Veterinap:v Microbiology, 25 ( 1990 ) 131-141 Elsevier Science Publishers B.V., Amsterdam

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Suppression of immunological responses in rabbits experimentally infected with bovine leukemia virus Misao Onuma*, Mayumi Wada, Yasuhiro Yasutomi, Masako Yamamoto, Hiroyuki M. Okada and Yoshimi Kawakami Department of Veterinary Microbiology, Rakuno Gakuen University, Ebetsu 069, Japan (Accepted 12 March 1990)

ABSTRACT Onuma, M., Wada, M., Yasutomi, Y., Yamamoto, M., Okada, H.M. and Kawakami, Y., 1990. Suppression of immunological responses in rabbits experimentally infected with bovine leukemia virus. Vet. Microbiol., 25: 131-141. Ten 2- to 4-month-old rabbits were inoculated subcutaneously with bovine leukemia virus (BLV)infected bovine or sheep cells. By 6 weeks after inoculation all ten rabbits had converted to BLV antibody-positive, and BLV or BLV antigen was detected in lympbocytes from most of the rabbits tested, although there were few antigen-producing cells. Three rabbits showed continuous respiratory symptoms after infection and one died with pneumonia. Humoral immune responses against mouse serum were significantly suppressed in BLV-infected rabbits compared with non-infected control rabbits. The lymphocyte blastogenesis response was also suppressed in BLV-infected rabbits. At the time of necropsy, six rabbits showed pulmonary lesions; however, none of the BLV-infected rabbits had tumors during an observation period of over 1 year.

INTRODUCTION

Bovine leukemia virus (BLV) is a causative agent of bovine leukosis. Several species of animals, including cattle (Miller et al., 1972 ), sheep (Olson et al., 1972), goats (Hoss and Olson, 1974), chimpanzees (Van Der Maaten and Miller, 1976 ) and rabbits (Bansal and Singh, 1980) can be infected with BLV. BLV is highly oncogenic in sheep, but was less oncogenic when inoculated into goats and cattle (Olson et al., 1970; Olson et al., 1981; Mammerickx et al., 1981 ). Attempts to infect rabbits with BLV have been successful, but no tumor developed during a short-term observation period of 2-3 months (Bansal and Singh, 1980). Previously Burny et al. ( 1985 ) reported that BLV*Present address: Department of Epizootiology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060, Japan.

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infected rabbits showed fluctuations in BLV antibody titers followed by a sudden fall of the titer to zero. They suggested that these rabbits were suffering from a condition resembling a BLV-induced immunodeficiency syndrome. Laboratory animals susceptible to BLV infection, suitable for develo p m e n t of a vaccine against BLV, are needed. The present study was carried out in an attempt to determine whether BLV-infected rabbits develop tumors a n d / o r immunodeficiency syndromes during a long-term observation period of over 1 year. MATERIALS A N D M E T H O D S

Experimental animals and inoculum Six 2-month-old rabbits and four 4-month-old rabbits were inoculated subcutaneously with lymphocytes from cattle with persistent lymphocytosis or with fetal lamb kidney (FLK) cells persistently infected with BLV as shown in Table 1. Four 2- to 4-month-old rabbits were not inoculated (controls). After inoculation, serum samples were obtained every 2 weeks for detection of serum BLV antibody. Hematological tests and body weight determination were performed every month. All rabbits were kept under conventional conditions.

Detection of BLV antibody The immunodiffusion (ID) test using glycoprotein antigen was done by the method described previously ( O n u m a et al., 1975 ). For detection of BLV antibody, the enzyme-linked immunosorbent assay (ELISA) was performed using sucrose gradient-purified BLV as antigen. 50 #1 of a predetermined concentration of purified BLV, which had been fixed with 2% glutaraldehyde, was adsorbed to individual wells at 4 °C overnight. The plates were washed three times with 0.1% Tween 80 in 0.05 M PBS, pH 7.2 (PBS-Tween). To eliminate non-specific reactions, 50/~1 of 1% gelatin was added, and the plates were incubated at 37°C for 1 h and then washed with PBS-Tween. 50/zl of 1:700 dilution of test serum, diluted with PBSTween, was incubated in antigen-adsorbed wells at 37°C for 1 h. U n b o u n d immunoglobulin (Ig) was removed from the wells by washing, and was then reacted with 50/tl of a 1:1000 dilution of peroxidase-labeled anti-rabbit IgG (Cappel Lab.) at 37°C for 1 h. After washing the plates with PBS-Tween, 100 /zl of a substrate solution containing 0.2 m M 2,2'-azino-di(3-ethylbenzthiazoline sulfonic acid) (Sigma Chemical, St. Louis, MO) and 0.004% H202 in 0.05 M citrate buffer, pH 4.0, was added to each well, and the plate was incubated at 37°C for 1 h. A4o 5 w a s scored by a Micro-ELISA automatic reader ( I m m u n o R e a d e r N J-2000, InterMed). Sera from preinoculated rabbits were used as a control; A4o 5 values for these were always less than 0.2. The ELISA endpoint titer was estimated from the dose-response curve of

