INFECTION AND IMMUNITY, Aug. 1973, p. 264-271Copyright © 1973 American Society for Microbiology

Vol. 8, No. 2Printed in U.S.A.

Pathogenesis of Aleutian Disease of Mink:Nature of the Antiglobulin Reaction and Elutionof Antibody from Erythrocytes and Glomeruli of

Infected MinkH. J. CHO AND D. G. INGRAM

Department of Veterinary Microbiology and Immunology, University of Guelph, Guelph, Ontario,Canada NIG 2WI

Received for publication 13 March 1973

Erythrocytes from mink chronically infected with Aleutian disease virus(ADV) gave positive antiglobulin reactions with rabbit anti-mink immunoglobu-lin (Ig)G, anti-mink C3, and anti-mink serum, but did not react with anti-minkIgM. The strongest reaction was observed with anti-mink C3. Immunoelectro-phoresis demonstrated that serum from rabbits injected with erythrocytes fromADV-infected mink gave a precipitin line with normal mink serum in thebeta globulin region corresponding to C3. When normal mink erythrocytes wereexposed to serum from ADV-infected mink, they were not sensitized, demon-strating that the antibodies in these mink sera were not directed againsterythrocyte antigens. Glycine-hydrochloride buffer treatment of erythrocytestromata and isolated glomeruli from ADV-infected mink yielded eluatescontaining serum proteins in the gamma globulin region which appeared to beIgG, and in the beta and alpha globulin regions which are probably complementcomponents. In both erythrocyte and glomerular eluates, anti-ADV antibody wasdemonstrated. These findings suggested that the positive direct antiglobulin testand glomerulonephritis in Aleutian disease is due to the persistence of ADV andformation and deposition of ADV antigen-antibody-complement complexes on

the erythrocyte surfaces and in glomerular capillaries.

Aleutian disease (AD) of mink is an immuno-logical disease caused by a persistent virusinfection. The disease is slowly progressive andinvariably fatal (9). AD was so named becauseit was first recognized in the Aleutian coat-col-ored mink; however, it is now known that thedisease occurs in all color phases of mink (4, 7).The disease is characterized by a systemicproliferation of lymphoid cells and generalizedplasmacytosis, hypergammaglobulinemia, glo-merulonephritis, arteritis, and hepatitis (7, 9,15, 16). It has been demonstrated that serumfrom AD virus (ADV) -infected mink have infec-tious virus-antibody complexes (17), and anti-ADV antibody titers were extremely high inhypergammaglobulinemic serum (2, 3, 13, 18).Glomerulonephritis in this disease was charac-terized by granular deposition of host immuno-globulin and complement along the glomerularcapillary walls and mesangia (7, 15, 18). Veryrecently (in 1973), ADV was isolated from

ADV-infected mink tissues (10; Cho and In-gram, Nature, in press).

In 1966, Saison and co-workers reported thaterythrocytes from ADV-infected mink ag-glutinated when reacted with rabbit anti-minkglobulins (19). They suggested that the positivedirect antiglobulin test (Coombs test) was dueto antibody, and that the antibody was either(i) autoantibody to normal erythrocyte mem-brane antigens, (ii) autoantibody to erythro-cyte membrane components made autoanti-genic through the direct or indirect effects of theviral infection, or (iii) antibody to an antigen(perhaps viral) attached or adherent to theerythrocyte membrane (20).The present investigation was undertaken to

identify and characterize the serum proteinsresponsible for the positive antiglobulin reac-tion in AD. In a second set of experiments,glomeruli were isolated from the kidneys ofADV-infected mink. Eluates of the glomeruli

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were prepared and the specificity of the anti-body in these eluates was determined.

MATERIALS AND METHODSExperimental animals. Aleutian (aa genotype),

standard dark (AA genotype), and pastel (AA or aAgenotype) mink, 3 months to 1 year of age, wereobtained from the AD-free herd of the Field Station ofthe Ontario Veterinary College. All of the mink weretested by immunoelectroosmophoresis (IEOP) (2; Choand Ingram, Can. J. Comp. Med., in press) and nonehad antibody against ADV antigen. Rabbits weighing2 to 3 kg were purchased commercially and used forthe preparation of antisera against mink serum pro-teins.

