The diagnosis of enzootic bovine leukosis

Comp. Immun. Microbiol. #l/bet. Dis. Vol. 8. No. 3/4, pp. 305 309, 1985 Printed in Great Britain

0147-9571/85 $3.00 + 0.00 Pergamon Press Ltd

THE DIAGNOSIS OF ENZOOTIC BOVINE LEUKOSIS M. MAMMERICKX~, D. PORTETELLE2 a n d A. BURNY2 ~National Institute of Veterinary Research, 99, Groeselenberg, B-1180 Brussels, Belgium 2Department of Molecular Biology, University of Brussels, B-1640 Rhode St-Gen~se, Belgium Abstract--This paper reviews the clinical and virological diagnostic procedures for enzootic bovine leukosis (EBL). The clinical diagnosis must be always confirmed by a specific laboratory test for Bovine Leukaemia Virus (BLV). Many virological tests were proposed. The sensitivity of all the diagnostic methods is sufficientto do an early detection of a BLV infection on an individual base. Advantages of the highly sensitive methods like RIA and ELISA appear when the samples to be tested have naturally very low antibody titers (individual milk, bulk milk, pooled sera).

Key words: enzootic bovine leukosis (EBL), Bovine Leukaemia Virus (BLV), diagnosis LE D I A G N O S T I C D E LA L E U C O S E B O V I N E E N Z O O T I Q U E R6sum6--Cet article passe en revue les m6thodes du diagnostic clinique et virologique de la leucose bovine enzootique (EBL). Le diagnostic clinique doit toujours ~tre confirm6 par un test de laboratoire sp6cifique du Virus de la Leucose Bovine (BLV). Beaucoup de tests virologiques ont 6t6 propos6s. La sensibilit6 de tous ces tests est suffisante pour faire une d+tection pr6coce d'une infection par le virus BLV sur une base individuelle. Les avantages des m6thodes hautement sensibles comme le RIA et I'ELISA, apparaissent lorsqu'on teste des 6chantillons qui ont des taux d'anticorps naturellement tr6s bas (laits individuels, laits de m6lange, m61anges de s6rums).

Mots-clefw leucose bovine enzootique (EBL), Virus de la Leucose Bovine (BLV), diagnostic INTRODUCTION Leukosis (leukemia or l y m p h o s a r c o m a ) , a neoplastic disease, occurs n a t u r a l l y in cattle. One form of bovine leukosis, called enzootic bovine leukosis (EBL), was identified early as a n infectious a n d transmissible disease, because it was very early observed that l y m p h o s a r c o m a preferably appeared in some herds a n d in some areas. In 1969, a C-type R N A virus was identified in cattle from leukotic herds. At the present time this virus, n a m e d bovine leukemia virus (BLV), is generally considered as the etiologic agent of EBL. BLV is able to induce an i m m u n o l o g i c a l response in the infected host which produces antibodies against the viral proteins a n d essentially against the m a i n internal p r o t e i n (p24) a n d the envelope glycoprotein (gp51). A n t i b o d i e s against gp51 have generally higher titers a n d a p p e a r earlier t h a n antibodies against p24 [1]. EBL should be distinguished from sporadic bovine leukosis (SBL), a cattle disease which occurs m a i n l y in y o u n g animals. In SBL, lesions have the same a p p e a r a n c e as in EBL, b u t n o virus could be retained as etiologic agent. EBL a n d SBL are two completely separate diseases [1]. CLINICAL DIAGNOSIS

Direct Method Tumor development M a l i g n a n t t u m o r s seldom appear in cattle p o p u l a t i o n , p r o b a b l y because a n i m a l s of this species are slaughtered at a relatively y o u n g age, a n d also perhaps because they are not 305

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submitted to environmental factors which are considered as important for the tumor development in other species, like humans and dogs. Many types of cancer have been described in cattle, but lymphosarcoma and leukemia seem to be the prevalent types in this species. Symptoms of the tumoral form of EBL vary widely from one animal to another and may be very discrete or not. Tumors generally appear in adult animals and may invade lymph nodes and all other internal or external tissues of the organism. Depending on the localization and the size of the tumor, a wide variety of disorders can be observed. Overt leukemia is sometimes the only obvious symptom encountered. Still more exceptional is the invasion of the bone marrow by tumor cells. Histopathological and hematological techniques are necessary to demonstrate the lymphoi'd nature of the neoplastic cell. Virological techniques (vide infra) allow to differentiate an EBL tumor from a SBL one. After death the presence of antibodies can be searched in the juice expelled from a tumor. The percentage of BLV-infected animals that develop a tumor in a leukotic herd depends on unknown genetic, environmental and immunological factors. However this percentage is always very low in cattle during the normal period of their productive life. In many BLV-infected herds no tumor cases could be observed, in other herds multiple tumor cases could be observed in a short period of time. The discovery of one or more tumor cases cannot give an exact idea of the infection rate in a herd. The discovery of a BLV induced tumor in an animal indicates that the herd of origin is probably infected by the same virus and that additional diagnostic tests must be performed on all the animals of the herd.

