Primary respiratory tract and lung tumors in animals have a lower incidence compared with other systems, as well as compared to human patients. In contrast, the lung is the preferential site of metastases in all animals [34]. In sheep, the most common tumor of the respiratory system is ovine pulmonary adenocarcinoma (OPA), also known as jaagsiekte or ovine pulmonary adenomatosis [32].
This work confirms the presence of JSRV-infected sheep in Romania. To the author’s knowledge, JSRV-related OPA has not been reported in other countries of Eastern Europe in the last decade.
For this study, the samples were collected from two slaughterhouses which are representative for Transylvania, not for the whole country. Unfortunately, due to specific slaughtering procedures, we collected the pulmonary tissue samples only at the end of slaughtering. As a consequence, an exact match between examined lungs and ear tag for each slaughtered sheep was not possible to be determined. Therefore, neither the exact origin and age of each sheep were not possible to be established. However, according to the records of the slaughterhouses, all slaughtered individuals were adult females and originated from different counties of Transylvania, including Cluj, Bistrita-Nasaud, Mures, Sibiu and Alba. Thus, the authors can relate that all the positive OPA cases originated from Transylvania, a historical region that is located in center of Romania. The present study shows that the prevalence of OPA is about 1.26%, and is higher compared to the previous reports regarding OPA situation in Romania, 0.8% and 0.5%, respectively [29, 30]. ExJSRV presence was not determined in any of these two previous studies. Furthermore, the prevalence of OPA in Romania is higher than in other European countries, including UK (0.9%) [18] and Ireland (0.5%) [17]. We can think about two possible explanations of this prevalence of OPA in Romanian Turcana sheep. First, it can be related with a high number of sheep that are raised in Romania. According to the Ministry of Agriculture data the number of sheep in 2019 was over 11 million heads. This places the Romanian sheep industry in the third position in Europe after Spain and UK. Another important causative factor could be related with sheep management in Romania. The majority of animals are raised in free pastures and there is a lot of sheep movements between regions to find fresh grasslands. This is a traditional sheep breeding management and it is a way of life for the sheep breeders that is still very present in Romania. These two factors might explain a higher OPA prevalence in Romanian sheep compared with other European countries. In contrast to the prevalence of OPA in Romania, the disease is quite common in South America, South Africa, and Scotland, where 5-20% of infected animals develop pulmonary tumors [35].
OPA represents a continuous issue regarding proper methods of diagnosis and prevention [36, 37]. Despite some specific clinical signs of classical OPA, (including dyspnea and tachypnea), in combination with progressive weight loss and nasal mucous discharge [38], and suggested imaging modalities (radiography, computed tomography and ultrasonographic examination) [39, 40] currently the gold standard diagnostic method for both classical and atypical OPA relies in gross and histology exams performed during post mortem evaluation [7]. In our study, data about clinical signs of slaughtered sheep with OPA were not available. Postmortem evaluation, including gross and histological exams of the affected sheep is essential for confirmation of OPA. Although gross changes are relatively specific for OPA, in some cases chronic bronchopneumonia should be included in the differential list. A recent study showed that numerous cases (78%) of suspected OPA, based on gross changes, were false positive lesions due to its similarities with chronic bronchopneumonia; moreover 67% were exJSRV-negative by RT-PCR and IHC. In some cases, OPA and bronchopneumonia can occur concurrently, and the pneumonic lesions may obscure the characteristic histological features of OPA [17]. Lesions of chronic bronchopneumonia in sheep are usually locally-extensive, dense, grey-dark red in colour, affecting the cranioventral region of the lung [35]. In our study, chronic suppurative bacterial bronchopneumonia without neoplastic lesions was histologically diagnosed only in four cases of suspected OPA, characterized by cranioventral consolidation of the pulmonary lobes. Our findings showed that the distribution of both classical and atypical forms of OPA was different than those caused by chronic bacterial bronchopneumonia, and the diaphragmatic lobe was the most affected part of the pulmonary parenchyma. In the other two cases, OPA had concurrent pulmonary necrosis, fibrosis, atelectasis and abscesses caused by Corynebacterium pseudotuberculosis, and these findings are in agreement with a previous study [18].
The histological features of OPA consist of proliferation of epithelial cells into acini, papillary and solid structures, supported by a fibrovascular stroma. In atypical form, in addition to the epithelial neoplasia, marked fibroblast proliferation, collagen deposition and mononuclear cell infiltration are characteristic microscopic changes [41]. These inflammatory changes may be caused by intercurrent infections or a specific immune response of the host [4]. Occasionally, small nodules of neoplastic loose mesenchymal tissue appear admixed with the neoplastic component [42]. Both epithelial and mesenchymal components showed rare mitoses and a ki67 index mean value of 11.4% [33], and this is in good agreement with our findings, where ki67 index of the epithelial component of classical OPA was higher (mean value of 10.87 %) than atypical OPA (mean value of 4.54 %).
Another subtype of solitary tumor associated with exJSRV in sheep is grossly characterized by round or elongated, white–grey nodules of bright gelatinous appearance, and sharply demarcated from the surrounding parenchyma [33]. These nodules are histologically classified as myxoid nodules or myxoid growths or mesenchymal growths (MGs) [33, 43]. MGs resemble characteristic features of benign mesenchymal tumors and have been described in a variable proportion of tumours, intermingled with the neoplastic epithelial component [43]; in some cases, they were identified in metastases [22]. The MGs are not an uncommon feature encountered in OPA, but interestingly, they were not reported in experimental studies [44].
In the current study, 8 out of 34 ovine pulmonary tumors presented MGs, accounting 23.53% of the samples, although there are reports of up to 40% of the OPAs that contain also the neoplastic mesenchymal component [45].
