PERSISTENT LYMPHOCYTOSIS IN CATTLE : ITS CAUSE, NATURE AND RELATION TO LYMPHOSARCOMA

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1 PERSISTENT LYMPHOCYTOSIS IN CATTLE : ITS CAUSE, NATURE AND RELATION TO LYMPHOSARCOMA J. F. Ferrer, R.R. Marshak, D. A. Abt, S. J. Kenyon To cite this version: J. F. Ferrer, R.R. Marshak, D. A. Abt, S. J. Kenyon. PERSISTENT LYMPHOCYTOSIS IN CAT- TLE : ITS CAUSE, NATURE AND RELATION TO LYMPHOSARCOMA. Annales de Recherches Vétérinaires, INRA Editions, 1978, 9 (4), pp <hal > HAL Id: hal Submitted on 1 Jan 1978 HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

2 PERSISTENT LYMPHOCYTOSIS IN CATTLE : ITS CAUSE, NATURE AND RELATION TO LYMPHOSARCOMA J. F. FERRER R. R. MARSHAK, D. A. ABT S. J. KENYON Section of Viral Oncology, Comparative Leukemia Studies Unit, School of Veterinary Medicine, University of Pennsylvania, New Bolton Center, Kennett Square, Pennsylvania Résumé LYMPHOCYTOSE PERSISTANTE CHEZ LES BOVINS : CAUSES, NATURE ET RELATION AVEC LE LYMPHOSARCOME. &horbar; Les études réalisées sur des populations bovines bien caractérisées suggèrent très fortement que le virus de la leucose bovine (BLV) est l agent causal de la forme adulte (enzootique) du lymphosarcome bovin et de la lymphocytose persistante (PL), et que les facteurs génétiques de l hôte jouent un rôle important dans l apparition de ces deux maladies. D autre part, les données existantes indiquent que les facteurs génétiques qui contrôlent le développement de la PL sont fréquemment indépendants de ceux qui contrôlent le développement du lymphosarcome. Il n est pas démontré que les bovins cliniquement normaux qui ont de la PL hébergent des cellules malignes ou ont quelque autre anomalie clinique. Chez ces animaux, la lymphocytose résulte de la multiplication de deux sous-populations distinctes de lymphocytes B non néoplasiques, dont l une ne contient pas de BLV. Des études de longue durée ont montré que la grande majorité des bovins à PL ne développent pas de lymphosarcome, même si on les garde jusqu à un âge avancé. Tout ceci montre que la PL n est ni une maladie, ni un stade préclinique du lymphosarcome. La PL pourrait plutôt être considérée comme une réponse bénigne à l infection par le BLV, et qui, bien que fréquemment associée au lymphosarcome, en est indépendante. Introduction. It is well known that herds with a high incidence of the adult (enzootic) form of bovine lymphosarcoma often contain clinically normal cattle with persistent lymphocytosis (PL). In many cases the development of lymphosarcoma is preceded by a period, usually several years, in which the animal has PL. Originally these observations were interpreted to mean that PL and lymphosarcoma are caused by the same infectious agent and that PL is a subclinical form of the disease (Bendixen, 1965). Thus, the two conditions are often referred to by the term «bovine leukosis». PL must be distinguished from the transient lymphocytosis associated with conditions that are unrelated to lymphosarcoma. Thus, the hematological keys to identify cattle with PL are based on the demonstration of an elevated lymphocyte count in at least 2 samples collected at intervals of no less than 3 months (Marshak, 1968 ; Abt et al., 1970). PL must also be distinguished from the lymphocytosis

3 which occurs in approximately one-third of the cases of lymphosarcoma as a clinical manifestation of the disease (Marshak et al., 1962). While this latter lymphocytosis is due to passage into the peripheral blood of aneuploid or neoplastic lymphocytes, cytogenetic studies have failed to reveal such abnormal lymphocytes in the peripheral blood of cattle with PL (Hare, W.C.D., personal communication). It should also be emphasized that, aside from lymphocytosis, cattle with PL have no clinically detectable abnormalities. Indeed, animals with PL reproduce normally, do not lose weight and show no curtailment in milk production. The discovery of the bovine leukemia virus (BLV) and the information on its distribution and natural mode of transmission has made it possible to more critically evaluate the nature of PL. In this paper we present data supporting the concept that PL and bovine lymphosarcoma are genetically determined and frequently