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Enzootic nasal adenocarcinoma in sheep: An update
Small Ruminant Research xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Enzootic nasal adenocarcinoma in sheep: An update ⁎
M. De las Heras , A. Ortín, M. Borobia, T. Navarro Departamento de Patología Animal, Universidad de Zaragoza.c/ Miguel, Servet 177, 50013, Zaragoza, Spain
A R T I C LE I N FO
A B S T R A C T
Keywords: Enzootic nasal adenocarcinoma Sheep Retrovirus
Enzootic nasal adenocarcinoma of sheep (ENAS) is a contagious tumor of the ethmoid turbinate mucosa. The disease occurs naturally in all continents except Australia and New Zealand. Similar disease has been described in goats. ENAS is aetiologically associated with enzootic nasal tumor virus 1 (ENTV-1) a type Beta retrovirus and closely related to jaagsiekte sheep retrovirus (JSRV) and enzootic nasal adenocarcinoma virus of goats (ENTV-2). Clinical signs include continuous nasal discharge, respiratory distress, exophtalmus and skull deformations. The tumors have been classified histologically as low grade adenocarcinoma. ENTV-1 can be demonstrated in tumors by immunohistochemistry using several antisera developed against JSRV antigens which cross-react with ENTV1 ones. ENTV-1 shares a oncogenic mechanisms with JSRV and ENTV-2 and active replication of the virus is required because oncogenic proprieties are associated with env gene products. Information about immune reactions in relation to ENTV-1 is scarce and controversial. Some early studies indicated absence of immune reaction although more recent studies have detected the presence of neutralizing antibodies in sheep with tumors and in contact sheep. However, the sensitivity and specificity of these tests are very low to be used in field studies. Several specific PRC techniques are available to detect viral genome in tissues and tumors. ENTV-1 can be detected in nasal secretion but is rarely found out of the tumor. The utility of PCR tests in control or eradication plans is discussed.
1. Introduction Enzootic nasal adenocarcinoma of sheep (ENAS, enzootic nasal tumor) was first described in Germany (Nieberle, 1940) and subsequently in many other countries since then. Thus the disease, which has been also reported in goats, has been recorded in all the major areas where sheep and goats are farmed with the exception of Australia and New Zealand. ENAS appears to be absent in the UK (reviewed in De las Heras et al., 2003). ENAS is a contagious neoplasm of gland cells of the ethmoid turbinate mucosa and aetiologically associated with a betaretrovirus. This retrovirus is known as enzootic nasal tumor virus 1 (ENTV-1) and it is closely related to jaagsiekte sheep retrovirus (JSRV) which causes the ovine pulmonary adenocarcinoma (OPA, jaagsiekte, ovine pulmonary adenomatosis), sheep endogenous retroviruses (Cousens et al., 1999) and the enzootic nasal tumor virus of goats (ENTV-2) (Ortín et al., 2003). The disease has been reproduced experimentally in goats (De las Heras et al., 1995) and in sheep (Walsh et al., 2013). In this brief review, we will try to summarize clinical and pathological features of the disease in sheep followed by pathogenic mechanisms of the ENTV-1. The last section will be dedicated to the
⁎
immune response against ENTV-1 in sheep and a discussion about diagnostic tests in vivo. 2. Clinical features and pathology Epidemiological data indicate that ENAS prevalence in affected flock is variable ranging from 0.1 to 15%. Preferentially young adults (3–5 years) (range 1–9 years old) are affected and several cases are always observed in the same flock (De las Heras et al., 1998). No genetic, sex or breed predisposition has been detected. The signs of the disease start with a small but continuous amount of sero-mucous fluid coming from the nostrils (unilaterally or bilaterally) (Fig. 1A). At this stage, the disease can be confused with other nasal pathologies like oestrosis, nasal polyps or bacterial infections. As the disease progresses, secretion is more abundant and causes the depilation on the area from the nostrils to the upper lips (Fig. 1B). This is a very particular clinical sing for this disease which can be used to differentiate from other nasal pathologies like oestrosis or salmonella infection. In advanced cases the disease shows characteristic clinical signs. Thus, snoring caused by stenosis of nasal passages, coughing, sneezing, head shaking together with softening and deformation of the skull bones (mainly frontal and
Corresponding author. E-mail address: [email protected] (M. De las Heras).
https://doi.org/10.1016/j.smallrumres.2019.04.018 Received 28 January 2019; Received in revised form 28 March 2019; Accepted 29 April 2019 0921-4488/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: M. De las Heras, et al., Small Ruminant Research, https://doi.org/10.1016/j.smallrumres.2019.04.018
Small Ruminant Research xxx (xxxx) xxx–xxx
M. De las Heras, et al.
Fig. 1. Clinical features of enzootic nasal adenocarcinoma of sheep. A: Young sheep showing early clinical signs. Unilateral sero- mucous clear fluid is coming continuously off the right nostril. B: Adult sheep showing advanced clinical signs. Nasal continuous sero-mucous discharge is characteristic together with softening of the region nasalis and fistula. Sero-mucous fluid also emerges from the fistula causing depilation of the close skin area below.