The MGs are also recognised of being associated to JSRV infection due to the positive constant virus immunolabeling [33 ,42, 46]. Our results revealed, in all cases of MGs, an intense immunoexpression using antibodies against JSRV-MA, and confirmed that the MGs represent JSRV-associated neoplastic growths.
The MGs were immunohistochemically characterized using mesenchymal and muscle cells markers, but their embryonic origin is still debated. IHC showed that these nodules are composed of true mesenchymal cells, and do not express epithelial cell markers [33]. The histological features of MGs are similar to myxomas that have a population of spindle or stellate cells embedded in a matrix rich in acid mucopolysaccharides [47]. Primary pulmonary myxoma not associated to JSRV was also reported in sheep [48, 49]. In our study, two cases of pulmonary nodules showed specific gross, histological and histochemical (AB-PAS matrix) features of pulmonary myxoma, and were confirmed through IHC. Pulmonary myxomas are immunopositive for vimentin and positive for S100 protein [49]. Our results, regarding the negative immunoreaction of pulmonary myxomas and MGs for S100 protein are in contrast with these previous evidence [49], but go towards other reported data [48].
A recent study demonstrated that the origin of mixed solitary OPA, containing both epithelial and mesenchymal components, consisted of local progenitor cells involved in lung repair [33]. It is well known that a small population of contractile fibroblasts are frequently found in normal alveolar septae, and the myofibroblast overactivity is directly correlated with lung fibrosis progression [50]. In lungs chronically affected with fibrosis, the mesenchymal cell phenotypes vary from proliferating fibroblasts to fully differentiated smooth muscle cells, and the predominant cell type is the myofibroblast, a fibroblast-like cell that expresses alpha-smooth muscle actin (alpha-SMA) [51]. Moreover, an immunohistochemical study of human pulmonary fibrosis revealed that myofibroblasts express SMA, vimentin, and desmin [51, 52].
Based on the immunohistochemical results of the present study and according to the aforementioned findings, our hypothesis suggests that the cell origin of this mesenchymal proliferative lesions or MGs is most likely contractile myofibroblasts.
IHC is also a useful method to identify small pulmonary lesions of OPA and to differentiate primary form metastatic pulmonary tumors, but is not routinely used as commercial test in veterinary practice [17]. IHC was recently used for evaluation of classical and atypical form of OPA in sheep [16, 53].
In the present study all tumors diagnosed as acinar and/or papillary predominate type of pulmonary adenocarcinoma, were MCK and TTF1 positive, and negative for vimentin, suggesting a primary epithelial origin of lung tissue. TTF1 (Thyroid transcription factor-1) is a 38 kDa nuclear protein member of the Nkx2 homeodomain transcription factor family, expressed by canine and feline type II alveolar pneumocytes, bronchiolar and thyroid epithelial cells [54, 55]. In this population, TTF-1 expression was intensely and diffusely positive in all epithelial cells of pulmonary adenocarcinoma, classical and atypical forms, and negative in the MGs, confirming that TTF-1 maintains a very high specificity for respiratory epithelium in sheep, as suggested by previous studies [56].
The results of this study also provide important evidence on the use of ICC and TEM on JSRV identification in postmortem collected lung tissue. The cellular composition of spontaneously-arising OPA tumours has been previously characterized by electron microscopy as round oval virions of 100–125 nm in diameter, with small bump-like structures existing on the surface of the virus particles [57], and are confirmed by our findings. ICC is a highly productive method used in biomedical research to identify various antigens in cells [58]. In this study, ICC showed the presence of intracytoplasmic JSRV antigen within neoplastic cells and fewer macrophages in all examined cases. Furthermore, all ICC positive cases were confirmed by IHC and PCR analyses. This method could have many practical applications but, to the author’s knowledge no other data supporting the utility of ICC in the detection of JSRV in sheep is reported. We consider that ICC could be used as a rapid and efficient test for JSRV identification from OPA, and it could have a possible application for intravitam diagnosis of JSRV, however this requires further studies.
PCR method is considered to be more sensitive than IHC for identification of exJSRV from lung tissue samples [17]. In this respect, we aimed to detect by PCR the presence of exJSRV proviral DNA by amplifying a region from the LTR sequence. ExJSRV was identified by PCR in 97.05% of analysed samples exhibiting tumors, showing a higher sensitivity than immunohistochemistry (94.11%). In all cases, the adjacent pulmonary parenchyma was negative for exJSRV. However, in one case, we could not prove the presence of JSRV in OPA neither by PCR nor IHC. The PCR results are in agreement with those described in other studies [17].
Bai et al., (1996) [3] demonstrated that exJSRVs derived from some endogenous JSRV loci following mutations in LTR or other regions of the proviral genome during evolution. The long terminal repeat (LTR) U3 sequence and the envelope gene (env) are the major factors of retroviral tropism. LTR contains the viral promoter and enhancer which interact with the cellular transcription machinery, and it is preferentially active in cell lines originated from Clara cells and alveolar type II pneumocytes [59, 60]. LTR is also considered the only element capable to distinguish exogenous JSRV associated with OPA from endogenous loci in the sheep genome [3]. In our report, the phylogenetic analysis showed that the Romanian isolates (JSRV-RO) belonged to exJSRV, type 2, and it was very similar to UK strain (accession number AF105220.1). These findings provide novel information on the geographic distribution of the genetic lineages of exJSRV in sheep.
Our findings confirm that exJSRV - related OPA represents a retroviral neoplastic disease that needs to be included in the list of possible differential diagnoses of chronic respiratory distress in the sheep population in Eastern Europe. The zoonotic potential of sheep exJSRV- OPA infection and its implications in human health remains to be investigated.