Vol. 40, No. 2INFECTION AND IMMUNITY, May 1983, p. 795-8050019-9567/83/050795-11$02.00/0Copyright C 1983, American Society for Microbiology

Rapid Induction of Hypothyroidism by an Avian LeukosisVirus

JEANNE K. CARTER AND R. E. SMITHt*

Department of Microbiology and Immunology, Duke University Medical Center, Durham, North Carolina27710

Received 12 October 1982/Accepted 25 January 1983

Infection of 10-day chicken embryos with an avian leukosis virus, RAV-7,resulted in hypothyroidism within 3 weeks posthatching. Histological examinationof the thyroids from infected chickens showed an extensive infiltration oflymphoblastoid cells by 7 days posthatching. Areas resembling germinal centerswere present in the thyroids of infected chickens by 3 weeks posthatching.Examination of circulating thyroid and pancreas hormones showed a significantreduction in T3 and T4 levels and a trend toward higher insulin levels after 16 daysposthatching. T4 supplementation of RAV-7-infected chickens alleviated some

aspects of the disease syndrome but did not abrogate all symptoms. Markedinvolution of both bursa and thymus glands was noted. RAV-7 had an RNAgenome of 8.2 kilobases and a polypeptide composition characteristic of an avianleukosis virus. The hypothyroidism followed a dose response to RAV-7 infection.

Avian retroviruses have long been used toprovide model systems for tumor formation.Induction of tumors in chickens by these virusesis either an acute response induced by viruseswhich carry their own oncogene, such as sarco-ma viruses or defective leukemia viruses, or achronic response induced by replication-compe-tent viruses which carry no detectable oncogene(14). The latter group of viruses, the avianleukosis viruses, induce frankly neoplasticgrowths, such as B-cell lymphomas (4), prolifer-ative disorders, such as osteopetrosis (30), andchronic degenerative diseases, such as anemia(25) and immunosuppression (34). Recent atten-tion has been focused on the chronic debilitativediseases, since they may represent hitherto un-recognized consequences of retrovirus infectionand provide model systems for diseases of hu-mans (35).

Chronic debilitation and stunting in animalscan be the result of metabolic or endocrinedysfunction, one example of which is obesityand hyperlipidemia in chickens. One modelwhich has received considerable attention isspontaneous autoimmune thyroiditis in chickens(8, 37). In this model, obesity and lipemia aredue to hypothyroidism which is caused by lym-phoid infiltration of the thyroid gland (38). Asecond metabolic disease in the chicken is thefatty liver and kidney syndrome observed inbroiler chickens at 10 to 30 days of age (20). This

t Present address: Department of Microbiology and Envi-ronmental Health, Colorado State University, Ft. Collins, CO80523.

disorder is characterized by fat deposition in theliver and kidney, hypoglycemia, reduced hepaticglycogenesis, and reduced hepatic glycogen (3,7). Fatty liver and kidney syndrome is believedto be associated with biotin deficiency (3), andsome features of the syndrome are induced inolder chickens by aflatoxin (17). A third disor-der, which remains uncharacterized, is the in-duction of lipemia in chicken embryos by Japa-nese encephalitis virus (16). In this disease,Japanese encephalitis virus appears to suppresslipid metabolic enzymes, and the circulatinglipid reflects the high lipid content of the yolk(16).We have discovered that Rous-associated vi-

rus 7 (RAV-7) causes a syndrome characterizedby hyperlipidemia and obesity in chickens (8).When RAV-7 is injected into 10-day-old embry-os, chickens show a frank lipemia and fatty liverby 21 days posthatching. The obese and stuntedsyndrome occurs at high frequency and withhigh mortality. Chickens which survive an initialhigh mortality exhibit a low incidence of lym-phoid leukosis, osteopetrosis, and nephroblas-toma (8). Only one previous report of RAV-7disease induction has been published (17), andRAV-7 is reported to cause a low incidence oflymphoid leukosis and other neoplasias wheninjected into hatched chickens (17).The purpose of the present study was to

examine the role of the thyroid and pancreas inthe RAV-7-induced disease syndrome. We ex-amined histological sections of the thyroid andpancreas and levels of circulating thyroid andpancreas hormones of RAV-7-infected chickens

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INFECT. IMMUN.796 CARTER AND SMITH

