A. GALLI

A. GHIDONI

L. CARBONI

English

Crossref

A cytogenetic investigation on peripheral blood lymphocytes
of cattle affected by enzootic bovine leukemia
A. GALLI L. CARBONI A. GHIDONI *
* Consorzio Provinciale Fecondazione Artificiale, Via Borgo Palazzo 128, 24100 Bergamo, Italia
.. Dipartimento di Cenetica e di Biologia dei Microrganismi, Universit9 degli Studi di Milano,
Via Celoria 26, 20133 Milano, Italia
Summary
This cytogenetic study involved 10 Agar-Gel Immunodiffusion (AGID) test-positive cows
without lymphocytosis, 6 similar subjects with lymphocytosis and 10 healthy subjects. No apprecia-
ble differences were found for aneuploid rates or chromosome aberrations. However, a higher
frequency of polyploid cells was consistently observed among subjects affected by enzootic bovine
leukemia without lymphocytosis (average : 4.8 p. 100) as compared with healthy controls (ave-
rage : 1.5 p. 1(N)). The lymphocytes in both groups were stimulated by phytohemagglutinin. A
higher frequency of polyploid cells was not found among subjects affected by enzootic bovine
leukemia with lymphocytosis, whose lymphocyte cultures were stimulated by concanavalin A.
Key words : Chromosome, enzootic bovine leukemia, Italian Friesian cattle.
Résumé
Etude cytogénétique de lymphocytes du sang périphérique
de bovins atteints de leucémie enzootique
L’étude cytogénétique concernait 10 vaches positives pour le test AGID (immunodiffusion en
gel d’agar) et exemptes de lymphocytose, 6 sujets similaires atteints de lymphocytose et 10 sujets
sains. On n’a pas trouvé de différence appréciable pour les taux d’ancupl6idie ou les aberrations
chromosomiques. Cependant, on a observé une fréquence plus élevée de cellules polyploïdes chez
les sujets atteints de leucémie bovine enzootique sans lymphocytose (moyenne : 4,8 p. 100) que
chez les témoins sains (moyenne : 1,5 p. 100). Dans les 2 groupes, les lymphocytes sont stimulés
par la phytohémagglutinine. On n’a pas trouvé une fréquence plus élevée de cellules polyploïdes
chez les sujets atteints de leucémie bovine enzootique avec lymphocytose, dont les cultures
lymphocytaires sont stimulées par la concanavaline A.
Mots clés : Chromosome, leucémie bovine enzootique, bovins Frisons d’Italie.
I. Introduction
Enzootic bovine leukemia (EBL), a lymphosarcomatous-like chronic disease caused
by a retroviral agent (Bovine Leukemia Virus, BLV) has been the object of few
cytogenetic investigations. A submetacentric supernumerary chromosome and other
sporadic anomalies concerning chromosome condensation were found in lymph nodes of
a Holstein bovine affected by lymphosarcoma (BASRUR et al. , 1964). Various non
specific chromosomal anomalies were also observed in peripheral blood lymphocytes
(GUSTAVSSON & ROCKBORN, 1964 ; GUSTAVSSON, 1966 ; HARE et al., 1967 ; MARTIN &
FLANAGAN, 1967). However, a specific robertsonian translocation involving chromo-
somes 1 and 29 was observed (GUSTAVSSON & ROCKBORN, 1964) in tumour cells of
cattle affected by lymphatic leukemia, and a higher frequency of chromosomal anoma-
lies were found (LOMBARD, 1968 ; FERRER, 1980) to be associated with this pathologic
situation. WEINHOLD (1970) demonstrated a relatively high percentage of hyperploid
cells in bovine leucosis. In a more recent study only sporadic structural chromosomal
anomalies were found {GRIMOLDIet al. , 1983) in peripheral blood lymphocytes of cows
clinically healthy, affected by EBL. Hypodiploid cells metacentric and submetacentric
chromosomes were found in cows affected by tumoral forms of bovine leukosis. These
findings suggest, in accord with another study (FERRER, 1980), that the persistent
lymphocytosis may be a benign response to the viral agent involved in this cattle
disease. Guo & Guo (1983) showed some chromosome anomalies in Bovine leucosis,
e.g. hyperploid and hypoploid cells, chromatid and isochromatid aberration, etc.,
especially significantly diminished nucleolus organizer regions (NORs), and significantly