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serial four-fold dilutions of serum. The ELISA endpoint titer was taken as the reciprocal of the m a x i m u m dilution of antibody giving A4o5 > 0.2. Detection o f BL V Rabbit peripheral blood lymphocytes (PBL) were separated by the FicollConray method. Separated lymphocytes (3 X 105/well) were cocultured with C81 cells (1 × 105/well) in a 2-cm 2 plate for 4-5 days. After incubation, the cells were fixed and stained with Giemsa. For the immunofluorescence (IF) test, separated lymphocytes ( 1 × 106/ ml ) in RPMI 1640 with 10% fetal calf serum (FCS) and concanavalin A (con A, 12.5/zg/ml) were cultured for 2 days. Cell smears were then fixed and stained with anti-BLV p24 monoclonal antibody (Aida et al., 1987 ). To study antigen expression during long-term culture, rabbit lymphocytes were cultured with con A for 2 days. The cells were then divided into two lots at 3- to 4-day intervals for about 1 m o n t h using RPMI 1640 containing 10% FCS and 10% interleukin 2 ( I L - 2 ) derived from monkey lymphocytes. During longterm culture, lymphocytes were tested for expression of BLV antigen using the 1F test. Humoral i m m u n e response against mouse serum protein To investigate the humoral i m m u n e response against foreign protein in BLVinfected rabbits, six BLV-infected rabbits (nos. 1,3,5,6,7 and 8 ) and 3 control rabbits (nos. 11,12 and 13) were injected with 0.84 mg mouse serum 5.5 months after BLV inoculation. After injection, serum samples were obtained at 1- to 2-week intervals for 15 weeks. Antibody titer against mouse serum was determined by ELISA as described above. Control A4o5 values were obtained in preimmunized sera; these values were less than 0.2. Blastogenesis test Blood samples in citrate-phosphate-dextran as anticoagulant were obtained from seven BLV-infected and three non-infected rabbits 9.5 months after BLV inoculation. Lymphocytes were suspended at 1 X 106 viable cells/ ml with R P M I 1640 containing 10% horse serum. The mitogenic response was determined using 200/tl of cells (2 × 105 cells/well) in triplicate wells of 96-well flat-bottomed microplates with 12.5/zg/10/A of con A in each well. Control wells contained the same volume (200 pl) of cells with 10/tl of medium. The plates were incubated at 37 °C for 72 h, and 1/zCi of [ 3H ] thymidine was added to each well. Cultures were harvested onto fiberglass filter disks 18 h later. The incorporation of [ 3H ] thymidine into cells was determined using a scintillation spectrophotometer, and results were expressed as the stimulation index (SI; Odawara et al., 1987).

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Detection of rabbit B and T cells To determine the percentage ofT and B cells in BLV-infected rabbits, membrane IF tests were performed. The monoclonal antibodies used were LI 1/ 135 and 2C4, against pan T and Ia antigens respectively (Jackson et al., 1983; Lobel and Knight, 1984). 50-70% of normal rabbit lymphocytes reacted with L11/135 and 30-40% with 2C4. Statistical analysis The statistical significances of the data were evaluated by Student's t test. RESULTS

Detection of BLV antibody As shown in Table 1 and Fig. 1, four rabbits (nos. 3,8,9 and 10) converted to antibody-positive at 13 days by the ID test, and the remaining six rabbits converted to positive by 46 days after inoculation. Six BLV-infected rabbits (nos. 2,3,5,6,7 and 8) were raised over 1 year. The antibody was continually detected in these six rabbits during the 416-day postinoculation period. 15.5 months after inoculation, four of six rabbits tested had converted to negative, TABLE 1

Rabbits used for the experiment and results of BLV infection Rabbit Age

Weight Inoculum Detection

no.

