Abstract
Ovine pulmonary adenocarcinoma (OPA) is a transmissible lung cancer caused by Jaggsiekte sheep retrovirus (JSRV). It is difficult to identify animals infected with JSRV but are clinically healthy. The virus does not induce a specific antibody response and, although proviral DNA sequences of JSRV can be found in mononuclear blood cells, the detection is inconsistent. The aim of this study was to investigate the presence of JSRV in the bone marrow of infected sheep and develop a more consistent screening method. Immunohistochemical examination of bone marrow samples from 8 asymptomatic JSRV-infected sheep revealed the presence of positively labelled cells. However, JSRV could not be detected by a highly sensitive polymerase chain reaction (PCR) in bone marrow aspirates periodically collected from these animals. Results suggest that JSRV-infected cells may be present in the bone marrow of symptomless animals, but the number is below the detectable level for PCR. Therefore, this technique does not seem to be helpful for preclinical diagnosis of OPA.
Résumé
L’adénocarcinome pulmonaire ovin (OPA) est un cancer pulmonaire transmissible causé par le rétrovirus ovin de Jaggsiekte (JSRV). Il est difficile d’identifier les animaux infectés par le JSRV mais qui sont cliniquement en santé. Le virus n’entraine pas la production d’anticorps spécifiques et, bien que des séquences d’ADN provirales de JSRV peuvent être retrouvées dans les mononucléaires du sang, la détection est inconstante. L’objectif de la présente étude était d’examiner la présence de JSRV dans la moelle osseuse de moutons infectés et de développer une méthode de tamisage plus constante. L’examen par immunohistochime d’échantillons de la moelle osseuse de huit moutons asymptomatiques mais infectés par JSRV a révélé la présence de cellules positivement marquées. Toutefois, le JSRV ne put être révélé par une épreuve d’amplification en chaine par la polymérase (PCR) très sensible à partir d’aspirations de la moelle osseuse récolées périodiquement à partir de ces animaux. Les résultats suggèrent que les cellules infectées par JSRV peuvent être présentes dans la moelle osseuse d’animaux asymptomatiques, mais le nombre se situe sous le seuil détectable pas PCR. Ainsi, cette technique ne semble pas utile pour le diagnostic préclinique d’OPA.
(Traduit par Docteur Serge Messier)
Ovine pulmonary adenocarcinoma (OPA), also known as sheep pulmonary adenomatosis or Jaagsiekte, is an infectious disease of sheep. It occurs naturally in almost all countries worldwide, with the exception of Australia, New Zealand, and Iceland. Ovine pulmonary adenocarcinoma is a transmissible lung cancer caused by Jaggsiekte sheep retrovirus (JSRV) (1) that induces transformation of secretory epithelial cells of the distal respiratory tract, specifically progenitors of type II pneumocytes (2). Diagnosis of OPA in vivo depends on the terminal clinical signs of affected animals, such as dyspnea, moist respiratory sounds, and copious secretion of lung fluid (3). At this stage, the presence of JSRV can be confirmed in lung fluid by immunoblotting (4), enzyme-linked immunosorbent assay (ELISA) (5), or polymerase chain reaction (PCR) (6). It is also possible to obtain confirmation of suspected clinical OPA at early stages, in the absence of excessive lung fluid, by PCR testing of bronchoalveolar lavage samples (7). However, it is difficult to identify infected animals during the long incubation period, when animals remain clinically healthy, due to the lack of a specific antibody response against JSRV (3,8,9). Jaggsiekte sheep retrovirus proviral DNA has been shown by PCR to be in peripheral blood mononuclear cells (PBMC) prior to the onset of OPA lesions in experimentally infected animals (10,11), and during the preclinical period of the natural disease (12–14). However, detection was inconsistent (13,14), probably as a result of the low proviral load in blood cells (10). The apparent poor sensitivity of PCR analysis on blood samples indicates this technique is unsuitable for screening individual animals, rather it may be appropriate at flock level (14). Therefore, for the detection of isolated cases and the implementation of OPA control programs, preclinical tests with greater sensitivity to detect JSRV-infected individuals would be very helpful.
Previous studies have shown the presence of different retroviruses in bone marrow cells, such as human immunodeficiency virus (15), feline immunodeficiency virus (16,17), and caprine arthritis- encephalitis virus (18). It has been proposed that infected bone marrow cells may represent a viral reservoir, maintaining viral infection and replication (15–18). Jaggsiekte sheep retrovirus transcripts and proviral DNA have also been detected in bone marrow samples of OPA affected sheep (6), but until now, infected animals have not been investigated. The objective of the present study was to investigate for the presence of JSRV in the bone marrow of asymptomatic infected sheep using immunohistochemical and PCR techniques, and analyze whether the use of bone marrow aspirates for PCR detection of JSRV infected animals would be an improvement over the sensitivity of the PCR blood test.
