cardona et al., 2000
). Producers of SPF flocks often report that chickens become seropositive after the onset of sexual maturity, but the source of infection is seldom identified (McNulty, 1991
).
The objective of this work was to determine the location of CAV in chronically infected chickens. Initial studies focussed on the detection of CAV in the reproductive tracts of breeder birds and determining the cell types that harbour virus. Once the virus was detected in selected birds, further studies determined that the virus could be transmitted to embryos.
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Methods |
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DNA extraction.
DNA was extracted from each tissue sample using standard techniques (Moore, 1988 ) with some modifications. Briefly, tissues were minced and incubated overnight at 37 to 41 °C in digestion buffer. Each sample was extracted with a mixture of phenolchloroformisoamyl alcohol (25:24:1) once and the DNA precipitated at -20 °C with 1 vol. of isopropyl alcohol and 0·1 vol. 3 M NaCl overnight. The DNA was pelleted by centrifugation at 14000 g at 4 °C for 20 min. The DNA was resuspended in TE pH 7·4, and quantified using a DU-50 Beckman spectrophotometer at 260 and 280 nm.
PCR.
A nested PCR method was used for tissue screening. Two µg of tissue DNA was amplified with primers O3F (CAAGTAATTTCAAATGAACG) and O3R (TTGCCATCTTACAGTCTTAT). The first PCR reaction consisted of 5 min at 94 °C followed by 35 cycles consisting of 1 min at 94 °C, 2 min at 45 °C and 1 min at 72 °C for 1 min, followed by 10 min at 72 °C. One µl of the first PCR reaction was amplified with primer N3 (CCACCCGGACCATCAAC) and primer N4(GGTCCTCAAGTCCGGCACATTC). After an initial denaturation for 5 min at 94 °C, the nested PCR reaction was performed for 30 cycles using the following program: two cycles of 30 s at 94 °C, 30 s at 60 °C and 30 s at 72 °C, followed by two cycles each with the annealing temperature decreasing by 1 °C down to 56 °C, and ending with 20 cycles of 30 s at 94 °C, 30 s at 55 °C and 30 s at 72 °C, followed by one extension step of 10 min at 72 °C. Testis DNA from the two P2a flocks was amplified using -actin primers (primer 5' actin, CCCCCGTGCTGTGTTCCCATCTATCG; primer 3' actin, GGGTGCTCCTCAGGGGCTACTCTCAG). A 5 min denaturation step was followed by 35 cycles of 30 s at 94 °C, 30 s at 55 °C and 30 s at 72 °C followed by a final step of 7 min at 72 °C.
Southern blotting.
PCR fragments were separated in 1·5% agarose gels, exposed to ultraviolet light for 2 min, denatured and transferred to a nylon membrane (Nytran; Schleicher and Schuell), using standard Southern blotting techniques (Sambrook et al., 1989 ). The DNA was cross-linked to the membrane in a Spectolinker XL-1500 UV cross-linker (Spectronics). The Genius probe labelling and detection kit (Boehringer Mannheim) was used for hybridization and detection according to the manufacturers instructions. The probe used was a nick-translation labelled clone of the CAV genome. Briefly, pCIA-AB (Soiné et al., 1993
) was digested with EcoRI and fractionated on a 0·75% agarose gel; the smaller band (2·3 kb) was cut out from the gel and the DNA was purified using the Concert gel purification kit (GibcoBRL) according to the manufacturers instructions.
Sequencing.
Primers to CIA-1 were used to amplify the hypervariable region of VP-1 (Renshaw et al., 1996 ) of the CAV present in the SPF flocks (SH-1 strain). Two µg of ovarian tissue DNA from a 97-0 hen was amplified with primers O1F (AGGTGTATAAGACTGTAAG) and PshA1R (GAACAGGTGCCAGCCCCCAAACAT). The PCR reaction was performed for 35 cycles using the same protocol as used in the first step of the nested PCR. The hypervariable regions of VP-1 and VP-3, generated with the O3F and O3R primers, were cloned into pCR TOPO 2.1 vector using the TOPO TA cloning kit (Invitrogen) according to the manufacturers instructions. DNA sequencing was carried out at the BioResource Center at CU on a Perkin Elmer Biosystems model 377-XL DNA sequencer using dye-terminator chemistry. The thermocycling was done in a Perkin Elmer Biosystems model 9600 thermocycler.
Sequence analysis.
Sequences were aligned with published CAV sequences using the program Multiple Sequence Alignment version 1.0.1.2 (Informax Vector NTI suite) software. GenBank accession numbers of the aligned sequences are as follows: CIA-1 is L14767, L-028 is U69549, Cux-1 is M55918, ConnB is U69548, 82-2 is D31965. The sequences for the virus isolated from the SPF flocks, SH-1, are AF21564 and AF21563 for VP1 and VP3, respectively.
Virus isolation.