Virus. Source of ADV and preparation of inoculumwere described elsewhere (Cho and Ingram, Can. J.Comp. Med., in press).

Preparation of rabbit antisera against minkserum proteins. Rabbits were immunized with minkantigens in Freund incomplete adjuvant. The follow-ing antisera were produced: anti-C3 (RAC3); anti-complement (RAC); anti-immunoglobulin (Ig)G(RAIgG), anti-IgM (RAIgM), and anti-mink serum(RAS).Mink antisera against Brucella abortus and Sal-

monella typhimurium "O" antigens were used toprepare antigen-antibody complexes. These antiseracollected at 1 week and 10 weeks after immunizationwith Salmonella and Brucella antigens, respectively,were used as the source of mink IgM and IgG.

Antigen-antibody complexes were prepared by in-cubating the antigens with the appropriate antisera inthe presence of 0.01 M ethylenediaminetetraaceticacid (EDTA) at 37 C for 2 h. The complexes werewashed four times with 0.85% NaCl containing 0.01 MEDTA and twice with 0.85% saline. The sedimentswere suspended in saline, emulsified in Freund in-complete adjuvant, and inoculated subcutaneouslyinto rabbits three times at 12-day intervals.

Zymosan-mink-complement complexes were pre-pared by the methods of Mardiney and Miiller-Eber-hard (14) and used as the source of mink antigens forthe production of RAC3. Salmonella typhimurium"H" antigen and specific rabbit antiserum were usedto prepare immune complexes. These complexes wereadded to fresh normal mink serum and incubated at 5C for 18 h and then at 37 C for 30 min. Theantigen-antibody-complement complexes werewashed six times with cold saline and used to immu-nize rabbits to produce rabbit anti-mink complement(RAC).

For the preparation of RAIgG, the rabbit antiserawere absorbed repeatedly with antigen-antibody com-plexes composed of Salmonella typhimurium "O"antigen and mink antiserum collected 1 week afterimmunization. RAIgM sera were absorbed with minkIgG fractions eluted with 0.0175 M phosphate buffersolution (pH 6.3) by diethylaminoethyl-cellulosechromatography.RAC and RAC3 were each absorbed four times with

immune complexes composed of Salmonellatyphimurium "O" antigen-mink antibody andBrucella abortus antigen-mink antibody.

The specificity of the reagents was tested byimmunoelectrophoretic analysis. After absorption,only precipitin lines for IgG, IgM, and ,Blc wereobserved with appropriate reagents and normal minkserum (Fig. la, lb, 2, 3, 4).

Each rabbit antiserum used in this investigationwas absorbed six times with washed normal red bloodcells pooled from Aleutian, pastel, pearl, and stand-ard dark mink.

Preparation of rabbit anti-ADV-infected minkerythrocytes serum. For the preparation of rabbitanti-ADV-infected mink erythrocytes, 6 ml of 50%mink erythrocyte suspension collected from six in-fected mink which gave positive antiglobulin reac-tions were injected intravenously into two rabbitstwice at an interval of 7 days, and 14 days after thesecond injection serum was collected and absorbedwith normal mink red blood cells as described above.

Direct antiglobulin test. Mink blood was collectedby cardiac puncture into equal volumes of Alseversolution. The cells were washed at least five timeswith 0.85% saline and made up to a 2% suspension in0.85% saline. One drop of each cell suspension wasadded to one drop of each antiglobulin reagent. Cellsand serum were thoroughly mixed and incubated at37 C for 1 h. At the end of the reaction, cells from eachtube were carefully removed with a Pasteur pipetteand spread on a slide for microscope examination.Preparation of erythrocyte stromata from ADV-

infected mink. The method of Kochwa and Rosen-field (11) was followed with some minor modifica-tions. For the preparation of erythrocyte stromata ofADV-infected mink, 50 ml of blood was collected fromfour infected mink showing strong direct antiglobulinreactions and thoroughly washed as described above.The packed cells were pooled and were made up to a10% suspension in saline. For the preparation of thered cell stromata, the red cells were lysed by adding2.5 ml of a 0.5% solution of digitonin for each 50 ml ofthe 10% red cell suspension and stirred for 1 min. Thismaterial was centrifuged at 52,800 x g for 30 min, andthe supernatant fluid was discarded. Erythrocytestromata were washed six times with large volumes ofcold isotonic saline. The pellets were reacted withphosphate-buffered saline (PBS, 0.01 M in 0.14 MNaCl, pH 7.4) for 1 h and after centrifugation at52,800 x g for 30 min, the supernatant fluid wascollected. The erythrocyte stromata were utilized forthe preparation of eluates.

