Int. J . Cancer:38, 139-143 (1986) 0 1986 Alan R. Liss, Inc.

RESISTANCE OF LINE 6 3 CHICKENS TO RETICULOENDOTHELIOSIS- VIRUS-INDUCED BURSA-ASSOCIATED LYMPHOMAS Aly M. FADLY and Richard L. WITTER US Department of Agriculture, Agricultural Research Service, Regional Poultry Research Laboratory, 3606 East Mount Hope Road, East Lansing, MI 48823, USA.

Chickens of lines 63 and 1515 x 7, were inoculated with the chick syncytial strain of reticuloendotheliosis virus (REV) or with the Rous-associated virus-I of avian leukosis virus (ALV) at hatching. At 4, 10, 16, and 36 weeks post inoculation (PI), chickens were tested for REV- and ALV-induced viremia and antibody. The in- cidence of REV- or ALV-induced bursa-associated lym- phomas in line 63 chickens was compared with that in line 1515 x 7, chickens. Inoculation of REV at hatching resulted in immunological tolerance to the virus in line 63 but not in line 1515 x 7, chickens. Between 70% and 100% of line 63 chickens remained viremic and lacked REV antibody throughout the experimental period of 36 weeks. In contrast, ALV-inoculated chick- ens of both lines had antibody by 16 weeks PI. The frequencies of REV- and ALV-induced bursa-associated lymphomas in line 63 chickens were significantly lower than in line 1515 x 7, chickens. Further, the incidence of bursa-associated lymphomas induced by REV in line 15t5 x 7, chickens was significantly lower than that induced by ALV. These results suggest that: (I) the genetic constitution of the host may influence the immunological response to REV infection; (2) chickens resistant to ALV-induced bursa-associated lymphomas are equally resistant to such lymphomas induced by another unrelated avian retrovirus, REV; and (3) ALV is a more potent inducer of bursa-associated lympho- mas than REV.

Reticuloendotheliosis virus (REV), an avian retro- virus distinguishable from all members of the avian leukosis-sarcoma group (Purchase et al., 1973; Pur- chase and Witter, 1975; Witter, 1984), was originally isolated from turkeys in 1958 (Robinson and Twie- haus, 1974). Natural outbreaks of REV-associated neoplastic disease in ducks, turkeys and quail have been reported (Grimes and Purchase, 1973; Carlson et al., 1974; Sarma et al., 1975; Paul et a l . , 1977; Solo- mon et al., 1976; Schat et al., 1976; McDougall et al., 1978; Li et al., 1983; Witter and Glass, 1984). Such neoplastic disease has not frequently been recognized in chicken flocks, although a transmissible tumor as- sociated with REV infection has been described (Rat- namohan et al., 1980). Further, virological and sero- logical evidence for REV infection in chickens has been documented (Aulisio and Shelokov, 1969; Witter et al . , 1982; Witter, 1984).

Depending on the strain of virus, inoculation of REV in chickens can induce diverse clinical and pathologi- cal manifestations. Replication-defective, transform- ing REV strain T (Hoeltzer et al., 1979) can cause acute neoplasia of the reticuloendothelial system, whereas non-defective strains can cause an immuno- suppressive runting syndrome characterized by abnor- mal feathering, enlarged nerves and atrophy of the bursa and thymus (Witter et al., 1970; Mussman and Twiehaus, 1971; Kawamura et al., 1976). These non- defective REV strains were only recently also shown to be capable of inducing chronic lymphoid neoplasia in chickens (Witter and Crittenden, 1979; Grimes et

al., 1979). These lymphomas resemble those induced by avian leukosis virus (ALV) as regards organ distri- bution, latency, surface IgM (Witter and Crittenden, 1979; Nazerian et al . , 1982), alteration of host onco- gene c-myc (Noori-Dalloii et al., 1981), and bursa dependency (Fadly and Witter, 1983). Most recently, non-bursa1 visceral lymphomas accompanied by en- larged nerves were observed in chickens inoculated with non-defective REV (data not shown). These lym- phomas are characterized by absence of bursa1 lesions, and by a latent period of 6-36 weeks. Further, the incidence of these non-bursa1 lymphomas was influ- enced by line of chickens and strain of virus.