independent response to BLV infection. Relationship between BLV infection, persistent lymphocytosis and lymphosarcoma. BLV can be detected in lymphocytes of infected cattle by either electron microscopy (Miller et al., 1969 ; Ferrer et al., 1971 ; Stock and Ferrer, 1972) immunologic techniques (Ferrer et al., 1972 ; McDonald and Ferrer, 1976) or an infectivity assay (Ferrer and Diglio, 1976 ; Ferrer et al., 1976a ; Diglio et al., 1978) which is based on the ability of the virus to induce syncytia (Diglio and Ferrer, 1976). Several serological techniques are available for the detection of BLV antibodies, including immunofluorescence (Ferrer et al., 1975a), immunodiffusion (FerreretaL, 1972 ; Ferrer et a/., 1975a ; Miller and Van der Maaten, 1977), virus neutralization (Ferrer and Diglio, 1976 ; Ferrer et al., 1976a), complement fixation (Miller and Van der Maaten, 1974), and radioimmunoassay (McDonald and Ferrer, 1976 ; Ferrer et al., 1976a ; Devare et al., 1976). The application of some of the techniques for the diagnosis of BLV infection has been critically evaluated by correlating the results with the presence and absence of the virus (Ferrer et a/., 1977b ; Ferrer et a/., 1977a). It has been demonstrated that BLV is rapidly spread by contact (Piper et al., 1975 ; Ferrer et a/., 1976b), particularly during the summer months (Bech-Nielsen et al., 1978). There is strong evidence that biting insects play a major role in the transmission of the virus (Bech-Nielsen et al., Prenatal transmission accounts for less than 20 % of the cattle that are infected as adults (Ferrer et al., 1976b ; Piper et al., During the past 19 years a number of multiple-case, contact and leukosis-free herds have been studied at the University of Pennsylvania by means of periodic blood counts, pedigree analyses, and clinical surveillance (Abt et al., 1970 ; Ferrer et al., 1974 ; Abt et a/., 1976). These cattle populations have provided an extraordinary opportunity for large scale, controlled studies on the relationships between BLV infection, PL and lymphosarcoma. The results of these studies ( Ferrer et al, 1974 ; Abt et al, 1976 ; Ferrer et a/., 1975b) have shown that BLV is present in

4 90 % or more of adult cattle with lymphosarcoma. As shown in Table 1, the incidence of BLV infection among clinically normal cattle in multiple-case and contact herds is high and moderate, respectively. In contrast, leukosisfree herds are either completely free of BLV infection or contain only a few infected animals. This close correlation between BLV infection and lymphosarcoma or lymphosarcoma risk (Ferrer et al, 1974) strongly supports the view that BLV is the etiologic agent of bovine lymphosarcoma. The results summarized in Table 2 show that virtually all clinically normal cattle with PL are infected with BLV. Furthermore, PL can apparently be induced in sheep by the inoculation of BLV-containing materials (Van der Maaten and Miller, 1976 ; Ressang et al., 1976). These observations support the view that BLV is also the causative agent of PL. The data in Table 2 demonstrate that 33 % of the cattle without PL in multiple-case and contact herds studied are infected with BLV. In fact, of the 477 BLV-infected cattle in Table 2, only 139 (29 %) had PL. In other words, more than 60 % of the BLV-infected cattle are asymptomatic virus carriers. Considering the incidence of lymphosarcoma in wellcharacterized study herds (Abt et al, 1970) on which data on the prevalence of BLV infection is available (Ferrer et al., 1974 ; Abt et al, 1976) we estimate that the incidence of lymphosarcoma among BLV-infected cattle is no greater than 5 %. Figure 1 shows a schematic representation of the available data on the relationships between BLV infection, PL and lymphosarcoma. From these data it is clear that in addition to BLV, other factors play a role in the development of PL and lymphosarcoma in cattle. Evidence for the participation of genetic factors in the development of persistent lymphocytosis and lymphosarcoma. Studies on a large number of multiple-case herds in the United States have clearly established that bovine lymphosarcoma tends to aggregate along familial lines (Croshaw et al., 1963 ; Marshak and Abt, 