(months)

(kg)

and cell number (XI04)

Detection o f B L V of antibody ~ SA IF (days)

1 2 3 4 5 6 7 8 9 10 11 12 13 14

2 2 2 4 4 2 2 4 2 4 4 2 4 2

1.3 1.5 1.4 2.2 1.6 1.3 1.5 1.8 1.55 1.8 2.3 1.4 1.6 1.2

PBL 2 PBL 2 PBL 2 PBL 10 PBL 10 FLK 2 F L K 10 FLK 10 FLK 10 FLK20 None None None None

28-289 46-416 13-576 21-165 35-416 21-416 21-471 13-416 13-173 13- 75 Negative Negative Negative Negative

+ + + + + + + NT -

+ + + + + + + NT -

Clinical remarks

RS C RS C RS

C RS RS

Observation periods (days)

killed 289 killed 516 died 2 576 died 165 killed 628 killed 516 killed 516 killed 516 killed 173 died 2 75 killed 326 killed 516 killed 60 killed 5t6

1Numbers indicate date of seroconversion of the last positive sample. 2Died from causes other than p n e u m o n i a . PBL, peripheral blood lymphocytes; ELK, fetal lamb kidney cells; SA, syncytium assay; IF, i m m u n o fluorescence; C, cough; RS, respiratory symptoms; NT, not tested.

13 5

IMMUNE RESPONSE OF RABBITS INFECTED WITH BLV

0.6

ti

0,4

8 0.2 0

!::I IIIII o

. . . .

~

. . . .

lb

T i m e a f t e r BLV I n o c u l a t i o n

.If 4s

'. .T

(Months)

Fig. 1. Detection of BLV antibody. Upper panel shows the mean + s.d. of OD values by ELISA. Lower panel shows the percentage of immunodiffusion (ID)-positive rabbits.

and two (nos. 3 and 7 ) were still antibody-positive. When the same six rabbits were tested at 17 months only one was found to be positive. Changes in OD values of BLV antibody by ELISA was determined using 1:700 dilutions of test samples and expressed as mean_+ s.d. of the A4o5 value. The mean + s.d. for preinoculated sera from the BLV-infected group was 0.134 + 0.034. After inoculation OD values gradually increased, and reached a peak value of 0.54 + 0.12 at 5 weeks; values then decreased slightly but were still 0.43 _+0.11 13 months later (Fig. 1 ). About 16 months later, the OD value dropped to 0.26 + 0.12, showing a good correlation with the ID results ( 2 / 6 ID-positive). Western blot analysis using NP40-disrupted BLV antigen, indicated that these rabbit sera reacted mainly with BLV p24 (data not shown).

Detection of BL V and BL V antigens and transmission of infection BLV and BLV antigens were detected in cultured PBL from BLV antibodypositive rabbits using the syncytium assay and the IF test, respectively. Using the syncytium assay, all rabbits were negative 1 m o n t h after inoculation, but two rabbits (nos. 3 and 4) were positive 2 months later. By 3 months, seven of nine rabbits tested were positive (Table 1, Fig. 2a). The results of the syncytium assay were not constant, because all rabbits tested at 4 and 5 months were syncytium-negative. We therefore performed the IF test after short-term

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Fig. 2a. Syncytia formation by cultured rabbit lymphocytes, b, Immunofluorescence antigenpositive cells ( × 700). c, Abcess in lung (rabbit no. 5, HE stain, × 30) d, Alveolar hemorrhages and pulmonary thrombi in lung of rabbit no. 4 (HE stain, × 300).

culture of PBL with con A at 5 months. As shown in Table 1, seven of ten BLV antibody-positive rabbits tested were positive, but fewer than 1% of cells were antigen-positive (Fig. 2b). At 9 months, the same seven rabbits were again tested; six were antigen-positive, but fewer than 1% of cells were positive. The PBL from these six rabbits were cultured with IL-2 for about 1 month and sequentially tested for antigen expression by the IF test. Fewer

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than 1% of the cells in long-term cultures were antigen-positive, a value similar to that for short-term cultures. To determine whether BLV-infected rabbit lymphocytes can transmit BLV infection to other rabbits, PBL (4 X 106/rabbit) from infected rabbits were injected subcutaneously into two l-year-old rabbits. These rabbits converted to antibody-positive 2 weeks later and continuously produced antibodies for at least 3 months. The same PBL (4X 106/rabbit) were also inoculated into four 5-day-old rabbits, but none of these rabbits converted to positive.