higher sister chromatid exchanges (SCE).
The present investigation was undertaken to obtain more cytogenetic information
on cows affected by EBL with or without persistent lymphocytosis.
II. Material and methods
The investigation concerned 26 cows of the Italian Friesian breed from 2 herds
located near Bergamo in The Northern Po valley (Italy). The herds are similarly
equipped and under similar management. All subjects were tested twice, with 12
months’ interval, by the agar gel immunodiffusion test (AGID test) for bovine leukosis
virus of BEX et al. (1979). On all subjects the leucocyte formula was determined twice,
with one month’s interval, on peripheral blood smears, after staining by the standard
May-Gruenwald-Giemsa method (BECCARI & MAZZI, 1966), in order to identify subjects
with or without relative persistent lymphocytosis.
The 26 cows under study were grouped as follows :
1. Ten healthy subjects as a control (Group H).
2. Ten AGID test-positive subjects without persistent relative lymphocytosis
(Group LI).
3. Six AGID test-positive subjects with persistent relative lymphocytosis (Group
L2).
Peripheral blood cultures were set up according to standard procedures (after
MOOREHEAD et ll. , 1960) with the following details : in each vessel suitable to 8 ml
cultures were mixed the lymphocyte fraction of 8 ml venous blood, 13 wg phytohemag-
glutinin (type M, Gibco), or 10 wg concanavalin A (IV-S, Sigma) and TC 199 medium
with 20 p. 100 autologous serum. The concanavalin A mitogen was used in place of
phytohemagglutinin for subjects -of Group L2 where the latter achieved only a poor
stimulation. Cultures were arrested at 72 hr. Two hours before collecting cells, colcemid
(Gibco) at .011Lg/ml (final concentration) was added to each culture. Slides were made
according to the standard air drying procedure and stained with a 4.5 p. 100 Giemsa
solution, buffered at pH 6.8. An average of about 125 metaphases were analyzed for
each subject. Cells lacking 2 or more chromosomes were not considered in the analysis.
The C and G banding patterns of cells with the robertsonian translocation 1/29
were obtained following the procedures of SuMNER (1972) modified by POPESCU (1975),
and of SEABRIGHT (1971) modified by HAGELTORN & GUSTAVSSON (1973), respectively,
studied by POPESCU (1975).
III. Results &dquo;
The leucocyte formulas of all subjects analysed, as obtained from peripheral blood
smears, are shown in table 1. While group H and Ll showed a typical leucocyte
formula, Group L2 had relatively higher values for lymphocytes (mostly -at the expense
of neutrophile leucocytes). This is a rather common finding among subjects with
lymphocytosis (STOEBER & GRUENDER, 1977). _
The results of the cytogenetic investigation are reported in table 2. By comparing
AGID test-positive and control subjects, significant differences at the chromosomal
level were not found except for a higher percentage of polyploid cells (4.8-9 p. 100)
among Group Ll (tabl. 3). Other observations concerning the frequency of aneuploid
cells, chromosomal breaks or gaps and obvious chromosomal rearrangements, failed to
disclose any significant difference between any of the groups examined in this regard.
IV. Discussion
Standard values regarding the frequency of chromosomal anomalies in bovine
leucocyte cultures stimulated in vitro are not available in the literature. The frequency
of numerical anomalies in the healthy subjects, Group H (polyploid cells 1.5 p. 100,
aneuploid cells 1.1 p. 100) is not higher than found with humans (LITTLEFIELD & Gott,
1973).
The finding of a higher percentage of polyploid metaphases in lymphocytes of
Group Ll subjects (AGID test-positive, without lymphocytosis) may be regarded as a
consequence of the viral infection, as was suggested by HARE et al. (1964).
The frequencies of cells with breaks (3.4-3.8 p. 100), after stimulation with
phytohemagglutinin, may be regarded as relatively high if compared with observations
made on human cultured lymphocytes, where most investigators reported values of 1 p.