Indirect Method Persistent lymphocytosis An animal infected by BLV may or may not develop persistent lymphocytosis (PL). This fact was not known before the discovery of the virus. At that time many a.uthors had observed a correlation between the presence of lymphosarcoma cases in a herd and the presence of numerous animals with PL in the same herd. This observation led to the establishment of the leukosis keys in cattle. These keys gave the values of the lymphocytes counts above which an animal was considered as dubious or frankly positive for EBL. With the virological methods now available, it was proved that PL appeared in only 50-60% of BLV infected animals. PL is a non-specific symptom of EBL which may theoretically be observed in other pathological situations. Practically the discovery of several animals with PL in a herd is a high presumption in favour of EBL. This diagnosis procedure is suitable at the level of the herd but is inapplicable on an individual basis. Today, virological diagnosis has replaced hematological diagnosis. VIROLOGICAL DIAGNOSIS

Detection of the Viral Antigens Direct methods Electron microscopy. C-type particles are released in short-term cultures of lymphocytes from BLV-infected animals and can be directly visualized by electron microscopy. This procedure allowed in 1969 the discovery of BLV. BLV is so far the only C-type virus

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described in cattle, but electron microscopy is not routinely used for diagnosis. This method remains a sophisticated, time consuming technique which requires skilled technicians and expensive equipment. Serological tests. All the serological tests used for the detection of antibodies against BLV (vide infra) require one or more viral antigens. In turn, these tests can be adapted for the detection of the viral antigens.

Indirect methods Syncytia-inducing activity. One peculiar property of BLV or BLV producing cells is to induce syncytia (or polykaryocytosis) in appropriate indicator cells from human, bovine, bat, caprine or feline origin. The syncytia-inducing activity is specifically neutralized by antibodies to BLV. Based on this observation, several tests were proposed and particularly the SIA (syncytia induction assay) and the EPT (early polykaryocytosis test). All these tests are relatively complicated and not easy to standardize. They require techniques of cell cultures and must be performed aseptically. Bio-assay on sheep. BLV is easily transmitted to sheep and it expresses in that species a highly oncogenic activity. Practically, all the sheep inoculated, by any way, with BLV containing material develop antibodies in two to three weeks post-inoculation. They all die with tumor within a few months to seven years. This observation points out the sheep as the best species to realize a bio-assay. The bio-assay is an expensive test which requires particular breeding facilities. Detection of the Antibodies Directed Against Viral Antigens Direct methods Agar-gel immunodiffusion. The agar-gel immunodiffusion test (AGID) was the first test used to detect specific antibodies directed against the viral antigens. It was first performed with ether-disrupted virion preparations which contained essentially the p24 antigen. Later an ether-sensitive antigen was identified. This antigen, the glycoprotein gp51, rapidly appeared as more efficient than the p24 antigen to detect animals infected by BLV. AGID test is a sensitive, simple and specific method which does not require special equipment and can be used in all laboratories. This technique is very popular and widely used all over the world. AGID test is the official method prescribed by the EEC regulations. Immunofluorescence. Indirect immunofluorescence (IIF) test can be used to detect seropositive animals infected by BLV. This technique is sensitive and specific, but laborious to perform. The use of a fluorescent microscope for examination of a large number of slides is a tiresome work. Complement fixation. Complement fixation test (CFT) can also be used to detect BLV antibodies. This method is inadequate for the examination of bovine sera because many of them have an anticomplementary activity. The method cannot be recommended for large scale survey. Radioimmunoassay. Radioimmunoassay (RIA) is a highly sensitive technique to detect antibodies in sera. This method is very sophisticated and requires radiolabeled reagents and special equipment which cannot be found and used in all laboratories. RIA is generally used for fundamental research works with BLV and not for routine examinations. Enzyme-linked immunosorbent assay. Enzyme-linked immunosorbent assay (ELISA) is ('IMID~3~

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based on the same principles as RIA. The techniques has the same sensitivity as RIA and does not require special equipment. Reagents to perform an ELISA are not dangerous to manipulate and the test can be used in all laboratories. To perform a classical ELISA for the detection of BLV antibodies, a non-purified BLV antigen is absorbed onto the wells of microplates. The sera to be tested are diluted and incubated with precoated microplates. The BLV bound antibody is detected by an enzyme coupled antibovine immunoglobulin reagent. This classical technique frequently gives high background values mainly due to the absorption of non-viral contaminants of the viral preparation onto the walls of the wells. To overcome this obstacle one can use a purified gp51 antigen, but the technique is laborious and expensive. Use of well selected monoclonal antibodies is an elegant manner to solve the problem and to reduce background values [2, 3, 4]. This variant of the classical ELISA technique seems to be the best sensitive method presently available for the diagnosis of EBL. Indirect methods Neutralization test. All the viriological methods used for the detection of the BLV viral antigens can include an inhibition step by specific antibodies. Based on this principle, many tests were proposed:

--virus neutralization test (VNT); syncytia inhibition test or syncytia induction inhibition test (SIT); ---early polykarycoytosis inhibition test (EPI); pseudotypes inhibition test (PI). All these tests require aseptic reagents and have thus drawbacks inherent to this requirement. CONCLUSIONS

The clinical diagnosis of EBL must be confirmed by a specific laboratory test for BLV. Several tests for the detection of the virus or the antibodies are not easy to use in routine work because they require carefully collected samples. Everybody knows that in the conditions of the veterinary practice, it is impossible to supply the laboratory with such samples. Other tests require equipment, reagents and sophisticated techniques which cannot be easily found in laboratories for routine examinations. Comparison of the different diagnosis techniques by different authors does not always lead to similar conclusions. This is due to techniques which are not always run in the right conditions, by trained operators and with adequate reagents. Consequently, appreciation of the efficacy of the various diagnosis tests proposed is a hazardous exercise. Nevertheless, in the wide variety of diagnosis techniques investigated, four seem to be widely used over the world: the bio-assay on sheep, RIA, A G I D test and ELISA. The first two methods are mainly used in research laboratories, the two last ones are widely used to detect and eradicate the disease in field conditions. In the enzootic bovine leukosis system, the advantage of the use of sensitive methods like RIA or ELISA for individual testing on sera, is limited to a short period of time in the life of an animal. In fact, after infection with BLV, the production of specific antibodies in the serum grows rapidly. Generally, after 2-3 weeks it reaches a level where practically all diagnosis procedures are effective. In other words, the sensitivity of all the diagnosis

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methods is sufficient to do an early detection of a BLV infection. The advantages of the highly sensitive methods like RIA and ELISA over the immunodiffusion gp51 technique are not sufficient to renounce to the A G I D test. AGID test remains the simplest diagnosis procedure with sufficient sensitivity to allow the rapid clean up of an infected herd. The high sensitivity of the RIA and the ELISA is useful when the samples to be tested have very low antibody titers. This is particularly true in two situations: (1) to do a diagnosis on biological fluids in which the antibody levels are naturally very low (individual milk samples) [5-7]; (2) to detect antibodies in bulk milk samples [6, 7] or in pooled sera [8, 9]. REFERENCES 1. Burny A., Bruck C., Chantrenne H., Cleuter Y., Dekegel D., Ghysadael J., Kettmann R., Leclercq M., Leunen J., Mammerickx M. and Portetelle D. Bovine leukemia virus: molecular biology and epidemiology. In Viral Oncology (Edited by Klein G.), pp. 231-289. Raven Press, New York (1980). 2. Portetelle D., Bruck C., Mammerickx M. and Burny A. Use of monoclonal antibody in an ELISA test for the detection of antibodies to bovine leukaemia virus. J. Virol. Meth. 6, 19-29 (1983). 3. Mammerickx M,, Portetelle D., Bruck C. and Burny A. Le diagnostic de la leucose bovine enzootique fi l'aide d'un test immunoenzymatique (ELISA) impliquant un anticorps monoclonal. Ann. Mbd. Mbt. 128, 55 63 (1984). 4. Mammerickx M., Portetelle D., Bruck C. and Burny A. Use of an ELISA involving monoclonal antibody for the detection of antibodies against bovine leukemia virus in a herd with high incidence of enzootic bovine leukosis. Zbl. vet. Med. B. 31, 210-218 (1984). 5. Ban J., Zajac V., Altaner C. and Cerny L. Early diagnosis of virus induced bovine leukosis in milk by a simple modified ELISA test. Zbl. vet. Med. B. 29, 591-595 (1982). 6. Toma B., Vuillaume A., Manet G., Duret C., Eloit M., Crespeau F., Chappuis G. and Parodi A. L. D6pistage de la leucose bovine enzootique par application du test ELISA sur le lait. Rec. Mbd. Vbt. 160, 53 60 (1984). 7. Mammerickx M., Portetelle D. and Burny A. Application of an enzyme-linked immunosorbent assay (ELISA) involving monoclonal antibody for detection of BLV antibodies in individual or pooled bovine milk samples. Zbl. vet. Med. B. 32, 526-533 (1985). 8. Mammerickx M., Portetelle D. and Burny A. La d~tection de la leucose bovine enzootique fi l'aide d'un test immuno-enzymatique (ELISA) effectu~ sur des m~langes de s~rums. Ann. M~d. V~t. 128, 203 209 (1984). 9. Mammerickx M., Portetelle D., Nys J. and Burny A. Le d6pistage de masse de la leucose bovine enzootique en Belgique fi l'aide d'un test immunoenzymatique (ELISA) effectu6 sur m~langes de s6rums. Ann. Mbd. Vbt. 128, 455465 (1984).