1968). Familial clusters of the disease have also been reported by European workers (Bendixen, 1965 ; Henricson and Olson, ). Detailed studies on the familial aggregation of bovine lymphosarcoma were conducted in a multiple-case herd (herd BF) which is maintained at the University of Pennsylvania. During the past 20 years, the BF herd has experienced 69 histologically confirmed cases of lymphosarcoma in a population at risk of approximately 350 animals. Owing to the availability of detailed pedigree data, it has been possible to show that virtually all the cases occurred only in certain well-defined families (Marshak and Abt, 1968 ; Abt D.A. Marshak R.R. and Ferrer J.F., Manuscript in preparation). BLV can be detected in virtually all adult cattle in the BF herd (Piper et al., 1978 ; Ferrer et al., 1974 ; Abt et al., 1976). However, only 18 % of these animals were infected before birth (Ferrer et a/., 1976b ; Piper et al., 1978). Since the incidence of lymphosarcoma in this herd is similar to that of prenatal (vertical) infection with BLV, the possibility was considered that the familial aggregation of the disease is the consequence of the vertical transmission of the virus. However, a retrospective study showed that many cattle with lymphosarcoma in the BF herd became infected with BLV after birth

5 All calves were colostrum-deprived and in contact with infected dams during a 10-week nursing period. After weaning, calves from all groups were mixed and raised together. (Kenyon S. and Ferrer J.F., manuscrit in preparation). Thus, it seems clear that the development of lymphosarcoma in response to BLV infection is determined by the host s genetic constitution rather than by the age at which it becomes infected with BLV. Studies on the BF and other herds have shown that PL also occurs in familial aggregations (Bendixen, 1965 ; Abt et al., 1970). The influence of host genetic factors on the development of PL was examined further in a group of colostrum-deprived calves from herds and families that had been carefully characterized in terms of risk of PL and lymphosarcoma. Only one of the calves was infected at birth and all were in contact with infected cattle during the nursing period of 10 weeks. After weaning, calves from the different groups were mixed and raised together. The results of this experiment are summarized in Table 3. At the conclusion of the experiment (55-73 months), the incidence of BLV infection among the 3 groups of calves ranged from 89 % to 92 %. This uniform distribution of BLV was in striking contrast to the distribution pattern for PL. Out of 34 calves with PL, 26 (76 %) originated in PL-positive families and only 8 (24 %) from PL-negative families. No cases of PL occurred in the group of calves from leukosis-free herds, despite the fact that 90 % were infected with BLV. From these studies it is clear that even in cattle populations with a high incidence of BLV infection, the occurrence of lymphosarcoma and PL is restricted to certain families. This strongly supports the concept that the ability of an animal to develop either PL or lymphosarcoma in response to BLV infection is genetically determined. However, the observation that PL and lymphosarcoma do not always aggregate within the same families (Abt et al., 1970) is evidence that the genetic factors determining susceptibility to these conditions are independent. Characteristics of the lymphocytes involved in persistent lymphocytosis. It has been established that in cattle with PL the large majority of the peripheral blood lymphocytes bear surface markers typical of B lymphocytes (Muscoplat et al., 1974 ; Piper and Ferrer, 1974 ; V1/eiland and Straub, 1975 ; Kenyon and Piper, 1977a). Using density gradient centrifugation techniques, the B lymphocytes of cattle with PL have been separated into a subpopulation that is infected with BLV, and a non-infected subpopulation that spontaneously incorporates tritiated thymidine after 3 days in in vitro culture (Kenyon and Piper, 1977b). The latter subpopulation, unlike the malignant lymphocytes that are found in the blood of cattle with lymphosarcoma, are sensitive to glucocorticoids, both in vitro and in vivo (Bloom, 1978) and can be specifically inhibited in vitro by sera containing BLV antibodies (Kenyon S., Manuscript in preparation). In addition, peripheral blood found in clinically normal cattle with PL do not show the cytogenetic abnormalities found in neoplastic bovine lymphocytes (Hare, personal communication ; Bloom, 1978). The avai-

