1 Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik EH26 0PZ, UK
2 Veterinary Laboratories Agency (VLA-Lasswade), Pentlands Science Park, Bush Loan, Penicuik EH26 0PZ, UK
3 Universidad de Zaragoza, Facultad de Veterinaria, 50013 Zaragoza, Spain
4 University of Edinburgh, Division of Veterinary Biomedical Sciences, Edinburgh EH9 1QH, UK
Correspondence
Daniela Salvatori
salvd{at}mri.sari.ac.uk
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ABSTRACT |
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MAIN TEXT |
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The neonatal lamb model has been very useful in determining the aetiological role of JSRV in OPA and some aspects of its pathogenesis, but is unsuitable for evaluating the efficacy of potential vaccine preparations and studying pathogenic mechanisms of JSRV infections that occur later in life. These studies also have been hampered by the absence of detectable antibodies to JSRV in affected animals (Sharp & Herring, 1983; Ortín et al., 1998
).
The aims of the present study were to investigate whether JSRV infection, OPA lesions and clinical disease could be induced in older lambs and to determine the dynamics of the infection by PCR examination of peripheral blood samples.
Four groups of Scottish Blackface lambs were inoculated with JSRV-containing LF, as confirmed by Western blot and RT-PCR (Palmarini et al., 1995). Ages at inoculation were: 1 week (n=5), 1 month (n=10), 3 months (n=10) and 6 months (n=10). All animals were from an OPA-free flock at Moredun Research Institute, UK. Six lambs from the same source were used as unchallenged controls.
LF collected from sheep that were naturally affected with OPA was clarified and stored at 70 °C. In order to remove variation in JSRV titres between batches of LF and to ensure that each lamb received the same inoculum, several batches of LF from different sheep were thawed, pooled together, aliquotted and stored at 70 °C. Immediately before inoculation of the lambs, the appropriate number of aliquots were thawed and concentrated to give a 12·5-fold concentrate of the original volume (Sharp et al., 1983). Each lamb in each age group received 5 ml of this concentrated LF pool by the intratracheal route, as described previously (Sharp et al., 1983
).
Peripheral blood samples were collected into EDTA tubes immediately before challenge and at intervals until necropsy. Peripheral blood leukocytes (PBLs) were obtained by centrifugation after lysis of erythrocytes (García-Goti, 1999) and the cell pellet was stored at 70 °C. DNA extraction was performed by using a DNeasy Tissue kit (Qiagen) following the manufacturer's protocol for animal blood. Blood samples from OPA-free Icelandic sheep were processed simultaneously as negative controls.
A single-round PCR to amplify the U3 region of JSRV was developed, based on the previously reported primers PI and PIII (Palmarini et al., 1996). The new PCR was validated by using DNA from the OPA cell line JS7 (Jassim et al., 1987
; DeMartini et al., 2001
), diluted in a background of 600 ng DNA from Icelandic sheep PBLs. Optimized PCR conditions were: 5 µl 10x PCR buffer (Qiagen), 200 µM dNTPs (Roche Diagnostics), 200 nM each primer (MWG) and 1·25 U HotStarTaq polymerase (Qiagen) were added to ultra-pure water (Sigma) to a final volume of 50 µl. To this pre-mix, 600800 ng test DNA was added and incubated at 94 °C for 16 min, followed by 35 cycles at 94 °C for 30 s, 59 °C for 30 s and 72 °C for 30 s, with a final extension step of 5 min at 72 °C. This JSRV-U3-PCR successfully amplified 10100 copies of JSRV template that were integrated into JS7 genomic DNA (data not shown). The test samples were analysed in triplicate and considered to be positive if one or more of the replicates gave a band of the correct molecular mass. To confirm the integrity of the DNA, samples that were negative in the JSRV-U3-PCR were tested for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (Palmarini et al., 1996
).