maxillary), exophthalmos and also skin fistula can be present (Fig. 1B). Body condition is gradually lost and animals eventually die due to bacteria complication of the tumor which ends with pneumonia or septicemia (reviewed in De las Heras et al., 2003). At necropsy, tumors are found in the nasal cavity, either unilaterally or bilaterally, arising from the ethmoidal mucosa and effacing the normal architecture of the ethmoidal conchae. Tumors are soft, grey or reddish-white in color with a fine granular surface and covered with mucus. They are found together with nasal polyps in some cases. The tumor grows compressing the close nasal conchae, cranial bones or invades paranasal or frontal sinuses (Fig. 2A). Necrosis or purulent inflammation of the tumors is also seen. Nasal tumors can be differentiate from other nasal pathologies like those associated with Salmonella diarizonae, because they are located in the ethmoidal conchae whereas salmonella associated lesions are found in the ventral turbinate and occasionally in the dorsal one. The microscopic architecture of the tumors is similar in all cases examined by us and joins the characteristics of a low grade adenocarcinoma of nasal glands (De las Heras et al., 2003). Light microscopy reveals epithelial secretory cells proliferating into an acinar, tubular, papillary or even solid patterns. Inner parts of the tumors are more tubular or acinar whereas external zones show a clear papillary pattern (Fig. 2B). Apart from some areas of cellular atypia or local invasion, no signs of malignancy and low mitotic index are seen. In addition, metastasis in regional lymph nodes or in other organs has never been recorded. The stroma is very scanty but infiltrated by, either scattered or grouped, lymphocyte/plasmocyte cells. Electron microscopy and histochemical studies revealed that neoplastic cells correspond to serous, mucous or mixed glands cells and seem to originate either in olfactory or respiratory mucosal glands (Reviewed in De las Heras et al., 2003). Several polyclonal and monoclonal specific antibodies to JSRV viral proteins, which cross-reacts with ENTV proteins, have been generated for virus demonstration on tissue sections by immunohistochemistry but they are not commercially available. They have been used as a research tool to differentiate ENA from other respiratory tumors and in other investigations.
naturally infected tumor bearing sheep (Eckstrand et al., 2013). Besides, North American and European genomes shared more than 96% sequence identity with high degree of aminoacid conservation among isolates (Walsh et al., 2010). Thus, it seems that ENTV-1 shows little genomic variations and it shares common pathogenic mechanisms with JSRV and ENTV-2. These three viruses interact with mammalian cells through HYAL-2 (hyaluronglucosaminase 2) receptor for virus attachment and entry and can infect different cell types. HYAL-2 is a ubiquitous membrane surface protein that belongs to hyaluronidases (Alberti et al., 2002; Dirks et al., 2002). However, ENTV-1 active replication is mostly restricted to nasal cavity epithelia where viral LTRs (long terminal repeat) are active (Yu et al., 2011). This active replication of the virus is important because their oncogenic properties are mainly on envelope proteins. Several in vitro experiments have indicated that JSRV and ENTV-1 share common mechanisms of cell transformation. JSRV and ENTVs env gene products clearly activates a number of proteins involved in signaling cascades controlling cell growth and fate such as Akt (Protein Kinase B) or MAPK (Mitogen activated kinase) signaling pathways, inducing permanent activation of these tyrosine kinase cell signaling routes disturbing de control of cell growth and survival leading to a growth advantage of deregulated cells. In spite of, other mechanisms leading to transformation, such as targeted integration of the virus genome cannot be totally ruled out (Alberti et al., 2002; Monot et al., 2015). A proviral clone of ENTV-1 has been constructed from which mature virions can be generated. This virus produced from this molecular clone will be very useful for future experiments to understand the pathogenesis of this disease (Walsh et al., 2016b).
4. Immune response and diagnostic tests The evaluation of the serological immune response in sheep with ENAS has been limited and controversial. Studies using Western blotting found reactivity to recombinant JSRV capsid protein (JSRV-CA), as a Glutathion S-transferase (GST) fusion protein, in sheep sera from diseased animals. However, this reaction could be abolished completely by absorption with the GST fusion partner but not with JSRV-CA. This suggested that the activity recognized against this recombinant JSRVCA was not specific (Ortín et al., 1998). In addition, Western blotting techniques also failed to detect any antibody against viral antigens from concentrated ENAS fluid (Ortín et al., 1998). A recent study has identified ENTV-1 reactivity by ELISA and Western blot in sheep serum from
3. ENTV-1 pathogenesis The comparison of the genomic sequence of the ENTV-1 with ENTV2 and JSRV concluded that these retroviruses are very similar (De las Heras et al., 2003). Like JSRV, ENTV-1 shows minimal sequence variation in the LTR(Long terminal repeat) non-coding region derived from
Fig. 2. Gross pathology and histopathology of enzootic nasal adenocarcinoma of sheep. A: Skull section showing tumor originated in the ethmoidal mucosa. Etmoidal nasal turbinates have been replaced by the tumor growing pressing dorsal and ventral conchae and causing obstruction of the nasal passages. B: Histopathology of the tumor. Nasal glands proliferate in an acinar and tubular pattern (asterisks). Nasal gland epithelia proliferates in tubulo-papillary pattern (star). Lympho-plasmocytic cells infiltrate the interstitium.