FIG. 1. Histological sections of thyroid glands from RAV-7-infected and uninfected chickens. Thyroid glandswere treated as described in the text. All sections were stained with hematoxylin and eosin. Bar, 30 p.m. (A)Thyroid from an uninfected chicken 2 days posthatching. The follicular epithelial cells are squamous, and thecolloid appears uniform. (B) Thyroid tissue from a RAV-7-infected chicken 2 days posthatching. The follicularepithelial cells, in some areas, show an increased height, and the colloid is pale and not uniform in appearance.(C) Thyroid tissue from a RAV-7-infected chicken 7 days posthatching. The follicular epithelial cells arecolumnar and contain numerous large vacuoles. The colloid is not uniform in appearance. Lymphoblastoid cellswith large nuclei and prominent nucleoli are infiltrating the tissue replacing the normal thyroid structure.

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RAV-7-INDUCED HYPOTHYROIDISM 797

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and uninfected chickens during the developmentof the disease syndrome. We show that RAV-7caused endocrine changes which accounted forthe hyperlipidemia and stunting observed ininfected chickens.

MATERIALS AND METHODSAbbreviations. RAV-7, Rous-associated virus 7;

MAV-2(0), myeloblastosis-associated virus of sub-group B inducing predominantly osteopetrosis; SAT,spontaneous autoimmune thyroiditis; OS, obese strainof chickens from the Cornell C strain; T3, triiodothy-roxine; T4, thyroxine.

Virus. RAV-7 was initially isolated from a stock ofBryan high-titer Rous sarcoma virus (14). The RAV-7used for this study was obtained from C. Moscovici,Veterans Administration Hospital, Gainesville, Fla.,and was grown in chicken embryo fibroblasts. Thevirus used in this study was biologically purified bytwo rounds of end-point purification on C/O chickenembryo fibroblasts and was stored at -70°C. RNAsizing and protein electrophoresis procedures werepreviously reported (33).

Chickens. Fertile eggs from the experimental inbredSC white leghorn line were obtained from HylineInternational, Dallas Center, Iowa. Ten-day-old chick-en embryos were inoculated by intravenous injectionof a chorioallantoic vein (1) with 104 infectious units ofRAV-7. After infection as embryos, chickens hatchedwith an efficiency of about 90%. Infected chickenswere hatched and reared in an isolation room of theAnimal Laboratory Isolation Facility of the DukeUniversity Comprehensive Cancer Center. Uninfectedchickens were hatched and reared in a separate roomof the same facility.

Histological examination. At selected times during

the course of the disease, chickens were sacrificed byintravenous administration of pentobarbital. Tissuespecimens were collected immediately and fixed in10% buffered formalin, dehydrated, embedded, sec-tioned, and stained with hematoxylin and eosin ormethyl green pyronin, following conventional proce-dures. Tissue specimens were collected from uninfect-ed hatchmates of RAV-7-infected chickens and wereprocessed identically. All histological sections wereprepared by Experimental Pathology Laboratories,Inc., Raleigh, N.C.Hormone assays. Circulating T3, T4, insulin, and

glucagon levels were determined, using radio-immunoassay kits (Cambridge Medical Diagnostics,Inc., Billerica, Mass.). Samples were collected follow-ing the protocols provided with the kits. All chickenswere fasted overnight before sample collection. Bloodsamples for glucagon assays were collected by jugularvenipuncture into sodium-EDTA (14 mg/10 ml ofblood). The samples were immediately placed on ice,and the protease inhibitor aprotinin was added at 1mg/10 ml of blood; samples remained on ice untilplasma was separated by centrifuging at 2,000 rpm for10 min. An ultracentrifugation step of 70,000 x g for60 min was performed on all samples to remove lipid,since many samples were visibly lipemic. Samples notassayed on the day of collection were frozen andstored at -20°C. Assays for chickens 2 weeks post-hatching or younger were run on pooled samples ofequal volume from 2 to 3 chickens. All samples wererun in duplicate. Samples from uninfected age-matched controls were collected, processed, andstored identically and were run simultaneously withsamples from infected chickens.T4 supplementation. Age-matched chickens, either

infected with RAV-7 as 10-day-old embryos or unin-fected, were separated into four groups. The groups

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798 CARTER AND SMITH

were divided as follows: group 1, eight uninfectedchickens which received saline; group 2, six uninfect-ed chickens which received T4; group 3, eight RAV-7-infected chickens which received saline; and group 4,five RAV-7-infected chickens which received T4.Chickens were given either 4 ,ug of Synthroid (levothy-roxine sodium; Flint Laboratories, Deerfield, Ill.) dis-solved in 0.9% NaCl (pH 10) per 100 g of body weightor an equivalent volume of 0.9% NaCl (pH 10) on adaily basis via subcutaneous injection. The chickenswere weighed weekly, and dosage was modified toadjust for weight gain. At 31 days posthatching, allchickens were sacrificed, serum samples were taken todetermine circulating hormone levels, and body andliver weights were taken and tissue samples wereobtained for histological examination.