Hematological and clinical evidence of BL V infection in rabbits The number of red blood cells and lymphocytes were sequentially tested for 10 months after inoculation; there were no significant differences between control and infected groups. Body weight was also determined every month. In most BLV-infected rabbits and all control rabbits, body weight increased each month. However, two rabbits, of which one died from p n e u m o n i a (no. 4) and the other showed severe respiratory symptoms (no. 5 ), did not show an increase in weight 2 and 4.5 months respectively after inoculation. Five rabbits (nos. 3, 4, 5, 8 and 9 ) showed coughing and respiratory symptoms from 2.5 months after BLV inoculation. These animals were therefore treated with antibiotics. Three rabbits (nos. 4, 5 and 8 ) continued with severe respiratory symptoms for 3, 9 and 4 months, respectively, and no. 4 died of p n e u m o n i a 5.5 months after inoculation. Rabbits 5 and 8 had abcesses in the lungs at the time of dissection (Fig. 2c). At 17 months, six BLV-infected rabbits were tested for percentages of T cells and Ia antigen-positive cells by the IF test. The percentages of T cells in six infected rabbits were 50-70, similar to values in non-infected rabbits. On the contrary, the percentages of Ia-positive cells were 5-15 in four rabbits and 20-30 in the other 2, showing a slight decrease in the Ia-positive cells.

Humoral and blastogenic responses in infected rabbits Some BLV-infected rabbits showed fluctuating antibody titers but finally converted to antibody-negative. Since these rabbits showed continuous respiratory symptoms and weight loss, we tested whether the BLV-infected rabbits had immunodeficiency compared with the uninfected control rabbits. 5.5 months after BLV inoculation, six BLV-infected rabbits (nos. 1, 3, 5, 6, 7 and 8 ) and three non-infected control rabbits were injected with mouse serum protein to determine the humoral i m m u n e response. Serum antibody titers were determined by ELISA. As shown in Fig. 3, the mean highest antibody titers in control and BLV-infected groups were 4033 + 1350 and 1278 _+ 1110, respectively. Statistically significant differences in antibody titer were observed between the two groups from 3 to 13 weeks after antigen injection. Serum antibody titers in rabbit no. 5, which showed continuous respiratory

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s

4

01)

,p-

10.011 >~ 0

10.001)

-

10.001)

2

.0011

°

T 1

2

4

6

8

10

12

14

Time atler Immunlzallon Iweeks)

Fig. 3. Humoral immune responses against mouse serum protein in BLV-infected and non-infected rabbits by ELISA. Solid lines show the antibody titer (mean +_s.d. ) in BLV-infected ( • ) and control ( © ) rabbits. Dotted line shows the mean antibody titer of rabbit no. 5. The numbers in parentheses represent the respective P values in the Student's t test for the difference between the mean antibody titers for two groups at each interval.

symptoms, were lower than those in the other BLV-infected rabbits, and antibody could not be detected 7 weeks later. The mitogenic responses of PBL from BLV-infected rabbits were also tested at 9.5 months, and the Sis were compared with those of control groups. The mean SI _+s.d. for three control rabbits and seven BLV-infected rabbits (nos. 1, 2, 3, 5, 6, 7 and 8) were 123+86.1 and 44.3_+51.8, respectively ( P < 0 . 1 ) . Mitogen response were also tested at 17 months, when a similar suppression of blastogenic activity in the infected group was observed.

Pathologicalfindings at the time of necropsy Rabbit no. 4, which died of pneumonia, was necropsied. Alveolar hemorrhages and pulmonary thrombi were found in the lungs (Fig. 2d). Bordetella bronchiseptica was isolated from the pulmonary lesions. All other BLV-infected rabbits were killed 21 months after inoculation for histopathological observations. Six of nine BLV-infected rabbits had pulmonary lesions; the remaining three had no lesions in any tissues. On the contrary, of four noninfected control rabbits tested, one had pulmonary lesions and the other three had no lesions in any tissues. None of nine BLV-infected rabbits had histologically identifiable tumor tissue. DISCUSSION

In the present experiment, rabbits were inoculated with BLV-infected bovine lymphocytes or with FLK cells that were persistently infected with BLV;