Semen samples were obtained from the 97-2a male on 6 days over a 3 month period. Tissues were obtained from selected chickens from the 96-1 and 96-2 flock, finely minced, frozen and thawed three times and extracted with chloroform. The supernatant fluids were passed through a 0·2 µm filter. A one-quarter volume of each sample was added to 2·5x105 CU-147 cells (Calnek et al., 2000 ) in 3 ml of LeibovitzMcCoy medium containing 10% foetal bovine serum. The cells were incubated at 41 °C and split every third day to a density of 1x106 cells/ml. Cells were checked daily for cytopathic effect and at 6, 10, 14 and 19 days p.i. examined for the presence of CAV protein VP3 using monoclonal antibody 51.3 in an indirect immunofluorescence assay (Chandratilleke et al., 1991
).
In situ PCR.
Sections of formalin-fixed tissues were embedded in paraffin, cut to 3 µm and placed on poly-L-lysine coated slides. The sections were deparaffinized and re-hydrated in graded ethanols. Methods for in situ PCR are a modification of techniques from Fares et al. (1998) and Nuovo et al. (1993)
. Briefly, tissues were permeabilized with 6 mg/ml proteinase K and 0·01% Triton X-100. After permeabilization, the PCR master mix (with primers N3 and N4) was added to the slides and the tissue DNA amplified for 50 cycles in a PTC-100 thermocycler (MJ Research) using the following protocol: 1 min at 95 °C, 2 min at 45 °C, 1 min at 72 °C and a final extension step for 10 min at 72 °C.
After PCR amplification, coverslips were removed and the slides were washed in 1x SSC for 5 min. A digoxigenin-labelled probe was heated for 10 min at 100 °C and placed on the section. The slide was then hybridized for 4 to 12 h at 50 °C. After hybridization the slide was washed in 1x SSC containing 0·05% BSA for 10 min at 42 °C. Anti-digoxigenin antibody conjugated with alkaline phosphatase (Roche) was diluted 1:5000 in a dilution buffer (0·1 M Tris, 0·1 M NaCl), added to the slide, and incubated for 30 min at 37 °C for 30 min. The secondary antibody was removed by two 5 min washes in wash buffer (0·1 M Tris, 0·1 M NaCl, 0·05 M MgCl2). The colour substrate (NBT/BCIP) was diluted at 20 µl/ml in detection buffer (0·1 M Tris, 0·1 M NaCl, 0·05 M MgCl2) and incubated at room temperature in the dark until colour development was observed after 1 to 4 h. The reaction was stopped in distilled water and the slides were counterstained with haematoxylin for 2 min followed by three washes of 2 min in distilled water. The sections were dehydrated in graded ethanols followed by xylenes, allowed to air dry, and covered permanently with a cover-slip.
A CAV-specific probe was generated by amplification of pCIA-1 with the O3F and O3R primers (Soiné et al., 1993 ) as previously described. A negative control probe was generated by amplification of purified duck enteritis virus (DEV) DNA using primers in the UL6 region as described (Plummer et al., 1997
).
Serial sections were amplified with CAV-specific primers as described above. One slide was hybridized with the CAV probe and one was hybridized with the DEV probe. Both slides were washed and exposed to the colour substrate as described.
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Results |
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CAV was often detected in the ovaries while virus could not be detected in the other organs. Excluding flock 97-2a, the percentage of hens with only ovaries positive ranged from 20% in flock 97-0 to 38% in flock 96-1. In 5 and 12% of the hens with positive ovaries in flocks 96-1 and 97-0, CAV was also detected in the infundibulum. All remaining hens with positive ovaries had positive spleens also (Table 1). Two of the three hens from flock 97-2a were positive in all three tissues tested and one was positive in infundibulum and ovary only.
Detection of CAV in the reproductive organs of roosters by nested PCR
The results for the detection of CAV in the reproductive tracts of roosters from flocks 96-1, 96-2, 97-1 and 97-0 are summarized in Table 2. Interestingly, the incidence in the vas deferentia was higher than in the testes in all flocks but 96-2.
The vas deferens was most frequently the positive tissue. It was the only positive tissue in 20 to 37% of the CAV-positive males in flocks 96-1, 97-0, 97-1 and 96-2 (Table 2). The testes were never positive in the absence of a positive spleen or vas deferens. Spleens and vas deferentia were both positive in 0 (flock 97-1) to 33% in flocks 96-1 and 97-0.
Detection of CAV in tissues from hens and roosters by in situ PCR
Selected samples from chickens positive for CAV were used to characterize the cells harbouring virus. The results are summarized in Table 3. In the spleen a few CAV-positive cells were detected. These positive cells were morphologically similar to small lymphocytes and were located in the white pulp. Positive cells were not detected when serial sections were hybridized with the DEV probe. Spleens that were negative in the nested PCR were consistently negative in the in situ PCR.