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ENAS affected and in contact sheep belonging to a flock with high incidence of ENAS. Serum samples were tested for reactivity against recombinant ENTV-1 capsid and surface subunit (Env) proteins produced using polyhistidine tag (His-tag) bacterial expression system and removing His-tag prior to use it as antigen. Results showed that reactive antibodies against both capsid and Env proteins could be detected in the serum of sheep either with or without tumor evidence using ELISA and Western blot analysis (Walsh et al., 2014). Both techniques detected immune reactive antibodies against Env protein from the flock with a history of ENAS but not in the naive sheep serum samples. Furthermore, some samples from these sheep were also positive to neutralization test of ENTV-1 Env-pseudotyped virions. These results suggest that these interactions were indeed specific and that sheep are able to develop antibodies against the ENTV-1. In spite of this, ELISA and virus neutralization test showed low specificity and sensitivity and they are not reliably to be used to diagnose ENTV-1 infection (Walsh et al., 2014). Similar results have been obtained in sheep experimentally infected with ENTV-1 (Walsh et al., 2016a). In any case, this is a very relevant finding because it would mean that sheep may be not immune tolerant to exogenous ENTV-1 infection as it was previously thought. Studies on cell-mediated immunity to ENTV-1 have yet to be reported (De las Heras et al., 2003). As we have indicated in previous sections, ENTV-1 has been sequenced completely (Cousens et al., 1999). Using this information and from other studies, several PCR-based diagnostic techniques have been generated which discriminated sequences of ENTV-1 from JSRV, ENTV2 and sheep endogenous retroviruses (Ortín et al., 2003; Walsh et al., 2014). The distribution of the ENTV-1 in several tissues has been investigated using a specific PCR test that amplifies part of the U3 region in the LTR of the ENTV-1 (heminested-PCR) to detect provirus in sheep genomic DNA (Ortín et al., 2003). This PCR can detect a low copy number of virus in a DNA sample (less than 20 copies in 500 ng of sheep DNA) and in order to increase the chances for the detection of the virus the test was triplicated on each sample (Ortín et al., 2003). These studies conclude that ENTV-1 is mostly confined in the tumor and it is rarely found in other tissues such as the lung. However, JSRV and ENTV-2 are found in other tissues and in the peripheral blood monocytes (PBMCs) and seem to establish a disseminated infection (Ortín et al., 2003, 2004). Using specific PCR techniques detection of JSRV or ENTV-2 in PMBCs samples is possible and allows identifying animals at preclinical stages and with no evidence of tumors (Gonzalez et al., 2001, De las Heras et al., unpublished observations). However, as ENTV-1 is never found in PMCs, blood PCR would not be suitable for detecting ENTV-1 infected animals. Specific RT-PCR (U5 and gag regions) from North American ENTV-1 isolates performed on nasal exudates have been proposed as a part of an ante-mortem diagnostic test for ENAS surveillance and eradication programs. In this study RT-PCR on nasal swabs is correlated with pathology (Walsh et al., 2014). However this technique has some weaknesses for the use of it in surveillance or eradication programs of the disease, particularly when this system tries to work with large flocks. Nasal swabbing/tampons may be useful for small size flocks but it becomes very difficult to manage in large ones. From the other hand, nasal tumors are originated in ethmoid turbinate and sampling using nasal swabs do not reach this area. Thus, despite the high specificity and sensitivity of the PCR technique the sampling technique may not obtain the proper material when the tumors are very small. In addition, RT-PCR on nasal swabs will detect animals with tumors but not already infected animals with no lesions. If the flocks dynamics of the ENTV-1 infection is similar to other retroviral infections like jaagsiekte sheep retrovirus infection (Gonzalez et al., 2001), nasal swabbing will not detect already infected animals but with no tumors. Another important factor which may have influence in the decision of an eradication plan is that the prevalence of the disease in affected flocks. In our geographic area, the prevalence is about 0.1−0.3% (De las Heras et al., 1998). In that case, removing animals with ENAS clinical signs and may be