Animals were monitored daily after experimental infection for clinical signs of respiratory disease that might be suggestive of OPA. As soon as these were identified, lambs were killed humanely. Lambs that did not show any clinical signs were kept for a maximum of 7 months post-infection (p.i.) and then culled. Unchallenged control animals were culled at the end of the experiment, at the same time as the last inoculated lambs. At necropsy, examination of the lungs was performed to identify any gross lesions that might be suggestive of OPA. Neoplastic-like areas or, in their absence, a range of tissue sites from all pulmonary lobes were taken for microscopic examination. Samples were fixed in 10 % neutral-buffered formalin, processed routinely, sectioned at 5 µm and stained with haematoxylin and eosin (HE). Sections from selected lung samples were subjected to immunohistochemical examination for JSRV capsid protein (JSRV-CA) and JSRV surface glycoprotein (JSRV-SU) by using a newly developed antiserum, which was prepared by immunizing rabbits with glutathione S-transferase-SU (GST-SU) and His-tagged SU proteins in Freund's incomplete adjuvant. Recombinant fusion proteins were made by cloning and expressing nt 55576483 of JSRV (GenBank accession no. M80216) following standard molecular biology procedures (Sambrook et al., 1989). The fragment inserted in pGEX1
T (Pharmacia) was expressed in Escherichia coli cells to generate GST-SU, which was purified by using glutathione/Sepharose beads (Invitrogen). The fragment inserted in pIVEX2.4a (Roche Molecular Biochemicals) was expressed as a His-tagged protein by using the cell-free translation system RTS-500 (Roche Molecular Biochemicals), purified by using ProBond nickel resin (Invitrogen) and eluted following denaturing conditions according to the manufacturer's protocol. Both antibodies were used at 1/1000 dilutions as described previously (Palmarini et al., 1995
). Samples of lung and mediastinal lymph node were taken for JSRV-U3-PCR analysis.
None of the unchallenged control lambs developed clinical signs of respiratory disease and their PBLs were negative by JSRV-U3-PCR throughout the experiment. When culled at 14 months of age, neither histopathological lesions of OPA nor proviral DNA was detected in lung or mediastinal lymph-node samples.
In four of five 1-week-old lambs, severe clinical signs that were suggestive of OPA were observed at 7074 days p.i. (Fig. 1), whereas the other lamb (1W1; Fig. 2
) died at 28 days p.i. of an unrelated illness. All five lambs showed gross and microscopic lesions of OPA (Fig. 2
) and were consistently positive after infection by JSRV-U3-PCR (Fig. 2
).
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Nine out of ten 3-month-old lambs developed signs of OPA, with incubation periods ranging from 159 to 192 days (mean, 185 days; Fig. 1). The other animal (3M10; Fig. 2
) was asymptomatic when culled at 192 days p.i. Macroscopic lung lesions were confirmed histologically as OPA in all ten lambs. Whilst JSRV viraemia was detected consistently in six lambs, including the one that remained asymptomatic, the other four (3M2, 3M3, 3M5 and 3M8; Fig. 2
) were JSRV-U3-PCR-negative on occasions during the experiment.
Five out of ten 6-month-old lambs developed clinical OPA with incubation periods ranging from 164 to 169 days (mean, 182 days; Fig. 1) and, amongst the other five sheep, three (6M8, 6M9 and 6M10) were asymptomatic when culled at 190192 days p.i. All five clinically affected sheep and two asymptomatic lambs showed pathological evidence of neoplasia, although in one of them (6M9; Fig. 2
), only microscopic lesions were detected. Two lambs (6M1 and 6M2; Fig. 2
) died early from OPA-unrelated conditions; in these and in one of the asymptomatic culls (3M10; Fig. 2
), neither gross nor microscopic lesions of OPA were found. Within this group of 6-month-old lambs, PBLs from all animals were JSRV-U3-PCR-negative at 3 weeks p.i. but, with the exception of a single sheep (6M9; Fig. 2
), they were consistently positive thereafter.
Regardless of age at challenge, all sheep with histologically confirmed OPA showed the same pattern of neoplasia, which consisted predominantly of papillary adenocarcinoma of the alveoli (Fig. 3a, b). Immunohistochemical examinations showed JSRV-CA protein in the cytoplasm of a proportion of ATII neoplastic cells and nodules with the features described in previous studies (Palmarini et al., 1995
; Platt et al., 2002
). Detection of immunohistochemical reactivity to JSRV-SU was consistent in epithelial cells lining neoplastic alveoli (Fig. 3c
) and in polypoid structures in the bronchioles (Fig. 3d
). Immunolabelling was strongest at the apical surface of the cells.