Clinical chemistry. Serum triglycerides and choles-terol levels were determined by procedures previouslyreported (8).

RESULTS

Histology. Preliminary evidence for thyroidinvolvement indicated a need to examine thesequence and extent of thyroid alteration.Therefore, the first experiment was designed toexamine thyroid tissue at various times duringRAV-7 infection. Thyroids of RAV-7-infectedchickens taken at 2 days posthatching showedslight alteration, with the appearance of in-creased activity of the follicular epithelial cells(Fig. 1). The follicular colloid in the thyroid ofRAV-7-infected chickens at 2 days posthatching(Fig. 1B) did not appear as uniform as thefollicular colloid in the thyroids from uninfectedhatchmates (Fig. 1A). By 7 days posthatching,approximately 50% of the normal thyroid tissuewas replaced by lymphoblastoid cells in RAV-7-infected chickens (Fig. IC). As the disease syn-

drome progressed, the infiltration obliteratedmost of the normal thyroid tissue. Areas resem-

bling germinal centers were noted by 3 weeksposthatching, and by 5 weeks posthatching, ger-

minal centers were prominent (Fig. 2B). Follicu-lar epithelial cells late in infection were colum-nar to cuboidal, which indicated increasedactivity in these cells. Cells infiltrating the thy-roid appeared to be immature members of thelymphoid series. The nuclei of the infiltratingcells were large, with prominent nucleoli, andcytoplasms were slightly eosinophilic. Few plas-ma cells were noted. At times, the thyroid-thymus barrier was indistinct. Lymphoblastoidcells of similar morphology were observed toinfiltrate the pancreases of RAV-7-infectedchickens, but the infiltration was localized, andnormal pancreatic tissue remained throughoutthe disease process.

Hormone assays. The morphological evidencepresented above suggested that the function ofthe thyroid and pancreas might be altered. Toestablish whether a functional alteration was

present in these organs, we determined circulat-ing levels of T4, T3, insulin, and glucagon on 19pairs of RAV-7-infected and uninfected hatch-mates over a 43-day period. The most dramaticeffect was on the level of T4 (Fig. 3). During thefirst 2 weeks, no difference was noted in T4levels, but subsequent samples showed that T4levels in RAV-7-infected chickens were signifi-cantly lower than in uninfected chickens (P <0.001); by 40 days posthatching, the T4 level in aRAV-7-infected chicken was 19% that of a nor-mal hatchmate (Fig. 3). The levels of circulatingT3 were also decreased after 14 days (Fig. 4), butthe decrease was not as pronounced (P < 0.01).

Insulin levels were above normal levelsthrough 43 days posthatching, and ranged from114 to 352% of normal. Exceptions to the highinsulin levels were the 16-, 30-, and 37-daysamples, which were below normal. Glucagonlevels remained close to normal, except at 20 to30 days posthatching when the levels dropped to34 to 44% of normal (Table 1). It is interesting tonote that 20 to 30 days after hatching was thetime of highest mortality (data not shown). Al-though trends were apparent with the glucagonand insulin levels, the values were not signifi-cantly different for the RAV-7-infected chick-ens. By 8 days posthatching, RAV-7-infectedchickens were stunted, as indicated by a bodyweight 78% of normal. The body weights re-mained below normal and were 34.7% of normalat 43 days posthatching (Table 1).T4 supplementation. To examine the role of

hypothyroidism in the RAV-7 disease syn-drome, the effect of T4 supplementation wasexamined. The results of this study are present-ed in Table 2. T4 supplementation led to in-creased body weight, decreased liver weightexpressed as percentage of body weight, in-creased T3 and T4 levels, and decreased insulinlevels in T4-treated RAV-7-infected chickens(group 4) versus saline-treated RAV-7-infectedchickens (group 3). Group 3 showed highlysignificant differences (P < 0.01) in body weight,liver weight expressed as percentage of bodyweight, and T3 and T4 values when comparedwith the saline-treated uninfected chickens(group 1). All parameters except body weightand liver weight expressed as percentage ofbody weight showed a shift toward normal,which was reflected by a reduced level of signifi-cance between groups 1 and 4 (Table 2). Bodyweight, liver weight expressed as percentage ofbody weight, and T4, insulin, and glucagon lev-els were significantly different (P < 0.05) be-tween the two RAV-7 groups, indicating a shifttoward normal with T4 supplementation (Table2). The uninfected chickens which received T4(group 2) had reduced T3 and T4 levels. Gluca-gon levels were decreased with T4 supplementa-