enough to control the disease. In our opinion, RT-PCR tests based on nasal sampling may be useful under experimental conditions or for small flocks with low numbers of animals, but it has many weaknesses when is considered as a part of bigger plan of control or eradication program in normal farming situations of the most sheep rearing countries. In conclusion, either early clinical diagnosis or detection of infected animals before the tumors develop is very difficult for this disease at present. More research about natural infection and pathogenesis of ENAS is very necessary if we want to improve current diagnostic test and methods. 5. Concluding remarks We may conclude that no diagnostic test is currently available to detect ENTV- 1 infected sheep, but we can detect, using specific PCR techniques, sheep with small tumors and some of them clinically blind. However, sampling and PCR techniques are costly, difficult to implement in most of the sheep flocks and do not ensure the animal is free of the infection. Conflict of interest statement The authors have nothing to disclose. Acknowledgements Authors acknowledge University of Zaragoza, Gobierno de Aragón, Gobierno de España and European Commission providing funds for research on this sheep disease. References Alberti, A., Murgia, C., Shan-lu, L., Mura, M., Cousens, C., Sharp, J.M., Miller, D., Palmarini, M., 2002. Envelope-induced cell transformation by ovine betaretroviruses. J. Virol. 76, 5387–5394. Cousens, C., Minguijón, E., Dalziel, R.G., Ortín, A., García, M., Park, J., González, L., Sharp, J.M., De las Heras, M., 1999. Complete sequenze of enzootic nasal tumor virus, a retrovirus associated with transmissible intranasal tumors of sheep. J. Virol. 73, 3986–3993. De las Heras, M., García de Jalón, J.A., Minguijón, E., Gray, E., Dewar, P., Sharp, J.M., 1995. Experimental transmission of enzootic intranasal tumors of goats. Vet. Pathol. 32, 19–23. De las Heras, M., Minguijón, E., Ferrer, L.M., Ortín, A., Dewar, P., Cebrián, L.M., Pascual, Z., García, L., García de Jalón, J.A., Sharp, J.M., 1998. Naturally occurring enzootic nasal tumor of sheep in Spain: pathology and associated retrovirus. Eur. J. Vet. Pathol. 4, 11–15. De las Heras, M., Ortín, A., Cousens, C., Minguijón, E., Sharp, J.M., 2003. Enzootic nasal adenocarcnioma in sheep and goats. Curr. Top. Microbiol. Immunol. 275, 201–223. Dirks, C., Duh, F.-M., Rai, S.K., Lerman, M.I., Miller, D., 2002. Mechanism of cell entry and transformation by enzootic nasal tumor virus. J. Virol. 76, 2141–2149. Eckstrand, C.C., Castillo, D., McDonnel, S.J., Hillman, C.N., Vapniarsky, N., Shanthalingam, S., De las Heras, M., Murphy, B.G., 2013. Genetic variability and in vitro transcriptional permissibility of primary ovine beta-retrovirus promoter isolates. Am. J. Vet. Res. 74, 1421–1427. Gonzalez, L., García-Goti, M., Cousens, C., Dewar, P., Cortabarria, N., Extramiana, A.B., Ortin, A., De las Heras, M., Sharp, J.M., 2001. Jaagsiekte sheep retrovirus can be detected in the peripheral blood during pre-clinical period of sheep pulmonary adenomatosis. J. Gen. Virol. 82, 1355–1358. Monot, M., Archer, F., Gomes, M., Mornex, J.F., Leroux, C., 2015. Advances in the study of transmissible respiratory tumours in small ruminants. Vet. Microbiol. 181, 170–177. Nieberle, K., 1940. Uber endemischen crebs im siebbein von schafen. Z. schr. Krebsforch 49, 137–141. Ortín, A., Minguijón, E., Dewar, P., García, M., Ferrer, L.M., Palmarini, M., Gonzalez, L., Sharp, J.M., De las Heras, M., 1998. Lack of specific immune response against a recombinant capsid protein of jaagsiekte sheep retrovirus in sheep and goats naturally affected by enzootic nasal tumor and sheep pulmonary adenomatosis. Vet. Immunol. Immunopathol. 61, 229–237. Ortín, A., Cousens, C., Minguijón, E., Pascual, Z., Pérez de Villareal, M., Sharp, J.M., De las Heras, M., 2003. Characterization of enzootic nasal tumor virus of goats: complete sequence and tissue distribution. J. Gen. Virol. 84, 2245–2252. Ortín, A., Pérez de Villareal, M., Minguijón, E., Cousens, C., Sharp, J.M., De las Heras, M., 2004. Coexistence of enzootic nasal adenocarcinoma and jaagsiekte retrovirus infection in sheep. J. Comp. Path. 131, 253–258.
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Walsh, S.R., Stinson, K.J., Wootton, S.K., 2016a. Seroconversion of sheep experimentally infected with enzootic nasal tumor virus. BMC Res. Notes 9, 15. Walsh, S.R., Rosales Gerpe, M.C., Wootton, S.K., 2016b. Construction of a molecular clone of ovine enzootic nasal tumor virus. Virol. J. 13, 209. Yu, D.L., Linnerth-Petrik, N.M., Halbert, C.L., Walsh, S.R., Miller, A.D., Wootton, A.K., 2011. Jaagsiekte sheep retrovirus and enzootic nasal tumor virus promoters drive gene expression in all airway epithelial cells of mice but only induce tumors in the alveolar region of the lungs. J. Virol. 85, 7535–7545.
Walsh, S.R., Linnerth-Petrick, N.M., Laporte, A.N., Menzies, P.I., Foster, R.F., Wootton, S., 2010. Full-length genome sequenze analysis of enzootic nasal tumor reveals unusually high degree of genetic stability. Vir. Res. 151, 74–87. Walsh, S.R., Linnerth-Petrik, N.M., Yu, D.L., Foster, R.A., Menzies, P.I., Diaz-Mendez, A., Chalmers, H.J., Wootton, S.K., 2013. Experimental trasnsmission of enzootic nasal adenocarcinoma in sheep. Vet. Res. 44, 66. Walsh, S.R., Stinson, K.J., Menzies, P.I., Wootton, S.K., 2014. Development of an antemortem diagnostic test for enzootic nasal tumor virus and detection of neutralizing antibodies in host serum. J. Gen. Virol. 95, 1843–1854.