A group of 8 JSRV infected ewes aged 4 to 8 years, with no clinical signs of disease, were selected from 2 flocks of the breed Rasa Aragonesa, with a long history of OPA, located in Zaragoza, Spain. The analysis of peripheral blood mononuclear cells (PBMC) by using a heminested PCR specific for JSRV U3 long terminal repeat sequences (U3hn PCR) (6), was positive in all of them and, consequently, they were considered JSRV infected. They were taken to the animal facilities at Zaragoza Veterinary Faculty where samples of bone marrow aspirates and blood were collected from each animal, at 30-day intervals, on 4 occasions. The bone marrow harvest procedure from sheep was approved by the Animal Research Committee of the University of Zaragoza. Briefly, animals were sedated with 0.2 mL 2% xylazine, IV. The wool from the sternal area was shorn, the skin was shaved and disinfected, and 2 mL lidocaine (a local anesthetic) was injected intradermally. The bone marrow was aspirated from the sternum using a 13 G biopsy needle (Osteobell “T”; Biopsybell Medical Devices, Mirandola, Italy) attached to a heparinized syringe. Approximately 5 mL of bone marrow was harvested from each animal, and a smear was prepared and examined for corroboration of the presence of bone marrow cells. Simultaneously, blood samples were obtained by venipuncture of the jugular vein using individual, heparinized tubes. The buffy coats, containing white cells, were obtained from the blood and bone marrow samples, as described by De las Heras, et al (14). Total DNA was extracted using a modified phenol-chloroform method (19) and DNA purity and concentration were estimated from the optical density at 260 and 280 nm. The highly sensitive U3hn PCR was used to check DNA from blood and bone marrow samples. Six replicates, each containing 500 ng of test DNA, were prepared for each sample. Appropriate controls, including positive and negative controls, were used at each step to detect contamination. A product of 133 bp was expected. Samples were considered positive when at least 1 of the replicates was found to be positive. Samples that gave a negative result were tested by PCR for glyceraldehyde-3-phosphate dehydrogenase (GAP-DH) (6) in order to confirm the DNA quality. At the end of the experimental period, animals were humanely slaughtered according to local euthanasia regulations. At necropsy, tissue samples from mediastinal lymph node were taken, snap frozen, and stored at −80°C until U3hn PCR analysis. The lungs were examined thoroughly, and samples were obtained from several defined lung locations and from any gross lesion suggestive of OPA. Samples of bone marrow from the sternum were also taken. These samples were processed routinely for histological examination and sections were evaluated immunohistochemically, by previously described procedures, using a rabbit polyclonal antibody against JSRV gag proteins (5).
The results obtained in this study are summarized in Table I. The 8 sheep selected for this experiment were PCR positive in the mediastinal lymph node, considered the best possible indicator of pre-clinical JSRV infection (14), confirming they were all infected. Jaggsiekte sheep retrovirus was detected intermittently by PCR of the PBMC during the experiment, with 5 out of the 8 (62.5%) sheep testing positive in at least 1 of the sample periods. Jaggsiekte sheep retrovirus detection by PCR of the bone marrow aspirates was negative in all the replicates, of all the samples, of all the animals tested (a total of 192 replicates). Nevertheless, immunohistochemical examination showed the presence of very few positively labelled cells in bone marrow sections (1 to 3 positive cells after complete examination of the section) from 3 sheep (Figure 1). As mentioned, none of the animals in this study showed clinical signs of OPA, but necropsy revealed small (1–2 cm diameter) single, atypical OPA lesions (20) in 3 of them, which were confirmed by immunohistochemistry.
Table I.
Polymerase chain reaction (PCR) results from blood, bone marrow, and mediastinal lymph nodes (MLN). Immunohistochemical (IHC) results from bone marrow and ovine pulmonary adenocarcinoma (OPA) lesions in the group of 8 sheep infected with Jaggsiekte sheep retrovirus (JSRV)
| PCR results | IHC results | ||||
|---|---|---|---|---|---|
|
|
|
||||
| Sheep number | Blood | Bone marrow | MLN | Bone marrow | OPA lesion |
| 1 | 0/0/0/1 | 0/0/0/0 | 4 | Negative | No lesion |
| 2 | 0/1/0/0 | 0/0/0/0 | 4 | Negative | No lesion |
| 3 | 0/0/0/0 | 0/0/0/0 | 3 | Positive | No lesion |
| 4 | 1/0/1/0 | 0/0/0/0 | 4 | Negative | Positive |
| 5 | 0/0/0/0 | 0/0/0/0 | 2 | Negative | No lesion |
| 6 | 0/0/1/0 | 0/0/0/0 | 2 | Positive | No lesion |
| 7 | 0/1/0/0 | 0/0/0/0 | 2 | Negative | Positive |
| 8 | 0/0/0/0 | 0/0/0/0 | 2 | Positive | Positive |
| Animals PCR positive (n = 8) | 5 | 0 | 8 | ||
| Total PCR positive | 62.5% | 0% | 100% | ||
| Animals IHC positive (n = 8) | 3 | 3 | |||
Four blood and bone marrow aspirate samples taken at 30-day intervals and samples of mediastinal lymph node (MLN) obtained after sacrifice were analyzed using PCR for each of the 8 JSRV-infected sheep. Six replicates were prepared for each sample and results are shown as number of positive replicates. Immunohistochemical results for bone marrow and OPA lesions are also noted. A rabbit polyclonal antibody against JSRV gag proteins was used.