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RAV-7-INDUCED HYPOTHYROIDISM 799

FIG. 2. Histological sections of thyroid glands from RAV-7-infected and-uninfected chickens at 5 weeksposthatching. Thyroids were treated as described in the legend to Fig. 1. Bar, 75 ,um. (A) Thyroid tissue from anuninfected chicken. The follicles are well defined, with uniform colloid and squamous epithelial cells. (B)Thyroid tissue from a RAV-7-infected chicken. The organ is extensively infiltrated by lymphoblastoid cells, andthe formation of areas resembling germinal centers is apparent (arrows). The follicles which remain intact appearto be normal and highly active.

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800 CARTER AND SMITH

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2000O

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l0 20 30Age (days)

40 50

FIG. 3. Levels of T4 in RAV-7-infected and unin-fected chickens. Hormone assays were performed asdescribed in the text on infected and uninfected chick-ens of the same age during a 43-day observationperiod. Values for RAV-7-infected chickens are shownby shaded bars, whereas values for uninfected chick-ens are shown by open bars. Each determination wasperformed in duplicate, and each bar represents aninfected-uninfected chicken comparison.

tion regardless of whether the group of chickenswas infected with RAV-7.

Virus. Rapid onset of disease induced byavian retroviruses is usually associated withsarcoma or defective viruses which have anRNA size of 9.7 kilobases or much smaller than8.2 kilobases, respectively. To establish thatRAV-7 is a leukosis virus, RNA sizing andprotein characterization of the virus was per-formed. RAV-7 had an 8.2-kilobase RNA, as

500

400-

300-

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FlG. 4. Levels of T3 in RAV-7-infected and unin-fected chickens. See the legend of Fig. 3 for symbolexplanation.

TABLE 1. Circulating hormone levels in RAV-7-infected chickensa

Age Virus Wt (g) Insulin Glucagon(days) (,U/ml) (ng/ml)

33

88

1010

1111

1616

1717

1919

2424

2828

2929

3030

3333

3636

3737

3838

4040

4343

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

3431 (91.2)C

5946 (78.0)

NDND

5039 (78.0)

7873 (93.6)

10180 (79.2)

10788 (82.2)

NDND

66 (100)

NDND

8.17.1 (87.7)

66 (100)

717.5 (250)

7.66.4 (74.4)

NDbND

3,8004,000 (105.3)

1,9504,000 (205.1)

NDND

2,7502,000 (72.7)

2,9003,550 (122.4)

6 2,1509.5 (158.3) 2,200 (102.3)

67.4 (123.3)

248 7.8168 (67.8) 17

233113 (48.5)

256149 (58.2)

250157 (62.8)

303135 (44.6)

340110 (32.4)

NDND

343189 (55.1)

577200 (34.7)

(217.9)

69.8 (163.3)

139.8 (75.4)

9.412.5 (133.0)

9.611.5 (119.8)

19.57 (35.9)

6.422.5 (351.6)

13.56 (143.6)

614.5 (241.7)

4,0001,750 (43.8)

3,1001,050 (33.9)

3,9003,900 (100)

3,4501,275 (37.0)

3,3003,000 (90.9)

1,5501,580 (101.9)

2,3751,675 (70.5)

3,3004,000 (121.2)

NDND

4,0003,700 (92.5)

a Samples were taken according to the protocolprovided by Cambridge Medical Diagnostics, Inc., foruse with their radioimmunoassay kits. Samples foryoung birds were pooled from 2 or 3 birds, usingequivalent amounts per bird. All samples were ultra-centrifuged before assay to remove lipid.

b ND, Not determined.Values expressed as percentage of control value.