4
Contents lists available at ScienceDirect
Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Enzootic nasal adenocarcinoma in sheep: An update ⁎
M. De las Heras , A. Ortín, M. Borobia, T. Navarro Departamento de Patología Animal, Universidad de Zaragoza.c/ Miguel, Servet 177, 50013, Zaragoza, Spain
A R T I C LE I N FO
A B S T R A C T
Keywords: Enzootic nasal adenocarcinoma Sheep Retrovirus
Enzootic nasal adenocarcinoma of sheep (ENAS) is a contagious tumor of the ethmoid turbinate mucosa. The disease occurs naturally in all continents except Australia and New Zealand. Similar disease has been described in goats. ENAS is aetiologically associated with enzootic nasal tumor virus 1 (ENTV-1) a type Beta retrovirus and closely related to jaagsiekte sheep retrovirus (JSRV) and enzootic nasal adenocarcinoma virus of goats (ENTV-2). Clinical signs include continuous nasal discharge, respiratory distress, exophtalmus and skull deformations. The tumors have been classified histologically as low grade adenocarcinoma. ENTV-1 can be demonstrated in tumors by immunohistochemistry using several antisera developed against JSRV antigens which cross-react with ENTV1 ones. ENTV-1 shares a oncogenic mechanisms with JSRV and ENTV-2 and active replication of the virus is required because oncogenic proprieties are associated with env gene products. Information about immune reactions in relation to ENTV-1 is scarce and controversial. Some early studies indicated absence of immune reaction although more recent studies have detected the presence of neutralizing antibodies in sheep with tumors and in contact sheep. However, the sensitivity and specificity of these tests are very low to be used in field studies. Several specific PRC techniques are available to detect viral genome in tissues and tumors. ENTV-1 can be detected in nasal secretion but is rarely found out of the tumor. The utility of PCR tests in control or eradication plans is discussed.
1. Introduction Enzootic nasal adenocarcinoma of sheep (ENAS, enzootic nasal tumor) was first described in Germany (Nieberle, 1940) and subsequently in many other countries since then. Thus the disease, which has been also reported in goats, has been recorded in all the major areas where sheep and goats are farmed with the exception of Australia and New Zealand. ENAS appears to be absent in the UK (reviewed in De las Heras et al., 2003). ENAS is a contagious neoplasm of gland cells of the ethmoid turbinate mucosa and aetiologically associated with a betaretrovirus. This retrovirus is known as enzootic nasal tumor virus 1 (ENTV-1) and it is closely related to jaagsiekte sheep retrovirus (JSRV) which causes the ovine pulmonary adenocarcinoma (OPA, jaagsiekte, ovine pulmonary adenomatosis), sheep endogenous retroviruses (Cousens et al., 1999) and the enzootic nasal tumor virus of goats (ENTV-2) (Ortín et al., 2003). The disease has been reproduced experimentally in goats (De las Heras et al., 1995) and in sheep (Walsh et al., 2013). In this brief review, we will try to summarize clinical and pathological features of the disease in sheep followed by pathogenic mechanisms of the ENTV-1. The last section will be dedicated to the
⁎
immune response against ENTV-1 in sheep and a discussion about diagnostic tests in vivo. 2. Clinical features and pathology Epidemiological data indicate that ENAS prevalence in affected flock is variable ranging from 0.1 to 15%. Preferentially young adults (3–5 years) (range 1–9 years old) are affected and several cases are always observed in the same flock (De las Heras et al., 1998). No genetic, sex or breed predisposition has been detected. The signs of the disease start with a small but continuous amount of sero-mucous fluid coming from the nostrils (unilaterally or bilaterally) (Fig. 1A). At this stage, the disease can be confused with other nasal pathologies like oestrosis, nasal polyps or bacterial infections. As the disease progresses, secretion is more abundant and causes the depilation on the area from the nostrils to the upper lips (Fig. 1B). This is a very particular clinical sing for this disease which can be used to differentiate from other nasal pathologies like oestrosis or salmonella infection. In advanced cases the disease shows characteristic clinical signs. Thus, snoring caused by stenosis of nasal passages, coughing, sneezing, head shaking together with softening and deformation of the skull bones (mainly frontal and
Corresponding author. E-mail address: [email protected] (M. De las Heras).
https://doi.org/10.1016/j.smallrumres.2019.04.018 Received 28 January 2019; Received in revised form 28 March 2019; Accepted 29 April 2019 0921-4488/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: M. De las Heras, et al., Small Ruminant Research, https://doi.org/10.1016/j.smallrumres.2019.04.018
Small Ruminant Research xxx (xxxx) xxx–xxx
M. De las Heras, et al.
Fig. 1. Clinical features of enzootic nasal adenocarcinoma of sheep. A: Young sheep showing early clinical signs. Unilateral sero- mucous clear fluid is coming continuously off the right nostril. B: Adult sheep showing advanced clinical signs. Nasal continuous sero-mucous discharge is characteristic together with softening of the region nasalis and fistula. Sero-mucous fluid also emerges from the fistula causing depilation of the close skin area below.