Figure 1.
Immunohistochemistry of a bone marrow section from a Jaggsiekte sheep retrovirus (JSRV)-infected sheep, showing a single positive cell with labelled cytoplasm (arrows). A rabbit polyclonal antibody against JSRV gag proteins was used. Bar: 25 μm.
In this study, data obtained from the analysis of blood samples are in agreement with previous studies showing intermittent JSRV detection and few positive replicates in animals with evidence of infection but with no lesions or very early lesions of OPA (13,14).
However, JSRV could not be detected by PCR in any of the 192 replicates of bone marrow aspirates analyzed, although immunohistochemical examination of the bone marrow sections showed positively labelled cells in 3 of the animals. As inhibition of GAP-DH PCR was not found, the DNA from these samples was understood to be in good condition and these results indicate that the proviral load was not high enough to allow detection by this highly sensitive heminested PCR method. This procedure detects a single copy of JSRV provirus in a background of normal genomic sheep DNA (6). Assuming 500 ng DNA is equivalent to 75 000 cells and 6 replicates were prepared for each sample, the frequency of infected cells in bone marrow of asymptomatic animals seems to be even lower than that estimated in the PBMC of OPA affected animals (1/240 000 cells) (10).
The results of this study show that JSRV infected cells may be present in the bone marrow of symptomless animals, although in very low numbers. They seem to be below the detectable level for PCR, even using a highly sensitive heminested method and analyzing a large number of replicates. Therefore, PCR testing for JSRV derived from bone marrow aspirates does not appear to be more sensitive than the PCR blood test and does not constitute a useful tool for preclinical diagnosis of OPA.
Acknowledgments
Marta Borobia and Aurora Ortín share primary authorship of this work. The authors are grateful to Dr. Massimo Palmarini for generously providing the antibody against JSRV gag proteins and to Dr. Christina Summers for critically reading and revising the manuscript. This study was supported by the Department of Science, Technology and University of the Aragón Government and the European Social Fund.
References
- 1.Palmarini M, Sharp JM, De las Heras M, Fan H. Jaagsiekte sheep retrovirus is necessary and sufficient to induce a contagious lung cancer of sheep. J Virol. 1999;73:6964–6972. doi: 10.1128/jvi.73.8.6964-6972.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Murgia C, Caporale M, Ceesay O, et al. Lung adenocarcinoma originates from retrovirus infection of proliferating type 2 pneumocytes during pulmonary post-natal development or tissue repair. PLOS Pathogens. 2011;7(3):e100201411. doi: 10.1371/journal.ppat.1002014. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.De las Heras M, González L, Sharp JM. Pathology of ovine pulmonary adenocarcinoma. Curr Top Microbiol Immunol. 2003;275:25–54. doi: 10.1007/978-3-642-55638-8_2. [DOI] [PubMed] [Google Scholar]
- 4.Sharp JM, Herring AJ. Sheep pulmonary adenomatosis: Demonstration of a protein which cross-reacts with the major core proteins of Masson-Pfizer monkey virus and mouse mammary tumour virus. J Gen Virol. 1983;64:2323–2327. doi: 10.1099/0022-1317-64-10-2323. [DOI] [PubMed] [Google Scholar]
- 5.Palmarini M, Dewar P, De las Heras M, et al. Epithelial tumour cells in the lungs of sheep with pulmonary adenomatosis are major sites of replication for Jaagsiekte retrovirus. J Gen Virol. 1995;76:2731–2737. doi: 10.1099/0022-1317-76-11-2731. [DOI] [PubMed] [Google Scholar]
- 6.Palmarini M, Holland MJ, Cousens C, Dalziel RG, Sharp JM. Jaagsiekte retrovirus establishes a disseminated infection of the lymphoid tissues of sheep affected by pulmonary adenomatosis. J Gen Virol. 1996;77:2991–2998. doi: 10.1099/0022-1317-77-12-2991. [DOI] [PubMed] [Google Scholar]