100 or less (EVANS et al. , 1979 ; LOYD et al. , 1980 ; ALICATA et al. , 1981). However,
these values were found for both AGID test-positive or healthy subjects, thus possibly
resulting from our culture conditions. The frequency of breaks in peripheral blood
lymphocytes of subjects in Group 2 (AGID test-positive, with lymphocytosis) was,
however, relatively lower ; in this case the stimulation was achieved with concanavalin
A. As an explanation, one could suggest that the different mitogens may have
stimulated different subsets of T-lymphocytes with varying effects of the consequent
stimulation on other subsets of B-lymphocytes, as suggested by Kx!sTErrsErr et al.
(1982). Until this point is made clear, the analysis of cytogenetic data should properly
consider the existence of this kind of heterogeneity.
The frequency of robertsonian translocations was not found to differ statistically
among the 3 groups examined, although GUSTAVSSON & ROCKBORN (1964) showed that
some such rearrangements may be typical of lymphoid leukemia. The observations
concerning robertsonian translocation made in the present investigation seem to exclude
the hypothesis that this type of rearrangements is relevant to the etiology or the
progression of enzootic bovine leucosis, at least in the subjects examined by us. All of
the robertsonian translocations observed by us involved apparently chromosomes 1 and
29.
There were no marked differences in the frequencies of aneuploid cells observed in
the healthy subjects (H) and in the AGID test-positive subjects (Ll).
An average of 1.2 p. 100 of aneuploid cells was found in the 3 tested groups. This
result is not comparable with that of BASRUR et at. (1964), who found a high rate of
aneuploid cells (above 60 p. 100) in lymph node leucocyte cultures from one subject
affected by lymphosarcoma.
Group H (healthy) and Group Ll (AGID test-positive, without lymphocytosis)
both were stimulated with phytohemagglutinin ; in this comparison notable differences
were not found except for polyploid cells rates.
The poor stimulation of lymphocytes by the phytohemagglutinin in AGID test-
positive subjects with persistent lymphocytosis is parallel to similar stimulation difficul-
ties in human leukemic subjects, perhaps as a consequence of a deficient immune
response that characterizes such patients (BERNARD et al. , 1964 ; CHIORAZZt et al.,
1979 ; UMEDA et al., 1982 ; GHIDONI et al., 1984).
V. Conclusion
A higher frequency of polyploid cells was observed in AGID test-positive subjects
without persistent relative lymphocytosis (Group Ll).
A high frequency of chromosomal anomalies was not found in AGID test-positive
cows with persistent relative lymphocytosis (Group L2). Persistent lymphocytosis is
therefore probably a benign reactive condition of a lymphoproliferative kind as was
suggested by FERRER (1980) and GRIMOLDI et al. (1983).
Received October 14, 1985.
Accepted September 3, 1986.
Acknowledgements
The authors wish to thank Dr. M. Bosisio and Prof. M. SARI for technical assistance.
References
ALICAT P., BIONDI 0., CANTARELLA T., DE LUCA B., GHIDONI A., MOTIA S., POLITI G., RADICE
P., SAMMARTANO F., 1981. VariabilitA individuale nella frequenza di aberrazioni cromosomiche
in soggetti professionalmente esposti al rischio di radiazioni ionizzanti. Atti Ass. Genet. Ital.,
28, 9-12.
BASRUR P.K., GILMAN J.P.W., MCSHERRY B.J., 1964. Cytological observations on a bovine
lymphosarcoma. Nature, 25, 368-371.
BECCARI N., MAZZI V., 1966. Studio speciale delle cellule dei tessuti. In : BECCARI N., MAZZI V.
(ed.), Manuale di tecnica microscopica, 129-222, Casa Editrice Dr. Francesco Vallardi -
Societa Editrice Libraria, Milano.
BERNARD C., GERALDES A., BOIRON M., 1964. Action de la phytohemagglutinine in vitro sur les
lymphocytes de leuc6mies lymphodes chroniques. Nouv. Revue Fr. Hemat. , 4, 69.
BEX F., BRUCK C., MAMMERICKX M., PORTETELLE D., GHYSDAEL J., CLEUTER Y., LECLERCQ M.,