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The present study demonstrates clearly that JSRV infection can be induced in 100 % of lambs aged 16 months at the time of inoculation and that a high proportion of these animals develop clinical signs (6290 %) and lesions (87100 %) of OPA. This is in contrast to a previous study on a much smaller scale, in which only a proportion of 10-week-old lambs developed neoplastic lesions (Rosadio et al., 1988). One explanation for this discrepancy and for the success of the current experiment would be a large amount of JSRV in the inoculum, as LF contains more JSRV than tumour extracts (Herring et al., 1983
) and JSRV dose and incubation period are correlated inversely (Verwoerd et al., 1980
). Nevertheless, some findings in the present study support the notion of an age-related resistance to OPA. Firstly, within those animals that developed clinical OPA, the incubation period was significantly shorter (P<0·001) for lambs that were inoculated at 1 week of age than for older lambs (Fig. 1
) and it was also shorter (P=0·03) for animals that were challenged at 1 month than for those inoculated at 3 months. Secondly, the development of lesions was delayed in older lambs, as indicated by three asymptomatic lambs in the oldest age group that had no evidence of OPA lesions and a fourth lamb with only microscopic lesions (Fig. 2
). Finally, detectable viraemia occurred later in the group of 6-month-old lambs than in the group of 1-week-old lambs (Fig. 2
), and possibly also later than in the 1- and 3-month-old animals, although straight comparisons cannot be made with these two groups, as their first PCR testing on PBLs was slightly delayed.
As the same inoculum was used for all 35 lambs, our results suggest that age at infection has an effect on the development of OPA; this is particularly noticeable between 1 week and 1 month of life.
Other examples of age-related susceptibility to retroviral infections include feline leukemia virus (Hoover et al., 1976; Flynn et al., 2000
), certain avian leukosis viruses (Pizer et al., 1992
; Stedman et al., 2001
), visna/maedi virus (Lairmore et al., 1986
; Andresson et al., 1993
) and MasonPfizer monkey virus (Fine et al., 1975
). Whilst, for some of these infections, differences in the ability of neonates and older animals to mount immune responses could account for an age-related susceptibility, this is unlikely for OPA, in view of the lack of a specific humoral immune response to JSRV (Sharp & Herring, 1983
; Ortín et al., 1998
).
A more plausible explanation for the age effect in OPA would be that the target cells for primary JSRV infection and oncogenesis, ATII pneumocytes and Clara cells of the bronchioles, are present in higher numbers in newborn lambs than in older animals. In rodents and ruminants, the ratio of type I pneumocytes to ATII cells at birth is around 1 : 2, with ATII pneumocytes accounting for approximately 60 % of alveolar cells (Weibel & Taylor, 1996; Kahwa et al., 1997
; Otto, 1997
). During the first month of life, the number of ATII cells declines progressively and dramatically, so that they represent only 6 % of alveolar cells at 7 days, 1 % at 3 weeks and 0·2 % at 1 month, with no further significant changes occurring later (Wright & Alison, 1984
). Similarly, Clara cells retain fetal characteristics in calves and goat kids that are up to 30 days old, but lose them and become fully differentiated after this time (Castleman & Lay, 1990
; Kahwa et al., 2000
). We hypothesize that following intratracheal inoculation of JSRV, primary virus replication would occur in the ATII and Clara cells and that the magnitude of such replication would depend on the availability of these target cells. Thus, higher viral production could be expected in newborn lambs, leading to earlier detection of viraemia and development of neoplastic lesions (and hence a shorter incubation period), and vice versa in older lambs.
In conclusion, we have developed a new experimental model to induce JSRV infection and OPA in aged lambs, which can be monitored by an efficient PCR technique in blood samples. Apart from providing new insights into the pathogenesis of the disease by confirming an age effect in the timescale of OPA-related events, the model will be useful to test the efficacy of potential vaccines. Finally, our PCR results reinforce previous studies on the detection of JSRV infection in subclinically infected sheep (García-Goti, 1999; González et al., 2001
), providing an opportunity for future epidemiological research.
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ACKNOWLEDGEMENTS |
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Received 1 June 2004;
accepted 3 August 2004.
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