maxillary), exophthalmos and also skin fistula can be present (Fig. 1B). Body condition is gradually lost and animals eventually die due to bacteria complication of the tumor which ends with pneumonia or septicemia (reviewed in De las Heras et al., 2003). At necropsy, tumors are found in the nasal cavity, either unilaterally or bilaterally, arising from the ethmoidal mucosa and effacing the normal architecture of the ethmoidal conchae. Tumors are soft, grey or reddish-white in color with a fine granular surface and covered with mucus. They are found together with nasal polyps in some cases. The tumor grows compressing the close nasal conchae, cranial bones or invades paranasal or frontal sinuses (Fig. 2A). Necrosis or purulent inflammation of the tumors is also seen. Nasal tumors can be differentiate from other nasal pathologies like those associated with Salmonella diarizonae, because they are located in the ethmoidal conchae whereas salmonella associated lesions are found in the ventral turbinate and occasionally in the dorsal one. The microscopic architecture of the tumors is similar in all cases examined by us and joins the characteristics of a low grade adenocarcinoma of nasal glands (De las Heras et al., 2003). Light microscopy reveals epithelial secretory cells proliferating into an acinar, tubular, papillary or even solid patterns. Inner parts of the tumors are more tubular or acinar whereas external zones show a clear papillary pattern (Fig. 2B). Apart from some areas of cellular atypia or local invasion, no signs of malignancy and low mitotic index are seen. In addition, metastasis in regional lymph nodes or in other organs has never been recorded. The stroma is very scanty but infiltrated by, either scattered or grouped, lymphocyte/plasmocyte cells. Electron microscopy and histochemical studies revealed that neoplastic cells correspond to serous, mucous or mixed glands cells and seem to originate either in olfactory or respiratory mucosal glands (Reviewed in De las Heras et al., 2003). Several polyclonal and monoclonal specific antibodies to JSRV viral proteins, which cross-reacts with ENTV proteins, have been generated for virus demonstration on tissue sections by immunohistochemistry but they are not commercially available. They have been used as a research tool to differentiate ENA from other respiratory tumors and in other investigations.
naturally infected tumor bearing sheep (Eckstrand et al., 2013). Besides, North American and European genomes shared more than 96% sequence identity with high degree of aminoacid conservation among isolates (Walsh et al., 2010). Thus, it seems that ENTV-1 shows little genomic variations and it shares common pathogenic mechanisms with JSRV and ENTV-2. These three viruses interact with mammalian cells through HYAL-2 (hyaluronglucosaminase 2) receptor for virus attachment and entry and can infect different cell types. HYAL-2 is a ubiquitous membrane surface protein that belongs to hyaluronidases (Alberti et al., 2002; Dirks et al., 2002). However, ENTV-1 active replication is mostly restricted to nasal cavity epithelia where viral LTRs (long terminal repeat) are active (Yu et al., 2011). This active replication of the virus is important because their oncogenic properties are mainly on envelope proteins. Several in vitro experiments have indicated that JSRV and ENTV-1 share common mechanisms of cell transformation. JSRV and ENTVs env gene products clearly activates a number of proteins involved in signaling cascades controlling cell growth and fate such as Akt (Protein Kinase B) or MAPK (Mitogen activated kinase) signaling pathways, inducing permanent activation of these tyrosine kinase cell signaling routes disturbing de control of cell growth and survival leading to a growth advantage of deregulated cells. In spite of, other mechanisms leading to transformation, such as targeted integration of the virus genome cannot be totally ruled out (Alberti et al., 2002; Monot et al., 2015). A proviral clone of ENTV-1 has been constructed from which mature virions can be generated. This virus produced from this molecular clone will be very useful for future experiments to understand the pathogenesis of this disease (Walsh et al., 2016b).
4. Immune response and diagnostic tests The evaluation of the serological immune response in sheep with ENAS has been limited and controversial. Studies using Western blotting found reactivity to recombinant JSRV capsid protein (JSRV-CA), as a Glutathion S-transferase (GST) fusion protein, in sheep sera from diseased animals. However, this reaction could be abolished completely by absorption with the GST fusion partner but not with JSRV-CA. This suggested that the activity recognized against this recombinant JSRVCA was not specific (Ortín et al., 1998). In addition, Western blotting techniques also failed to detect any antibody against viral antigens from concentrated ENAS fluid (Ortín et al., 1998). A recent study has identified ENTV-1 reactivity by ELISA and Western blot in sheep serum from
3. ENTV-1 pathogenesis The comparison of the genomic sequence of the ENTV-1 with ENTV2 and JSRV concluded that these retroviruses are very similar (De las Heras et al., 2003). Like JSRV, ENTV-1 shows minimal sequence variation in the LTR(Long terminal repeat) non-coding region derived from
Fig. 2. Gross pathology and histopathology of enzootic nasal adenocarcinoma of sheep. A: Skull section showing tumor originated in the ethmoidal mucosa. Etmoidal nasal turbinates have been replaced by the tumor growing pressing dorsal and ventral conchae and causing obstruction of the nasal passages. B: Histopathology of the tumor. Nasal glands proliferate in an acinar and tubular pattern (asterisks). Nasal gland epithelia proliferates in tubulo-papillary pattern (star). Lympho-plasmocytic cells infiltrate the interstitium.