DEKEGEL D., BURNY A., 1979. Humoral antibody response to bovine leukemia virus infection
in cattle and sheep. Cancer Res. , 39, 1118-1123.
CHIORAZZI N., Fu S.M., MONTAZEN G., KUNKEL H.G., RAF R., GEE T., 1979. T cell help defect
in patients with chronic lymphocytic leukemia. J. Immun., 122, 1087-1090.
EVANS H.J., BUCKTON K.E., HAMILTON G.E., CAROTHERS A., 1979. Radiation-induced chromosome
aberrations in nuclear-dock-yard workers. Nature, 277, 531-534.
FERRER J.F., 1980. Bovine lymphosarcoma. Adv. Vet. Sci. Comp. Med., 24, 1-68.
GHIDONI A., PRIVITERA E., RAiMONDi E., ROVINI D., ILLENI M.T., CASCINELL N., 1984. Sister
chromatid exchanges in phytohemagglutinin-stimulated lymphocytes of nonfamilial cutaneous
malignant melanoma patients. In : RAYMOND R.T., HOLLANDER A. (ed.), Sister chromatid
exchanges, 855-858, Plenum Publishing Corporation, New York.
GRIMOLDI M.G., POLI G., SARTORELLI P., CALDORA C., OLDANI L., LOCATELLI A., 1983. Karyotype
analysis of lymphocytes from cattle at different stages of bovine leukemia virus infection. Br.
Vet. J., 139, 240-246.
Guo A., Guo D., 1983. The chromosomes of bovine leukemia. Acta Vet. et Zootech. Sinica, 14,
201-207.
GUSTAVSSON 1., 1966. Chromosome abnormality in cattle. Nature, 211, 865-866.
GusTvsSON I., ROCKBORN G., 1964. Chromosome abnormality in three cases of lymphatic
leukemia in cattle. Nature, 203, 990.
HAGELTORN M., GUSTAVSSON 1., 1973. Giemsa staining patterns for identification of the pig mitotic
chromosomes. Hereditas, 75, 144-146.
HARE W.C.D., McFEELY R.A., ABT D.A., FEIRMAN J.R., 1964. Chromosomal studies in bovine
lymphosarcoma. J. Natl. Cancer Inst. , 33, 105-118.
HARE W.C.D., YANG Tsu-Ju, McFEELY R.A., 1967. A survey of chromosomes finding in 47 cases
of bovine lymphosarcoma (leukemia). J. Natl. Cancer Inst., 38, 383-392.
KRISTENSEN F., KRISTENSEN B., LAZARY S., 1982. The lymphocyte stimulation test in veterinary
immunology. Vet. Immun. Immunop., 3, 203-277.
LITTEFIELD L.G., GOH K.O., 1973. Cytogenetic studies in control men and women. I. Variations
in aberration frequencies in 29,709 metaphases from 305 cultures obtained over a three-year
. period. Cytogenet. Cell Genet. , 12, 17-34.
LOYD D.C., PURROT R.J., REEDER E.J., 1980. The incidence of unstable chromosome aberrations
in peripheral blood lymphocytes from unirradiated and occupationally exposed people. Mutat.
Res., 72, 523-532.
LOMBARD C., 1968. Les leucoses des mammif6res. /n : LOMBARD C. (6d.), Les leucoses animales, 9-
127, I.N.R.A., Paris.
MARTIN W.B., FLANAGAN M., 1967. Karyotype analysis of leucocytes from normal and lymphosar-
comatous cattle (Bos Taurus). Br. J. Cancer, 21, 137-143.
MOORHEAD P.S., NOWELL P.C., MELLMAN W.J., BATTIPS M., HUNGERFORD D.A., 1960. Chromo-
somes preparations of leukocytes cultured from peripheral blood. Exp. Cell Res., 20, 613-616.
PoPEscu C.P., 1975. L’étude du caryotype bovin (Bos Taurus L.) par les mdthodes de bandes.
Ann. Biol. Anim. Bioch. Biophys., 15, 751-756.
SEABRIGHT M., 1971. A rapid banding technique for human chromosomes. Lancet, 2, 971-972.
STOEBER M., GRUENDER H.D., 1977. Apparato circolatorio. ll1 : ROSENBERGER G. (ed.), L’esame
clinico del bovino, 114-182, Editrice Essegivi, Piacenza.
SUMNER A.T., 1972. A simple technique for demonstrating centrochromatin. Exp. Cell. Res., 75,
304-306.
UMEDA J., HARA T., KOSEKI K., KOBAYASHI K., AICnz H., NIUIMA T., 1982. Diminished helper-T-
lymphocyte function in cancer patients. Cell. Mol. Biol. , 28, 67-71.
WEINHOLD E., 1970. Chromosomenuntersuchungen bei der Rinderleukose. Ith Europ. Kolloqu.
Zytogerietik in Veterinarmedizin u. Saugetierkd., 12-13 Oct., Giessen. 119-126. Inst. fur
Erpathol. und Zuchthygiene: ’


A cytogenetic investigation on peripheral blood lymphocytes  of cattle affected by enzootic bovine leukemia

http://www.gse-journal.org/10.1051/gse:19870101/pdf

A cytogenetic investigation on peripheral blood lymphocytes of cattle affected by enzootic bovine leukemia

Abstract

Similar works

Full text

Available Versions

Crossref

Crossref