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the rabbits were positive for antibodies against BLV 2 weeks after inoculation, and by 6 weeks all inoculated rabbits were positive. There was no direct correlation between the time of first seroconversion and the number of cells in the inoculum. BLV was detected in most of the seroconverted rabbits by 3 months after inoculation; however, there were few antigen-positive cells and syncytia compared with BLV-infected cattle and sheep. Thus we were unable to decide whether the antigen-producing cells (target cells) in rabbits were T or B cells. The reason why there were so few antigen-positive cells among the cultured PBL from BLV-infected rabbits is not yet clear. However, it is possible that BLV causes cell lysis in infected rabbit cells, and such an effect may prevent an increase in antigen-positive cells in long-term cultures. Although there were few antigen-positive cells, inoculation of lymphocytes ( 1 X 106/ animal) from BLV-infected rabbits resulted in the successful transmission of BLV infection to two noninfected 1-year-old rabbits. Interestingly, when the same number of rabbit lymphocytes from the same source was inoculated into four 5-day-old rabbits, none of these rabbits converted to seropositive. This may have been because of lower susceptibility to BLV infection in newborn rabbits. A similar difference in susceptibility to BLV infection in rabbits has been reported previously (Sentsui et al., 1988). Infection with retroviruses has been associated with various parameters of clinical immunosuppression in cats, mice and chickens (Dent, 1972; Cotter et al., 1975 ). Feline leukemia virus (FeLV) frequently causes death by predisposing the host to acute infections by other pathogens rather than by inducing leukemia (Essex et al., 1975 ). The humoral antibody response in FeLVinfected cats was both delayed and greatly reduced compared with that seen in uninfected control cats (Trainin et al., 1983). In BLV-induced leukemia in cattle and sheep, the natural cytotoxic activity of lymphocytes was significantly suppressed compared with that of healthy control animals and that of BLV-infected, but clinically healthy animals (Yamamoto et al., 1984). The humoral antibody response to various antigens, however, was not significantly altered in leukemia cattle (Ressang et al., 1980). In general, experimentally and naturally BLV-infected cattle and sheep do not show any immunodeficiency syndromes (Burny et al., 1986). In contrast, five BLVinfected rabbits showed respiratory symptoms that started 2.5 months after inoculation and continued for 3-6 months. Only one rabbit (no. 4), which lost weight, died of pneumonia; the other four rabbits lived for long periods. At the time of dissection, six of nine BLV-infected rabbits had lung lesions. Antibody titers against mouse serum in BLV-infected rabbits were found to be significantly depressed when compared with an uninfected control group. Furthermore, the mitogen response of PBL was also significantly depressed in infected rabbits. The present experiments did not clarify whether the suppression of immunological responses observed in BLV-infected rabbits was due to inoculation with BLV itself. Immunological suppression in BLV-in-

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fected rabbits may cause respiratory symptoms and lung lesions. Further studies are needed to confirm whether BLV infection causes immunosuppression and respiratory symptoms in rabbits. Rabbits are used as an animal model in the study of h u m a n T lymphotropic viruses (HTLV) such as HTLV-I and human immunodeficiency virus (HIV) infections (Miyoshi et al., 1983; Filice et al., 1988; Seto et al., 1988). Lymphocytes from rabbits infected with either HTLV-I or HIV produced more virus than lymphocytes from BLV-infected rabbits (Miyoshi et al., 1983; Seto et al., 1988 ). We attempted to use rabbits as laboratory animal models of BLV infection for the development of a vaccine against BLV. BLV-infected rabbits showed good antibody responses, but detection of BLV or the antigen was not consistent because of the low percentage of virus-producing cells among PBL from BLV-infected rabbits. Interestingly, since BLV-infected rabbits showed an immunodeficiency syndrome, BLV-infected rabbits might be a good animal model for AIDS in humans. The development of a similar i m m u n e dysfunction has been reported recently in BLV-infected rabbits (Wyatt et al., 1989). ACKNOWLEDGEMENTS

The authors are grateful to Miss M. Utsumi and Miss Y. Ogawa for assistance in the transmission experiments.