6 lable data suggest that the spontaneously incorporating lymphocytes of cattle with PL are immunologically comitted cells. Results of published (Kenyon and Piper, 1977b) and current studies in our laboratory indicate that, in cattle with PL, both B cell subpopulations are expanded. Discussion. The data discussed in this report indicate that PL and the adult form of bovine lymphosarcoma are induced by BLV, thus explaining the long-recognized association between the occurrence of these conditions. On the other hand, it is clear that genetic constitution of the host plays an important role in the development of both PL and lymphosarcoma. That the genetic factors controlling the development of PL and lymphosarcoma are independent is supported by the observation that 35 % of cattle with lymphosarcoma do not have histories of PL (Marshak and Abt, Even more conclusive is the fact that within the same herd PL and lymphosarcoma may occur in different families (Abt et al., 1970). Long term studies (Abt et al., 1970 ; Ferrer et al., 1974 ; Abt et al., 1976) have shown that the large majority of cattle with PL do not develop lymphosarcoma even when kept well beyond the Cean age at which the disease appears. There is no evidence that cattle with PL have clinical abnormalities other than lymphocytosis or that they harbor neoplastic cells (this is not to say that such cells may not appear in the blood of those few animals with PL that develop lymphosarcomal. The studies on the cells involved in PL strongly support the view that PL results from a benign proliferation of B lymphocytes. Thus, the inescapable conclusion of these findings is that, contrary to a commonly held opinion, PL is neither a disease nor a subclinical form of bovine lymphosarcoma. Rather, PL can be regarded as an independent benign response to BLV infection that occurs frequently in association with lymphosarcoma. The use of the term «bovine leukosis» to encompass both lymphosarcoma and PL could be justified by the epidemiological and causal relationships between the two,%conditions. While the term «leukosis» may be useful in a general sense, an accurate interpretation of the experimental and epidemiological data requires a nomenclature that clearly distinguishes between PL and lymphosarcoma. imprecise terminology, many Because of reports on the disease, particularly those on transmission, are difficult to evaluate or have led to overinterpretation. Some authors, for example, claim to have transmitted lymphosarcoma to cattle (Olson, 1974) based on data in which the recipient animals developed only lymphocytosis (Miller et al., The results discussed here and those reported previously (Ferrer et al., 1975a ; Ferrer et a/., 1977a ; Ferrer et al., 1974 ; Abt et al., 1976) show that, while BLV can be detected in virtually all cattle with PL, more than 60 % of BLV-infected cattle do not have PL. Therefore, il is obvious that hematological keys are inadequate as indicators of BLV infection and have little value in control and eradication programs. Since BLV is transmitted mainly by contact, each infected animal is a potential focus for spread of the virus. Consequently, the success of an eradication or control program will depend, in large measure, upon the application of highly sensitive diagnostic tests for BLV infection. In summary, a critical evaluation of the available data leads to the conclusion that PL is neither a disease nor a subclinical form of bovine lymphosarcoma. These data strongly support the concept that PL and lymphosarcoma are separate responses to BLV infection, each under the control of independent, although frequently coincidental, genetic factors. Acknowledgement. This work was supported by U.S. Public Health Service Grant 2 P01-CA14183, Pennsylvania Department of Agriculture Grant ME 4, U.S.D.A. Cooperative Agreement , 767, and a Grant from the Wetterberger Foundation. Summary Studies in well-characterized cattle populations strongly support the view that the bovine leukemia virus (BLV) is the causative agent of the adult (enzootic) form of bovine lymphosarcoma and persistent lymphocytosis (PL), and that host genetic factors play an important role in the deve-