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ENAS affected and in contact sheep belonging to a flock with high incidence of ENAS. Serum samples were tested for reactivity against recombinant ENTV-1 capsid and surface subunit (Env) proteins produced using polyhistidine tag (His-tag) bacterial expression system and removing His-tag prior to use it as antigen. Results showed that reactive antibodies against both capsid and Env proteins could be detected in the serum of sheep either with or without tumor evidence using ELISA and Western blot analysis (Walsh et al., 2014). Both techniques detected immune reactive antibodies against Env protein from the flock with a history of ENAS but not in the naive sheep serum samples. Furthermore, some samples from these sheep were also positive to neutralization test of ENTV-1 Env-pseudotyped virions. These results suggest that these interactions were indeed specific and that sheep are able to develop antibodies against the ENTV-1. In spite of this, ELISA and virus neutralization test showed low specificity and sensitivity and they are not reliably to be used to diagnose ENTV-1 infection (Walsh et al., 2014). Similar results have been obtained in sheep experimentally infected with ENTV-1 (Walsh et al., 2016a). In any case, this is a very relevant finding because it would mean that sheep may be not immune tolerant to exogenous ENTV-1 infection as it was previously thought. Studies on cell-mediated immunity to ENTV-1 have yet to be reported (De las Heras et al., 2003). As we have indicated in previous sections, ENTV-1 has been sequenced completely (Cousens et al., 1999). Using this information and from other studies, several PCR-based diagnostic techniques have been generated which discriminated sequences of ENTV-1 from JSRV, ENTV2 and sheep endogenous retroviruses (Ortín et al., 2003; Walsh et al., 2014). The distribution of the ENTV-1 in several tissues has been investigated using a specific PCR test that amplifies part of the U3 region in the LTR of the ENTV-1 (heminested-PCR) to detect provirus in sheep genomic DNA (Ortín et al., 2003). This PCR can detect a low copy number of virus in a DNA sample (less than 20 copies in 500 ng of sheep DNA) and in order to increase the chances for the detection of the virus the test was triplicated on each sample (Ortín et al., 2003). These studies conclude that ENTV-1 is mostly confined in the tumor and it is rarely found in other tissues such as the lung. However, JSRV and ENTV-2 are found in other tissues and in the peripheral blood monocytes (PBMCs) and seem to establish a disseminated infection (Ortín et al., 2003, 2004). Using specific PCR techniques detection of JSRV or ENTV-2 in PMBCs samples is possible and allows identifying animals at preclinical stages and with no evidence of tumors (Gonzalez et al., 2001, De las Heras et al., unpublished observations). However, as ENTV-1 is never found in PMCs, blood PCR would not be suitable for detecting ENTV-1 infected animals. Specific RT-PCR (U5 and gag regions) from North American ENTV-1 isolates performed on nasal exudates have been proposed as a part of an ante-mortem diagnostic test for ENAS surveillance and eradication programs. In this study RT-PCR on nasal swabs is correlated with pathology (Walsh et al., 2014). However this technique has some weaknesses for the use of it in surveillance or eradication programs of the disease, particularly when this system tries to work with large flocks. Nasal swabbing/tampons may be useful for small size flocks but it becomes very difficult to manage in large ones. From the other hand, nasal tumors are originated in ethmoid turbinate and sampling using nasal swabs do not reach this area. Thus, despite the high specificity and sensitivity of the PCR technique the sampling technique may not obtain the proper material when the tumors are very small. In addition, RT-PCR on nasal swabs will detect animals with tumors but not already infected animals with no lesions. If the flocks dynamics of the ENTV-1 infection is similar to other retroviral infections like jaagsiekte sheep retrovirus infection (Gonzalez et al., 2001), nasal swabbing will not detect already infected animals but with no tumors. Another important factor which may have influence in the decision of an eradication plan is that the prevalence of the disease in affected flocks. In our geographic area, the prevalence is about 0.1−0.3% (De las Heras et al., 1998). In that case, removing animals with ENAS clinical signs and may be