Preparation of acid eluates from the erythrocytestromata. Glycine-hydrochloride buffer, half volumeof whole blood (0.1 M, pH 3.0), was added to thesedimented erythrocyte stromata and mixed thor-oughly. The stromata were incubated at room temper-ature for 20 min and centrifuged at 52,800 x g for 30min. The supernatant fluids were collected and neu-tralized with 1 N NaOH to pH 7.0. The eluates wereconcentrated with polyethylene glycol. Anti-ADV an-tibody was assayed by IEOP, and immunoelectro-phoresis was carried out to examine the serum pro-teins contained in the eluates. Erythrocytes fromnormal mink were treated similarly and served as acontrol.

Isolation of glomeruli and preparation of glo-merular eluates from normal and ADV-infected

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CHO AND INGRAM INFECT. IMMUNITY

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FIG. 1-4. Immunoelectrophoretic patterns formed by rabbit antibodies to mink serum proteins. Abbrevia-tions: S, normal mink serum; RAIgG, rabbit anti-mink IgG (la, b); ARAIgG, rabbit anti-mink IgG absorbedwith mink IgG (la); RAIgM, rabbit anti-mink IgM (2); RAC3, rabbit anti-mink C3 (3); RAC, rabbit anti-minkcomplement (4).

mink kidney. Kidney cortex collected from 10 ADV-infected mink 4 months or later after infection was cutinto small pieces and washed five times with 0.85%saline. For the isolation of glomeruli, the method ofGreenspon and Krakower (6) was followed with minormodifications. Fragments of the cortex were forcedthrough a stainless steel sieve of 60 mesh (0.25 mm).The paste passed through the mesh, was collected andwashed with 0.85% saline five times by centrifuging at1,100 x g for 15 min. The sediment was mixed with an

82% sucrose solution. It was then centrifuged at 39,000x g for 3 min. The intact isolated glomeruli were thusbrought to the top. This top layer was collected andwashed with 0.85% saline five times, with centrifuga-tion at 70 x g for 10 min and finally at 2,500 x g for 15min. Glomeruli of more than 90% purity (Fig. 5) were

suspended in 0.85% saline and stored at 5 C for 18 h toremove soluble proteins. The glomerular suspensionwas frozen and thawed twice and washed twice with0.85% saline at 2,500 x g for 15 min. The pellet was

reacted with PBS for 1 h, and after centrifugation at

2,500 x g for 15 min the supernatant fluid was

collected as the PBS eluate. Glycine-hydrochloridebuffer (50 ml/g of pellet) (0.1 M, pH 3.0) was added tothe pellet and incubated for 1 h at room temperature.After centrifugation at 2,500 x g for 15 min, thesupernatant fluid was collected and neutralized with1 N NaOH to form the acid eluate.

Both PBS and acid eluates were concentrated withpolyethylene glycol. Antibody activity was assayed byIEOP, and immunoelectrophoretic analysis was car-ried out to examine the serum proteins eluted fromthe isolated glomeruli. Eluates from glomeruli col-lected from normal mink kidney served as a control.

Immunofluorescent test for detection of immunecomplexes in kidneys. RAIgG and RAC3 were conju-gated with fluorescein isothiocyanate (FITC). Frozenkidney blocks were cut with a cryostat. To remove

soluble proteins, the sections were washed in PBS for30 min at room temperature and were then stainedwith FITC-conjugated RAIgG or RAC3 for 40 min atroom temperature. After staining, all the specimens

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PATHOGENESIS OF ALEUTIAN DISEASE OF MINK

were washed in PBS for 30 min with several changes ofthe buffer, rinsed with distilled water, mounted inbuffered glycerine, and examined.