Differences between chicken lines in susceptibility to ALV-induced lymphomas have been described (Crittenden et al., 1972; Purchase and Gilmour, 1975; Baba and Humpheries, 1984). Further, differences be- tween chicken lines in mortality and lesion responses, induced presumably by defective REV strain T, have been described (Scofield et al., 1978). However, resis- tance of chicken lines to REV infection or REV-in- duced bursa-associated lymphomas has not been documented. This report describes differences be- tween two chicken lines, 63 and 1515 X 71, in their susceptibility to REV-induced viremia, antibody, and bursa-associated lymphomas. It also compares the re- sponse of these two lines of chickens to REV and ALV infection.

MATERIAL AND METHODS

Chickens Chickens were from line 6, subline 3 (63) of the

Regional Poultry Research Laboratory (RPRL). Chick- ens of this line are known to be highly susceptible to ALV infection, but relatively resistant to ALV-induced lymphomas (Purchase and Gilmour, 1975). Progeny of a cross between males of line 151, subline 5 (1515) and females of line 7, subline 1 (7,) of the RPRL were also used. The cross is highly susceptible to ALV- and REV-induced viremia and lymphomas (Crittenden et al., 1972; Purchase and Gilmour, 1975; Witter and Crittenden, 1979; Witter et al., 1981; Fadly and Wit- ter, 1983). The breeder flocks are maintained in isola- tion and are free from many avian pathogens including REV and subgroups A, B, C and D of ALV as deter- mined by routine tests for the respective antibodies. All experimental chickens were maintained in plastic- canopy isolators with filtered air under positive pres- sure from hatching until the end of the experiment.

Received: November 18, 1985, and in revised form February 10, 1986

140 FADLY AND WITTER

Viruses The chick syncytial (CS) strain of REV, a non-

defective strain (Cook, 1969), was used. The virus stock was previously clone-purified in chicken embryo fibroblasts and is known to be free of exogenous ALV (Witter and Crittenden, 1979). The Rous-associated virus-I (RAV-1) (Hanafusa, 1965), a subgroup A ALV, was propagated and titrated in C/E chicken embryo fibroblasts (Crittenden et al., 1984). REV and ALV were given intra-abdominally at 1 day of age. Each chick received lo4 infectious units of the respective virus. All chickens were vaccinated with the turkey herpesvirus at 1 day of age for protection against MD (Marek’s disease).

Virus and anrihody assays Plasma samples were assayed for REV and antibody

by the complement-fixation avian reticuloendotheliosis (COFAR) (Smith et al., 1977) and immunofluores- cence tests (Witter et al., 1970), respectively. The samples were also assayed for ALV by the comple- ment-fixation test for avian leukosis (COFAL) (Sarma et al., 1964; Smith, 1977) and for ALV antibody by the focus-reduction test (Ishizaki and Vogt, 1966).

Experimental design One-day-old chicks from lines 63 and 151s X 71

were divided into two groups per line; each group contained 100 chicks. Chicks in group 1 of each line were inoculated with the CS strain of REV and chicks in group 2 were inoculated with the RAV-1 strain of ALV. At 4, 10, and 16 weeks PI, plasma samples from 10 chickens from each group were tested for both REV and ALV and their respective antibodies. The chickens were killed and bursa of Fabricius, spleen and thymus were collected for histological evaluation. The remain- ing chickens in all groups were observed for lymphoma development for 36 weeks. At termination, plasma samples from representative chickens of all groups were also tested for both viruses and their respective antibodies.

Pathology All chickens that died during the experiment and

those that were killed at various intervals PI and at termination were autopsied. Lymphomas including those with bursal involvement were diagnosed on the basis of gross lesions or microscopic examination of tissue preparations stained with hematoxylin and eosin.

Statistical analysis Significance of differences in percentages was deter-

mined by Chi-square analysis. Statistical significance was determined at the 0.05 level of probability.

RESULTS

Virology and serology Tables I and I1 show viremia and antibody responses

of chickens inoculated with REV or ALV, respec- tively. At 4, 10, 16 and 36 weeks PI, the rates of REV- induced viremia in line 63 chickens were significantly (p < 0.05) higher than those in line 1515 X 71 chickens (Table I) . Further, the rates of antibody detection in line 63 chickens were significantly (p < 0.05) lower than those in line 1515 X 71 chickens. The rates of REV antibody in line 1515 X 71 chickens varied from 67% to loo%, compared to 0%-22% in line 63 chick-

ens. Thus, tolerant infection, defined here as persistent viremia with absence of antibody, was detected in REV-infected line 63 chickens, but not in line 1515 X 7] chickens.