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RAV-7-INDUCED HYPOTHYROIDISM 801

determined by agarose gel electrophoresis. Theprotein banding pattern on sodium dodecyl sul-fate-polyacrylamide gel electrophoresis was typ-ical of avian leukosis virus, and bands corre-sponding to gp85, gp37, p27, p19, p12, and p15/plO were observed (data not shown).

Effect of viral dilution. To further establish therole of RAV-7 in the induction of the diseaseprocess, we performed an experiment in whichchicken embryos were infected with dilutions ofthe virus, and chickens were sacrificed andexamined 39 days after hatching. Viral dilutionsof 1:20 to 1:200 had the most pronounced effect,in that the levels of triglycerides and cholesterolwere markedly elevated (Fig. 5). Body weightsand T4 levels were decreased at every dilutionemployed, whereas insulin levels and liverweights expressed as percentage of body weightwere elevated at most viral dilutions (Fig. 5). T3levels were decreased when higher RAV-7 dilu-tions (1:200 to 1:200,000) were injected (Fig. 5).These results suggest that RAV-7 interactedwith chicken embryos in a dose-dependent man-ner and that relatively high dilutions of virus (upto 1:200,000) were effective in causing the dis-ease syndrome.

Effect of RAV-7 on lymphoid organs. Avianleukosis viruses of subgroups A and B have beenexamined for the ability to cause lymphoid invo-lution and immunosuppression, and subgroup Bviruses cause lymphoid involution (34) and im-munosuppression (29, 34) whereas subgroup Aviruses cause neither (26, 29). It was therefore ofinterest to determine whether RAV-7, a leukosisvirus of the C subgroup, caused lymphoid organinvolution. It was found that RAV-7 infectioncaused a marked reduction in the size of thebursa and thymus, whereas the spleen size re-mained essentially unchanged in 3-week-oldRAV-7-infected chickens (Table 3). Examina-tion of the spleens of chickens older than 3weeks revealed that splenic involution was pre-sent from 4 weeks onward (data not shown).

DISCUSSIONThyroid tissue from chickens 2 days post-

hatching appeared to show some alteration withRAV-7 infection, but the change was not pro-nounced. Examination of thyroid tissue fromRAV-7-infected chickens revealed lymphoblas-toid infiltration as early as 7 days posthatching.Follicular epithelial cells at this time had largevacuoles and increased eosinophilia of the cyto-plasm, which was indicative of cellular degener-ation. By 16 days posthatching, the T3 and T4levels in RAV-7-infected chickens were signifi-cantly below normal, a finding which may reflectthe extensive infiltration of the thyroid and lackof normal thyroid structure. Continued low T3

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FIG. 5. Dose response to RAV-7. RAV-7 was given to 10-day-old chicken embryos by the intravenous route(0.1 ml of the indicated dilution), and the chickens were hatched and examined at 39 days of age. Values shownare means ± the standard error of the mean. C, Uninfected hatchmates; 1, 1:2 dilution of RAV-7 stock; 2, 1:20dilution of RAV-7 stock; 3, 1:200 dilution of RAV-7 stock; 4, 1:2,000 dilution of RAV-7 stock; 5, 1:20,000 dilutionof RAV-7 stock; 6, 1:200,000 dilution of RAV-7 stock. The original titer of the RAV-7 stock was 106 infectiousunits per ml; the 1:200,000 dilution therefore contained approximately 5 infectious units of virus.

and T4 levels resulted in increased activity of theremaining follicular epithelial cells, as indicatedby columnar-to-cuboidal appearance and in-creased basophilia of these cells. The changes inthyroid appearance and circulating thyroid hor-mones occurred before the appearance of hyper-lipidemia at approximately 20 days posthatching(8). The apparent hypothyroidism in RAV-7-infected chickens may therefore cause the hy-perlipidemia manifested later in the course of thedisease.

Hyperlipidemia is a common occurrence inhypothyroidism, an occurrence which can be

explained by alterations in the metabolism oflipids and phospholipids (13, 28). Correze andco-workers (12) reported changes in hexokinaseI, glucose 6-phosphate dehydrogenase, gluco-nate 6-phosphate dehydrogenase, and cyclicAMP accumulation in hypothyroidism in rats.These changes result in shunting glucose intofatty acid synthesis. Severson and Fletcher (31)reported a decrease in activity of acid cholester-ol ester hydrolase in rat liver and fat pad cellsfrom thyroidectomized rats with a net reductionin cholesterol catabolism. The net result is thatthe decrease in lipid utilization or catabolism