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Filice, G., Cereda, P.M. and Varnier, O.E., 1988. Infection of rabbits with human immunodeficiency virus. Nature (London), 335: 366-369. Hoss, H.E. and Olson, C., 1974. Infectivity of bovine C-type (leukemia) virus for sheep and goats. Am. J. Vet. Res., 35: 633-637. Jackson, S., Chused, T.M., Wilkinson, J.M., Leiserson, W.M. and Kindt, T.J., 1983. Differentiation antigens identify subpopulations of rabbit T and B lymphocytes. Definition by flow cytometry. J. Exp. Med., 157: 34-46. Lobel, S.A. and Knight, K.L., 1984. The role of rabbit la molecules in immune functions as determined with the use of an anti-la monoclonal antibody. Immunology, 51: 35-43. Mammerickx, M., Portetelle, D. and Burny, A., 1981. Experimental cross-transmission of bovine leukemia virus (BLV) between several animal species. Zentralbl. Veterinaermed, Reihe B., 28: 69-81. Miller, L.D., Miller, J.M. and Olson, C., 1972. Inoculation of calves with particles resembling C-type virus from cultures of bovine lymphosarcoma. J. Natl. Cancer Inst., 48: 423-428. Miyoshi, I., Yoshimoto, S., Taguchi, H., Kubonishi, I., Fujishita, M., Ohtsuki, Y., Shiraishi, Y. and Akagi, T., 1983. Transmission of rabbit lymphocytes with T-cell leukemia virus. Jpn. J. Cancer Res. (Gann), 74: 1-4. Odawara, T., Onuma, M., Yoshikawa, H., Yoshikawa, T. and Izawa, H., 1987. Circulating immune complex levels in cows with enzootic bovine leukosis. Jpn. J. Vet. Sci., 49: 697-702. Olson, C., Miller, L.D., Miller, J.M. and Gillette, K.G., 1970. Progress on transmission of bovine lymphosarcoma. In: Comparative Leukemia Research, 1969. Karger, Basel, Bibl. Haematol., 36: 476-492. Olson, C., Miller, J.M. and Hoss, H.E., 1972. Transmission of lymphosarcoma from cattle to sheep. J. Natl. Cancer Inst., 49: 1463-1467. Olsen, C., Kettmann, R., Burny, A. and Kaja, R., 1981. Goat lymphosarcoma from bovine leukemia virus. J. Natl. Cancer Inst., 67: 671-675. Onuma, M., Olson, C., Baumgartener, L.E. and Pearson, L.D., 1975. An ether-sensitive antigen associated with bovine leukemia virus infection. J. Natl. Cancer Inst., 55:1155-1158. Ressang, A.A., Rumawas, W., Rondhuis, P.R., Haagsma, J. and Bercovich, Z., 1980. Studies on bovine leukosis. VIII. The serologic response ofleukotic cattle to injections with Aujeszky's disease and Bordetella vaccines, tetanus toxoid and porcine erythrocytes. The cutaneous reaction following administration of Mycobacterium microti and the effect of pokeweed and phytohemagglutinin stimulation on leukotic lymphocytes. Zentralbl. Veterinaermed, Reihe B., 27: 576-588. Sentsui, H., Kono, Y., Itohara, S. and Ishino, S., 1988. Experimental infection of bovine leukemia virus in small laboratory animals. Jpn. J. Vet. Sci., 50: 1245-1251. Seto, A., Kawanishi, M., Matsuda, S., Ogawa, K. and Miyoshi, I., 1988. Adult T cell leukemialike disease experimentally induced in rabbits. Jpn. J. Cancer Res. (Gann), 79: 335-341. Trainin, Z., Wernicke, D., Ungar-Waron, H. and Essex, M., 1983. Suppression &the humoral antibody response in natural retrovirus infections. Science, 220: 858-859. Van Der Maaten, M.J. and Miller, J.M., 1976. Serological evidence of transmission of bovine leukemia virus to chimpanzees. Vet. Microbiol., 1: 351-357. Wyatt, C.R., Wingett, D., White, J.S., Buck, C.D., Knowles, D., Reeves, R. and Magnuson, N.S., 1989. Persistent infection of rabbits with bovine leukemia virus associated with development of immune dysfunction. J. Virol., 63: 4498-4506. Yamamoto, S., Onuma, M., Kodama, H., Mikami, T. and Izawa, H., 1984. Suppression of natural cytotoxic activity of lymphocytes from cattle and sheep during the progress of bovine leukosis. Vet. Microbiol., 9:105-111.

Suppression of immunological responses in rabbits experimentally infected with bovine leukemia virus.

Ten 2- to 4-month-old rabbits were inoculated subcutaneously with bovine leukemia virus (BLV)-infected bovine or sheep cells. By 6 weeks after inocula...
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