7 lopment of these two conditions. On the other hand, the available information indicates that the genetic factors controlling the development of PL are frequently independent of those controlling the development of lymphosarcoma. There is no evidence that clinically normal cattle with PL harbor malignant cells or have any other clinical abnormality. In these animals lymphocytosis results from the expansion of two distinct subpopulations of non-neoplastic B lymphocytes, one of which is free of BLV. Long-term studies have shown that the large majority of cattle with PL do not develop lymphosarcoma even when kept to advanced age. These data indicate that PL is not a disease nor a preclinical stage of lymphosarcoma. Rather PL should be considered as a benign response to BLV infection which, although frequently associated with lymphosarcoma, is independant of it. References ABT D.A., MARSHAK R.R., FERRER J.F., PIPER C.E., BHATT D.M., Studies on the development of persistent lymphocytosis and infection with the bovine C-type leukemia virus (BLV) in cattle. Vet. Microbiol., 1, ABT D.A., MARSHAK R.R., KULP H.W., POLLOCK R.J., Studies on the relationship between lymphocytosis and bovine leukosis. Proceedings of the Fourth International Symposium on Comparative Leukemia Research. Bibliotheca Haematol., 36, BECH-NIELSEN S., PIPER C.E., FERRER J.F., Natural mode of transmission of the bovine leukemia virus ; role of blood-sucking insects. Am. J. Vet. Res., 39, BENDIXEN H.J., Studies on leukosis enzootica bovis with special regard to diagnosis, epidemiology and eradication. Ph. D. Thesis, Copenhagen, (Virology Res. Resources Branch, Natl. Cancer Inst., Washington, D.C. 1965). BLOOM J.C., Glucocorticoid effect on peripheral blood lymphocytes in bovine leukemia virus infected cattle. Ph.D. Thesis, University of Pennsylvania. CROSHAW J.E. Jr, ABT D.A., MARSHAK R.R., HARE W.C.D., SWITZER J., IPSEN J., DUTCHER R.M., Pedigree studies in bovine lymphosarcoma. Ann. N. Y. Acad. Sci., 108, DEVARE S.G., STEPHENSON J.R., SARMA P.S., AARONSON S.A., Bovine lymphosarcoma : development of a radioimmunologic technique for detection of the etiologic agent. Science, 194, DIGLIO C.A., FERRER J.F., Induction of syncytia by the bovine C-type leukemia virus. Cancer Res., 36, DIGLIO C.A., PIPER C., FERRER J.F., An improved syncytia infectivity assay for the bovine leukemia virus. In Vitro, 14, FERRER J.F., ABT D.A., BHATT D.M., MARSHAK R.R., Studies on the relationship between infection with bovine C-type virus, leukemia, and persistent lymphocytosis in cattle. Cancer Res., 34, FERRER J.F., AVILA L., STOCK N.D., Serological detection of type C viruses found in bovine cultures. Cancer Res., 32, FERRER J.F., BALIGA V., DIGLIO C., GRAVES D., KENYON S.J., MC DONALD H., PIPER C., WUU K., 1976a. Recent studies on the characterization of the bovine leukemia virus (BLV) ; development of new methods for the diagnosis of BLV infection. Vet. Microbiol., 1, FERRER J.F., BHATT D.M., ABT D.A., MARSHAK R.R., BALIGA V.L., 1975a. Serological diagnosis of infection with the putative bovine leukemia virus. Cornell Vet., 65, FERRER J.F., 3HATT D.M., MARSHAK R.R., ABT D.A., 1975b. Further studies on the antigenic properties and distribution of the putative bovine leukemia virus. Proceedings of the Sixth International Symposium on Comparative Leukemia Research, Nagoya/Ise-Shima, Japan, Biblioiheca Hematol., 40, FERRER J.F., DIGLIO C.A., Development of an in vitro infectivity assay for the C-type bovine leukemia virus. Cancer Res., 36, FERRER J.F., PIPER C.E., ABT D.A., MARSHAK R.R., 1977a. Diagnosis of bovine leukemia virus infection ; evaluation of serological and hematological tests by means of a direct infectivity assay. Am. J. of Vet. Res., 38, FERRER J.F., PIPER C.E., ABT D.A., MARSHAK R.R., BHATT D.M., 1976b. Natural mode of transmission of the bovine C-type virus (BLVI. Bibliotheca Haematol., 43,

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