In contrast, inoculation of chickens with ALV at hatching failed to induce tolerant infection in either line (Table 11). By 16 weeks PI, ALV antibody was detected in 100% of chickens tested. No differences were detected between the two lines of chickens in rate of ALV-induced viremia. None of the chickens tested from the groups inoculated with REV showed any evidence of ALV infection and, similarly, none of the chickens tested from the groups inoculated with ALV showed evidence of REV infection (data not shown). Thus, there was no evidence of accidental exposure of chickens to either virus.

Histogenesis of REV- and ALV-induced lymphomas Sequential gross and microscopic evaluation of lym-

phoid organs collected at 4, 10 and 16 weeks PI re- vealed no evidence of lymphoma formation in the spleen and thymus of chickens tested from any group. However, by 16 weeks PI, gross bursal lymphomas were detected in line 151s X 71, but not in line 63 chickens, regardless of the virus used (Table 111). Mi- croscopic evidence of transformation of bursal follicles became apparent earlier in ALV-inoculated than in REV-inoculated line 151s x 71 chickens. At 4 and 10 week PI, 1 of 10 chickens tested from REV-inoculated line 63 chickens had evidence of lymphomas in the heart, proventriculus and nerves.

Incidence of REV- and ALV-induced lymphomas Data in Table IV show the incidence of lymphomas

and other neoplasms induced in line 63 and 1515 X 71 chickens by inoculation of REV or ALV at hatching. The incidence of both REV- and ALV-induced lym- phomas in line 63 was significantly (p < 0.005) lower than that in line 1515 X 71 chickens. Only 16% of line 63 chickens inoculated with either REV or ALV died with lymphomas or had lymphomas at termination. Further, none of the line 63 chickens that developed REV-induced lymphoma showed evidence of bursal involvement. In contrast, 6 of 7 chickens (86%) of this line that developed ALV-induced lymphomas showed evidence of bursal involvement. Most of the line 1515 x 71 chickens that developed REV- or ALV-induced lymphomas had evidence of bursal involvement (79 % , REV-inoculated group; 96 % , ALV-inoculated group). Interestingly, the rate of REV-induced bursa-associ- ated lymphomas in line 1515 X 71 chickens was signif- icantly (p < 0.005) lower than that induced by ALV (53% vs. 98%). Five of 8 chickens from line 63 that developed REV-induced lymphomas had gross or mi- croscopic evidence of nerve involvement. This type of lesion was not detected in line 63 chickens inoculated with ALV.

DISCUSSION

Data from this study clearly show that the two lines of chickens, RPRL 63 and 1515 X 7,, responded dif- ferently to REV infection with regard to lesion inci- dence and development of viremia and antibody. Further, the results suggest that line 63 chickens, which are known to be relatively resistant to ALV-induced bursal lymphomas (Purchase and Gilmour, 1975), are also resistant to such bursal lymphomas induced by

BURSA-ASSOCIATED LYMPHOMA RESISTANCE A N D REV 14 1

TABLE I ~ RETICULOENDOTHELIOSIS VIRUS (REV) AND ANTIBODY IN LINE 6, AND 1.51, x 7 , CHICKENS AT VARIOUS INTERVALS POST INOC1JI.ATION'

Age al testing (weeks) Chicken line

4

10

Viremia number Antibody number poa.1 p 0 s . l

nuniher rested' ( W j numbcr trstcd' t % j

63 ioiio 0/10 (O)** 15ISX7, o/ 10 (0) lo/ 10 ( 100) 63 lO / lO (loo)** 0/10 (O)** 1515x7, o/ 10 (0) 101 10 (100)

16 63 8/10 (80)** 1/10 (lo)** 1515x7, o/ 10 (0) 10/10 (loo)

36 63 7/10 (70)* 2/ 10 (20)" 15IsX7, 219 (22) 6/9 (67)

'Each chick was inoculated intra-abdominally at hatching with lo4 infectious units of the chick syncytial strain of REV.-'Samples of whole blood were assayed for REV by the complement-fixation avian reticuloendotheliosis (COFAR) test which detects REV group-specific antigen in inoculated chicken embryo lihrohlasts.~'Indirect immunoiluorcscence test on pla~ma.-~Aaterisks indicate significant difference by Chl-square analysis compared with 1 S 1 s ~ 7 1 chickens (* = p < 0.05: * * = p < 0.005).