TABLE 3. Lymphoid organ involution caused by RAV-7

Virusa No. of birds Body wt Bursa (as % Thymus (as % Spleen (as %observed body wt)b body wt) body wt)

5 135.4 ± 5.4 0.675 ± 0.030 0.736 ± 0.057 0.159 ± 0.013+ 7 97.4 ± 6.8 0.214 ± 0.125 0.203 ± 0.116 0.178 ± 0.078

Level of P < 0.001 P < 0.001 P < 0.001 NSsignificancec

a RAV-7 was given to 10-day-old embryos by the intravenous route at 104 infectious units per embryo.Chickens were sacrificed and examined at 20 days of age.

b Lymphoid organ weights are expressed as a percentage of the body weight.c Levels of significance according to Student's two-tailed t test. NS, No significant difference.

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RAV-7-INDUCED HYPOTHYROIDISM 803

overshadows the slight decline or unchangedlevels in lipid synthesis in hypothyroid animals(19).There is a striking similarity between RAV-7-

infected chickens and OS chickens, both pheno-typically and by the presence of hypothyroid-ism. SAT is a genetically determinedcharacteristic in Cornell C strain chickenswhich, after inbreeding, led to an obese chickenline (OS) (10, 11). The OS chickens exhibitobesity, high levels of antibodies to thyroglobu-lin, antibodies to thyroid microsomal compo-nents, infiltration of the thyroid by lymphoidcells, and a deficiency in T3 and T4 (37, 39).Appearance of obesity, reduced skeletal struc-ture, and long, silky feathers are usual by 3 to 5weeks posthatching, although a reduction in T4levels and an increase in 1311 uptake are reportedin newly hatched OS chickens (R. Sundick, M.Livezey, T. Brown, and N. Bagchi, Fed. Proc.35:512, 1976). The basis of SAT is hypothesizedto be multigenic and involves a primary thyroiddefect and defects in immune regulation (38).Although early work reports lymphoid infiltra-tion of OS thyroids at 1 to 3 weeks posthatching(21), most work emphasizes the appearance oflymphoid and plasma cells at 3 to 5 weeksposthatching (37). The onset of the RAV-7 syn-drome and infiltration of the thyroids appearedslightly earlier than that reported for SAT. Al-though the thyroids of RAV-7-infected chickenswere infiltrated by lymphoblastoid cells, therewas little evidence for the presence of largenumbers of plasma cells characteristic of SAT(37), and the infiltrating cells in RAV-7-infectedchicken thyroids appeared to be more immaturethan those seen in SAT (G. Wick, personalcommunication). Experiments are in progress todetermine the identity of the cells which infil-trate RAV-7-infected thyroids. It will be of fun-damental interest to establish whether a relation-ship exists between the infiltrating cell and thethymus and bursa gland involution which wasobserved in RAV-7-infected chickens (Table 3).There was no evidence of antithyroglobulin inRAV-7 infection (unpublished observations),and the presence of other autoantibodies has notbeen determined. These observations appear todistinguish the RAV-7 syndrome from SAT. Inwork with rats which exhibit spontaneous thy-roiditis, Kotani and co-workers (22) report abetter correlation between the extent of thyroid-itis and the presence of anti-follicular cell anti-bodies than between thyroiditis and antithyro-globulin. The presence of autoantibodies otherthan antithyroglobulin in the disease syndromeinduced by RAV-7 is an interesting possibilitywhich has not been examined.

Chickens which are hypothyroid and havegoiters induced by tapazole exhibit near normal

growth characteristics when given daily subcu-taneous injections of T4 at a dosage of 3 to 4,ug/100 g of body weight (32). The early diagnosisof SAT as a hypothyroid condition was made bysupplementation of the diet of OS chickens withT4, which results in abrogation of the syndrome(36). In the present study, supplementation ofRAV-7-infected chickens with Synthroidshowed some alleviation of disease symptoms.In comparison with the saline-treated RAV-7-infected chickens, T4-supplemented RAV-7-in-fected chickens showed a shift in all parameterstoward normal. However, T4 supplementationdid not abrogate the disease because significantstunting and alteration of circulating hormonelevels were still present when saline-treated un-infected chickens were compared with T4-treat-ed RAV-7-infected chickens. The decreased T4levels in the uninfected chickens which receivedSynthroid were not surprising because the exog-enous hormone could result in a negative feed-back on stimulation of in vivo T4 production bythe thyroid. The T4-treated uninfected chickensexhibited mild thyroid hypoplasia that was ap-parent upon histological examination of theirthyroid glands.Our attention has been drawn to avian retrovi-