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Discussion |
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The finding that ovaries and to a lesser degree the infundibulum are sites for persistence of CAV in hens is surprising. CAV replicates mostly in lymphoid tissues after infection of susceptible chickens (Adair et al., 1993 ; Hoop & Reece, 1991
; Smyth et al., 1993
). The spleen was therefore selected as a tissue representing target cells for lymphocytic infection in adult birds. Unexpectedly, CAV was often detected in ovaries in the absence of splenic virus. Examination of a limited number of positive ovaries by in situ PCR showed that very few cells were virus positive. The presence of CAV in intrathecal cells in ovaries and epithelial cells in the infundibulum suggests strongly that CAV, although lymphotrophic during the early pathogenesis, can infect other cell types.
Interestingly, the testes of chickens with CAV detectable in the vas deferentia were frequently negative (56 and 63% in flocks 96-1 and 97-0, respectively). The presence of PCR inhibitors in testis DNA was eliminated as a possible explanation because actin was amplified from these DNA samples. The vas deferens is the major storage organ for sperm in the chicken. The spermatozoa progress from efferent ductules to the epididymis and finally to the vas deferens and seminal vesicle and in the process mature and become motile (Etches, 1996 ). It is possible that CAV is present in precursor spermatozoa in the testes but at levels below the detection limits of the nested PCR. CAV may begin replication during the maturation process leading to detectable levels in the vas deferens. Alternatively, CAV may be found in the buffers and proteins added to the seminal fluid as spermatozoa progress through the male reproductive tract (Etches, 1996
).
It is not clear how CAV persists in the cells of the reproductive tract. The tissues have not been examined for the presence of viral proteins, but such studies are likely to be inconclusive in view of the few positive cells detected by in situ PCR. Therefore, it cannot be excluded that CAV establishes a chronic persistent or latent infection in the female reproductive tract. Alternatively, it is possible that CAV is present as episomal DNA or that it is integrated in chromosomal DNA. Independent of the question of the form of CAV in the reproductive organs these results differ dramatically from previous reports on the persistence of CAV in infected chickens and demonstrate clearly that CAV specifically persists in the reproductive tissues of males and females.
The detection of virus in embryos from matings using a positive sire and positive dams indicates that CAV can be vertically transmitted to embryos long after infection of immunocompetent dams and sires. These findings contrast with earlier reports in which the appearance of CIA lesions in chicks and virus isolation in MSB-1 cells were used to detect vertical transmission (Hoop, 1992 , 1993
; Hoop et al., 1992
; Yuasa & Yoshida, 1983
). The nested PCR method used in this work to detect CAV in breeder birds is substantially more sensitive than virus isolation. It may be that the presence of maternal antibodies reduced the titre of virus transmitted to chicks to levels that were undetectable by virus isolation in MSB-1 cells. In addition, the presence of maternal antibodies may have reduced virus replication thus preventing expansion of virus and decreasing the possibility of isolation.
The localization of the positive cells in 11-day-old embryos is of considerable interest. The origin of the mesenchymal cells near the developing vertebrae is not clear, but it is likely that these cells are still pluripotential and able to migrate to several tissues including gonads, bone marrow and liver. CAV could be isolated from whole embryos, embryonal liver, yolk sac and chorioallantoic membranes after inoculation in the yolk sac at 5 days of incubation, but other tissues were not examined (Von Bülow & Witt, 1986 ). K. A. Schat & K. A. Ealy (unpublished data) examined tissues of CAV-positive embryos using the nested PCR and found that most tissues including gonads, bone marrow and liver were positive. However, VP3 could not be detected in bone marrow smears even when positive with PCR, suggesting that virus replication was either limited and below the detection limits of the assay or absent. In view of the limited evidence for virus replication in embryos and the observation by Cardona et al. (2000)
that seroconversion occurred during sexual development it is proposed that viral DNA is transferred to the embryo in the absence of virus replication followed by activation of the virus during sexual development.
Partial sequence information on the PCR products indicates that CAV strain SH-1 has minor differences from two strains, Cux-1 and CIA-1, for which extensive pathogenesis studies have been reported (Hu et al., 1993a , b
; Smyth et al., 1993
). However, these studies did not use nested PCR assays for the detection of CAV and did not determine if virus persisted in gonad tissues. Although we cannot exclude the possibility that the pathogenesis of infection by SH-1 is different from that by Cux-1 or CIA-1, it is more likely that there are no differences. Based on these findings it is postulated that (1) vertical transmission of CAV may occur far more frequently and over a longer period of time than previously indicated, and (2) that the presence of antibodies will neither eliminate the virus from reproductive tissues nor prevent vertical transmission.
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Acknowledgments |
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Footnotes |
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c Present address: Eijkman-Winkler Institute for Microbiology, Infectious Diseases and Inflammation, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands.
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Received 4 February 2000;
accepted 10 May 2000.