- 7.Voigt K, Brügmann M, Huber K, et al. PCR examination of bronchoalveolar lavage samples is a useful tool in pre-clinical diagnoses of ovine pulmonary adenocarcinoma (Jaagsiekte) Res Vet Sci. 2007;83:419–427. doi: 10.1016/j.rvsc.2007.02.001. [DOI] [PubMed] [Google Scholar]
- 8.Ortín A, Minguijón E, Dewar P, et al. Lack of specific immune response against a recombinant capsid protein of Jaagsiekte sheep retrovirus in sheep and goats naturally affected by enzootic nasal tumour or sheep pulmonary adenomatosis. Vet Immunol Immunopathol. 1998;61:229–237. doi: 10.1016/s0165-2427(97)00149-9. [DOI] [PubMed] [Google Scholar]
- 9.Summers C, Neil W, Dewar P, et al. Systemic immune responses following infection with Jaagsiekte sheep retrovirus and in the terminal stages of ovine pulmonary adenocarcinoma. J Gen Virol. 2002;83:1753–1757. doi: 10.1099/0022-1317-83-7-1753. [DOI] [PubMed] [Google Scholar]
- 10.Holland MJ, Palmarini M, García-Goti M, et al. Jaagsiekte retrovirus is widely distributed both in T and B lymphocytes and in mononuclear phagocytes of sheep with naturally and experimentally acquired pulmonary adenomatosis. J Virol. 1999;73:4004–4008. doi: 10.1128/jvi.73.5.4004-4008.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Salvatori D, González L, Dewar P, et al. Successful induction of ovine pulmonary adenocarcinoma in lambs of different ages and detection of viraemia during the preclinical period. J Gen Virol. 2004;85:3319–3324. doi: 10.1099/vir.0.80333-0. [DOI] [PubMed] [Google Scholar]
- 12.González L, García-Goti M, Cousens C, et al. Jaagsiekte sheep retrovirus can be detected in the peripheral blood during the preclinical period of sheep pulmonary adenomatosis. J Gen Virol. 2001;82:1355–1358. doi: 10.1099/0022-1317-82-6-1355. [DOI] [PubMed] [Google Scholar]
- 13.Caporale M, Centorame P, Giovannini A, et al. Infection of lung epithelial cells and induction of pulmonary adenocarcinoma is not the most common outcome of naturally occurring JSRV infection during the commercial lifespan of sheep. Virology. 2005;338:144–153. doi: 10.1016/j.virol.2005.05.018. [DOI] [PubMed] [Google Scholar]
- 14.De las Heras, Ortín A, Salvatori D, et al. A PCR technique for the detection of Jaagsiekte sheep retrovirus in the blood suitable for the screening of ovine pulmonary adenocarcinoma in field conditions. Res Vet Sci. 2005;79:259–264. doi: 10.1016/j.rvsc.2005.02.003. [DOI] [PubMed] [Google Scholar]
- 15.Canque B, Marandin A, Rosenzwajg M, et al. Susceptibility of human bone marrow stromal cells to human immunodeficiency virus (HIV) Virology. 1995;208:779–783. doi: 10.1006/viro.1995.1211. [DOI] [PubMed] [Google Scholar]
- 16.Tanable T, Yamamoto JK. Phenotypic and functional characteristics of FIV infection in bone marrow stroma. Virology. 2001;282:113–122. doi: 10.1006/viro.2000.0822. [DOI] [PubMed] [Google Scholar]
- 17.Sandy JR, Robinson WF, Bredhauser B, Kyaw-Tanner M, Howlett CR. Productive infection of bone marrow cells in feline immunodeficiency virus infected cats. Arch Virol. 2002;147:1053–1059. doi: 10.1007/s00705-001-0790-2. [DOI] [PubMed] [Google Scholar]
- 18.Grossi P, Giudice C, Bertoletti I, et al. Immunohistochemical detection of the p27 capsid protein of caprine arthritis-encephalitis virus (CAEV) in bone marrow cells of seropositive goats. J Comp Path. 2005;133:197–200. doi: 10.1016/j.jcpa.2005.01.009. [DOI] [PubMed] [Google Scholar]
- 19.Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Vol. 2. Woodbury, New York: Cold Spring Harbor Laboratory Press; 1989. Isolation of DNA from mammalian cells; pp. 9.16–9.19. Book. [Google Scholar]
- 20.García-Goti M, González L, Cousens C, et al. Sheep pulmonary adenomatosis: Characterization of two pathological forms associated with Jaagsiekte retrovirus. J Comp Path. 2000;22:55–65. doi: 10.1053/jcpa.1999.0344. [DOI] [PubMed] [Google Scholar]