rus debilitation because stunting accompaniesseveral disease syndromes. For example, infec-tion of chickens with an avian osteopetrosisvirus, MAV-2(0), results in chickens which growat a rate 26% of that of uninfected hatchmates(2). In the case of MAV-2(0)-induced stunting,bone cell proliferation probably contributes asubstantial tumor load to the infected chicken.However, it is clear that viral plaque isolateswhich lead to little or no bone proliferationnevertheless cause a decrease in body weight(33). In the case of another avian leukosis virus,ring-necked pheasant virus, a failure of infectedchickens to grow accompanies the appearanceof lung tumors (9). Work is in progress to look atthe subgroup specificity of the RAV-7 syn-drome. In the case of both virus infections citedabove, it is clear that stunting is most severewhen a cellular proliferation is present, butanimals with no obvious growths are frequentlydebilitated. RAV-7 induced significant stuntingwithout evidence of cellular proliferation (Table1). In previous studies, RAV-7-induced stuntingis significant by 14 days posthatching, andgrowth rates of 3.9 g/day and 16.3 g/day arepresent in RAV-7-infected- and uninfectedchickens, respectively (8).Hypothyroidism is reported to cause in-

creased insulin release and decreased insulinutilization, which leads to a net increase ininsulin levels (5, 6). T4 is also postulated tofunction by increasing the numbers of receptorsfor insulin, glucagon, and P-adrenergic catechol-

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804 CARTER AND SMITH

amines (23). The increased number of hormonereceptors increases the turnover in these com-pounds and lowers their levels in circulation.The decreased utilization of insulin in hypothy-roidism can be the result of decreased receptorsfor insulin. Localized lymphoblastoid infiltrationof the pancreas in RAV-7 infection has beenreported (8) and suggests a primary effect of thevirus on pancreatic function. A secondary effecton pancreatic hormone levels could be producedby the hypothyroid condition, which could ex-plain the slightly increased insulin levels seenwith decreased T4 levels (Table 1). The reduc-tion of insulin levels with T4 supplementationwould indicate an interaction between the hor-mones (Table 2). The reduced levels of glucagonat 20 to 30 days posthatching may be an expres-sion of specific infiltration of alpha islets in thechicken pancreas. The avian pancreas containspredominantly islets with alpha cells or withbeta cells rather than islets with both alpha andbeta cells (18, 24). Some evidence of fibrosis inthe pancreases of RAV-7-infected chickens isfound with electron microscopy, but the fibrosishas not been localized to a specific islet type(unpublished observation). The transient natureof the glucagon decrease could be a result ofresolution of the damage in the pancreas. It isinteresting to note that the largest glucagondecrease corresponded with the time of highestmortality. Removal of the splenic lobe of theavian pancreas, which contains a majority ofalpha islets, results in hypoglycemia and death(24) and indicates an important role of glucagonin normoglycemia and survival in the chicken.The evidence presented here indicates that

infection of chickens by an avian leukosis virus,RAV-7, induced hypothyroidism. Thyroid alter-ation was supported by morphological evidencefor lymphoblastoid cell infiltration and biochem-ical evidence for an alteration of circulating T3and T4 levels in RAV-7-infected chickens. Evi-dence for alteration in pancreatic function wasindicated by morphological studies, but the cir-culating insulin and glucagon levels did not givea clear indication of functional alteration. Themechanism of alteration was infiltration of theendocrine glands by lymphoblastoid cells and, inthe case of the thyroid, resultant disappearanceof normal tissue. The disease syndrome ex-pressed, stunting and hyperlipidemia, was ex-plained by the hypothyroidism. The identifica-tion of the cell infiltrating the thyroid andpancreas, why that cell is homing to these spe-cific targets, and the subgroup specificity of thesyndrome remains to be elucidated.

ACKNOWLEDGMENTS

We thank Cheryl Harward and Ted Wheeler for diligenttechnical assistance, Jim Proctor of Experimental Pathology

Laboratories, Inc., for assistance with histological sections,and Dean Barnett for helpful discussion and consultation.

This research was supported by Public Health Servicegrants CA12323 and CA14236 from the National CancerInstitute. J.K.C. was a predoctoral trainee supported byPublic Health Service grant GM07184.

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