TABLE I1 - AVIAN LEUKOSIS VIRUS (ALV) AND ANTIBODY IN LINE 63,AND 1515 x7, CHICKENS AT VARIOUS INTERVALS POST INOCULATION

Age at testing (weeks) Chicken line

4

10

16

Viremia number p0s.i

nuniberitested' (%)

Antibody number pos.1

number tested' (%)

63 9/ 10 (90)4 0110 (0) t51SX7, 101 10 (too) 0/10 (0)

63 4/10 (40) 10/10 (loo) 1515x71 2/10 (20) 10/10 (loo)

63 4/10 (40) 6/10 (60) 1515x71 3/10 (30) 8/ 10 (80)

36 63 5110 (50) 10/ 10 ('002 1515x71 011 (0)5 111 (loo)

'Each chick was inoculated intra-abdominally at hatching with lo4 infectious units of the Rous-associated virus- I (RAV-I) of ALV.-'Samples of whole blood were assayed for ALV by the complement-fixation avian leukosis test (COFAL) which detects ALV group-specific antigen in inoculated chicken embryo fibroblasts.-'Samples of plasma were assayed for ALV antibody by the focus-reduction te~t.-~Differences between the 2 lines within the same time interval were not significant by Chi-square analysis.-'One chicken survived to 36 weeks of age.

TABLE 111 - BURSAL LYMPHOMAS IN LINE 6, AND 1515 x7, CHICKENS AT VARIOUS INTERVALS POST INOCULATION WITH RETICULOENDOTHELIO~IS VIRUS (REV) OR AVIAN

LEUKOSIS VIRUS (ALV) AT HATCHING

Lymphoma incidence VlNS Chicken line number pxinumber tested Age at testing

(weeks) Gross Microscopic'

4 REV 63 01 10 o/ 10 1515 X 71 o/ 10 o/ to

ALV 63 o/ 10 o/ 10 1515x7, o/ 10 0/10

10 REV 63 01 10 0110 151s x 7, Of 10 o/ 10

ALV 63 Of 10 01 10*

16 REV 63 0110 Oi 10* 1515x71 01 10 4/ 10

1515x7, 3/10 4/10

1515x71 21 10 6/ 10 ALV 63 o/ 10 0/10**

'Each chick was inoculated intra-abdominal1 with lo4 infectious units of the chick syncytial strain of REV or

indicate significant difference by Chi-square analysis compared with 1515 x 7, chickens within the SdmC inoculum group at the same time interval (* = p < 0.05; ** = p 4 0.005).

the Rous-associated virus- I (RAV- 1) of ALV.- Y Formalin-fixed, hematoxylin-eosin-stained preparations; asterisks

142 FADLY AND WITTER

TABLE IV - INCIDENCE OF LYMPHOMA IN LINE 63 AND 151,X7, CHICKENS INOCULATED WITH RETICULOENDOTHELIOSIS VIRUS (REV) OR AVIAN LEUKOSIS VIRUS (ALV) AT HATCHING

Number dying with Number with Frequency of neoplasms other

than Ivmohomas3 number at risk4 (5%) Ivmohornas' 1 y rn p h o m a I huraal Number inoculated

Virus Chicken line a! hatching2

REV 6, 100 0 8/49 (16)6,a 0" 1515 X71 100 1 33/62 (53)b 79b

ALV 6 3 100 3 7145 (16)' 86b 1515 X7, 100 3 49/50 (98)' 96h

'Each chick was inoculated intra-abdominally with lo4 infectious units of the chick syncytial strain of REV or the Rous-asociated virus-I atrain of ALV.-'At 4 , 10. and 16 weeks post inoculation, 10 chickens were killed; blood, bursa, thymu3 and spleen were collected.-'Chickens that died with neoplasms other than lymphoma such as erythroblastosis and hernangi~mas.-~Number of chickens at risk = number that survived to termination at 36 weeks ofagc + number that died with lymphonia.-5Percent of lymphoma-bearing chickens that had hursal lymphoma~.-~Values followed by a different letter within the Same column are significantly (p < 0.005) different.

another unrelated avian retrovirus, REV. Differences between lines of chickens in susceptibility to ALV- or MD-induced lymphomas have been reported (Critten- den et al., 1972; Purchase and Gilmour, 1975; Baba and Humpheries, 1984). Further, Scofield et al. (1978) reported that the MD-resistant N line was more sus- ceptible than the MD-susceptible P line to acute neo- plasia induced by REV stocks that contained both defective and non-defective viruses.