enough to control the disease. In our opinion, RT-PCR tests based on nasal sampling may be useful under experimental conditions or for small flocks with low numbers of animals, but it has many weaknesses when is considered as a part of bigger plan of control or eradication program in normal farming situations of the most sheep rearing countries. In conclusion, either early clinical diagnosis or detection of infected animals before the tumors develop is very difficult for this disease at present. More research about natural infection and pathogenesis of ENAS is very necessary if we want to improve current diagnostic test and methods. 5. Concluding remarks We may conclude that no diagnostic test is currently available to detect ENTV- 1 infected sheep, but we can detect, using specific PCR techniques, sheep with small tumors and some of them clinically blind. However, sampling and PCR techniques are costly, difficult to implement in most of the sheep flocks and do not ensure the animal is free of the infection. Conflict of interest statement The authors have nothing to disclose. Acknowledgements Authors acknowledge University of Zaragoza, Gobierno de Aragón, Gobierno de España and European Commission providing funds for research on this sheep disease. References Alberti, A., Murgia, C., Shan-lu, L., Mura, M., Cousens, C., Sharp, J.M., Miller, D., Palmarini, M., 2002. Envelope-induced cell transformation by ovine betaretroviruses. J. Virol. 76, 5387–5394. Cousens, C., Minguijón, E., Dalziel, R.G., Ortín, A., García, M., Park, J., González, L., Sharp, J.M., De las Heras, M., 1999. Complete sequenze of enzootic nasal tumor virus, a retrovirus associated with transmissible intranasal tumors of sheep. J. Virol. 73, 3986–3993. De las Heras, M., García de Jalón, J.A., Minguijón, E., Gray, E., Dewar, P., Sharp, J.M., 1995. Experimental transmission of enzootic intranasal tumors of goats. Vet. Pathol. 32, 19–23. De las Heras, M., Minguijón, E., Ferrer, L.M., Ortín, A., Dewar, P., Cebrián, L.M., Pascual, Z., García, L., García de Jalón, J.A., Sharp, J.M., 1998. Naturally occurring enzootic nasal tumor of sheep in Spain: pathology and associated retrovirus. Eur. J. Vet. Pathol. 4, 11–15. De las Heras, M., Ortín, A., Cousens, C., Minguijón, E., Sharp, J.M., 2003. Enzootic nasal adenocarcnioma in sheep and goats. Curr. Top. Microbiol. Immunol. 275, 201–223. Dirks, C., Duh, F.-M., Rai, S.K., Lerman, M.I., Miller, D., 2002. Mechanism of cell entry and transformation by enzootic nasal tumor virus. J. Virol. 76, 2141–2149. Eckstrand, C.C., Castillo, D., McDonnel, S.J., Hillman, C.N., Vapniarsky, N., Shanthalingam, S., De las Heras, M., Murphy, B.G., 2013. Genetic variability and in vitro transcriptional permissibility of primary ovine beta-retrovirus promoter isolates. Am. J. Vet. Res. 74, 1421–1427. Gonzalez, L., García-Goti, M., Cousens, C., Dewar, P., Cortabarria, N., Extramiana, A.B., Ortin, A., De las Heras, M., Sharp, J.M., 2001. Jaagsiekte sheep retrovirus can be detected in the peripheral blood during pre-clinical period of sheep pulmonary adenomatosis. J. Gen. Virol. 82, 1355–1358. Monot, M., Archer, F., Gomes, M., Mornex, J.F., Leroux, C., 2015. Advances in the study of transmissible respiratory tumours in small ruminants. Vet. Microbiol. 181, 170–177. Nieberle, K., 1940. Uber endemischen crebs im siebbein von schafen. Z. schr. Krebsforch 49, 137–141. Ortín, A., Minguijón, E., Dewar, P., García, M., Ferrer, L.M., Palmarini, M., Gonzalez, L., Sharp, J.M., De las Heras, M., 1998. Lack of specific immune response against a recombinant capsid protein of jaagsiekte sheep retrovirus in sheep and goats naturally affected by enzootic nasal tumor and sheep pulmonary adenomatosis. Vet. Immunol. Immunopathol. 61, 229–237. Ortín, A., Cousens, C., Minguijón, E., Pascual, Z., Pérez de Villareal, M., Sharp, J.M., De las Heras, M., 2003. Characterization of enzootic nasal tumor virus of goats: complete sequence and tissue distribution. J. Gen. Virol. 84, 2245–2252. Ortín, A., Pérez de Villareal, M., Minguijón, E., Cousens, C., Sharp, J.M., De las Heras, M., 2004. Coexistence of enzootic nasal adenocarcinoma and jaagsiekte retrovirus infection in sheep. J. Comp. Path. 131, 253–258.
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Walsh, S.R., Stinson, K.J., Wootton, S.K., 2016a. Seroconversion of sheep experimentally infected with enzootic nasal tumor virus. BMC Res. Notes 9, 15. Walsh, S.R., Rosales Gerpe, M.C., Wootton, S.K., 2016b. Construction of a molecular clone of ovine enzootic nasal tumor virus. Virol. J. 13, 209. Yu, D.L., Linnerth-Petrik, N.M., Halbert, C.L., Walsh, S.R., Miller, A.D., Wootton, A.K., 2011. Jaagsiekte sheep retrovirus and enzootic nasal tumor virus promoters drive gene expression in all airway epithelial cells of mice but only induce tumors in the alveolar region of the lungs. J. Virol. 85, 7535–7545.
Walsh, S.R., Linnerth-Petrick, N.M., Laporte, A.N., Menzies, P.I., Foster, R.F., Wootton, S., 2010. Full-length genome sequenze analysis of enzootic nasal tumor reveals unusually high degree of genetic stability. Vir. Res. 151, 74–87. Walsh, S.R., Linnerth-Petrik, N.M., Yu, D.L., Foster, R.A., Menzies, P.I., Diaz-Mendez, A., Chalmers, H.J., Wootton, S.K., 2013. Experimental trasnsmission of enzootic nasal adenocarcinoma in sheep. Vet. Res. 44, 66. Walsh, S.R., Stinson, K.J., Menzies, P.I., Wootton, S.K., 2014. Development of an antemortem diagnostic test for enzootic nasal tumor virus and detection of neutralizing antibodies in host serum. J. Gen. Virol. 95, 1843–1854.
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