Data from virological and serological assays show that chickens of both lines were susceptible to REV infection. However, line 63 chickens were more sus- ceptible to REV-induced tolerant infection than line 1515 x 7, chickens following inoculation with virus at hatching. Infection of embryos with non-defective strains of REV regularly induces tolerant infection (Ianconescu and Aharonvici, 1978; Witter et al., 1981), whereas infection at hatching of line 1515 X 71 chick- ens rarely induces tolerant infection (Witter, 1984). Results from this study suggest that the genetic consti- tution of the host may influence the serological re- sponse to REV, but not to ALV. The high susceptibility of line 63 chickens to REV-induced tolerant infection appears to be unrelated to the immunosuppressive ef- fects of REV, since the virus did not induce tolerant infection in line 1515 X 71 chickens. Further, chickens inoculated with the CS strain of REV at hatching mount an immune response against particulate antigens comparable to that of control uninfected chickens (Witter et al., 1981). It is likely that this REV-induced tolerant infection in line 63 chickens is the result of a specific interaction between virus and line of chickens. The serological response of chickens of both lines to ALV infection was similar to that reported previously (Purchase and Payne, 1984).

Results from sequential gross and microscopic ex- amination of lymphoid tissues indicate that the inci- dence of transformed bursal follicles during the early stages of lymphomagenesis correlates with the suscep- tibility of chickens to REV- or ALV-induced lympho- mas. In this experiment, none of the line 63 chickens examined had gross or microscopic evidence for trans- formed bursal follicles by 16 weeks PI. In contrast, 20%-30% and 40%-60% of chickens tested from line 1515 X 71 had gross and microscopic bursal lympho- mas, respectively. Baba and Humpheries (1985) de- tected transformed bursal follicles in 82% and in ll % of chickens that had been characterized as susceptible and resistant, respectively, to ALV-induced lympho- mas. The finding that transformed bursal follicles were

detected as early as 10 weeks PI in ALV-inoculated and at 16 weeks PI in REV-inoculated line 1515 X 71 chickens suggests that lymphomas induced by ALV in susceptible chickens develop faster than those induced by REV. The latent period for REV-induced bursa- associated lymphomas in susceptible chickens is rela- tively longer than that induced by ALV (Fadly er al., 1981 ; Fadly and Witter, 1983).

Although both lines of chickens were susceptible to REV infection, there were significant differences in the incidence of lesions and in the frequency of bursal involvement. Only 8 of 49 (16%) of line 63 chickens inoculated with REV at hatching developed lympho- mas by 36 weeks; none of these 8 chickens had evi- dence of bursal involvement upon gross or microscopic examination. These non-bursa1 lymphomas induced by REV in line 63 are considered to be unique. In con- trast, 33 of 62 (53%) of REV-inoculated line 1515 X 71 chickens developed lymphomas; 26 of these 33 chickens (79%) had evidence of bursal involvement. Differences between lines of chickens in their suscep- tibility to REV-induced bursa-associated lymphomas have not been described previously. Whether this re- sistance of line 63 chickens to REV-induced bursa- associated lymphomas is related to their unique sus- ceptibility to virus-induced tolerant infection is not known. Witter et a/. (1981) reported that line 1515 X 7 I chickens non-tolerantly infected with REV (inocu- lated at 1 day of age), had a significantly higher inci- dence of lymphomas than those that were tolerantly infected with the virus (inoculated as embryos). This contrasts with observations on ALV-induced lym- phomagenesis, since chickens tolerantly infected with ALV usually have a higher incidence of lymphomas than non-tolerantly-infected chickens (Purchase and Payne, 1984).

Finally, our results show that a line of chickens, characterized as resistant to ALV-induced bursal lym- phomas, was equally resistant to such bursal lympho- mas induced by another unrelated avian retrovirus, REV. The molecular mechanism involved in bursa cell transformation by REV is similar to that induced by ALV (Hayward et al., 1981; Noori-Dalloii et a]., 1981). However, the molecular basis for resistance of chickens to lymphoma development has not been cleariy defined.

ACKNOWLEDGEMENTS

We thank Mr. R . Brown and Ms. B. Riegle for ca- pable technical assistance.

BURSA-ASSOCIATED LYMPHOMA RESISTANCE AND REV

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143

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