RESULTSAntiglobulin test with red blood cells from

ADV-infected mink. Direct antiglobulin testswere run on the red blood cells from fiveAleutian and two standard dark mink infectedwith ADV. Red blood cells were collected at 5months after infection, and tests were con-ducted to determine whether these cells werecoated with IgM, IgG, C, or C3. Erythrocytesfrom ADV-infected mink gave positive reactionswith RAC3 and RAC. RAIgM gave negativereactions, whereas RAIgG gave positive reac-tions in four and negative reactions in threeinfected mink (Table 1). At 5 months after ADVinfection, anti-ADV antibody titers in theserum ranged from 10,000 to 40,000 (Table 1).One drop of normal mink serum mixed with onedrop of antiglobulin serum and incubated for 1h at 37 C failed to cause a direct antiglobulinreaction with erythrocytes from ADV-infectedmink, which showed a positive reaction with theantiglobulin reagents before absorption withnormal mink serum.As a control, four Aleutian mink were injected

with spleen suspension from a normal Aleutian

mink. Five weeks after immunization, directantiglobulin tests were run on red blood cellswith RAIgG, RAIgM, RAC, RAC3, and RAS.All cells gave negative results (Table 1).

Indirect antiglobulin test with serum fromADV-infected mink. To determine if serumfrom ADV-infected mink contains antibodiesagainst normal mink erythrocytes, 1 vol of a10% suspension of normal mink red blood cellswas treated with 4 vol of undiluted ADV-infected mink serum for 1 h at 37 C. Afterthorough washing, RAS was added. No aggluti-nation of red blood cells was observed. Thisexperiment showed that infected mink serumdid not sensitize normal mink erythrocytes.Production of antibody against serum

proteins on the surface of erythrocytes ofADV-infected mink. Two rabbits were injectedwith ADV-infected mink erythrocytes whichshowed positive direct antiglobulin reactions,and 14 days after the second injection sera werecollected and tested for antibody activityagainst normal mink serum. Immunoelectro-phoretic analysis, using these antisera, showeda very faint precipitin line with normal minkserum in the beta globulin region correspondingto C3. The position of the precipitin line wassimilar to Fig. 3.Demonstration of anti-ADV antibody in

FIG. 5. Isolated glomeruli from Aleutian disease virus-infected mink kidney prepared for antibody elution.

267VOL. 8, 1973

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TABLE 1. Direct antiglobulin reactions of red blood cells from ADV-infected and normal mink

Mink Serum anti-ADV Types of antiglobulinGroup | no. antibodyr titers RAIgG RAIgM RAC RAC3 RAS

ADV-infected A2 20,000 + + ±+ +++ 320cA4 10,000 ++ _ _ +++ 80A12 40,000 _ _ _ + 320A13 20,000 + - + + + + 160A18 10,000 _ - + + ++ 40S10 40,000 + - + + + 320S12 10,000 - - + + + 160

Normal A21 0 - - - - -

A22 0 - - - - -

Si 0 -

S2 0

aAnti-ADV antibody was assayed by immunoelectroosomophoresis.b Degree of agglutination of erythrocytes. Undiluted antiglobulins were used.c Reciprocals of the highest dilutions of rabbit anti-mink serum which

erythrocytes.

the acid eluates from ADV-infected minkerythrocyte stromata. By the treatment withdigitonin, red blood cells were lysed. The result-ant stromata were intact and were free ofhemoglobin after several washings. Anti-ADVantibody was eluted by glycine-hydrochloridebuffer treatment of the red cell stromata fromADV-infected mink (Fig. 6). PBS treatment ofthe same erythrocyte stromata failed to yieldany antibody activity. Erythrocyte stromatafrom normal mink did not show anti-ADVantibody after treatment with either glycine-hydrochloride buffer or PBS (Table 2). Im-munoelectrophoretic analysis showed that theacid eluate of erythrocyte stromata from ADV-infected mink yielded several precipitin lineswhen developed with RAS (Fig. 7). In additionto the slower migrating fractions of gammaglobulins which appeared to be IgG, lines ap-peared in the alpha and beta globulin regions,which were consistent with the suggestion thatthe acid eluates contained complement compo-nents.Elution of proteins in glomerular deposits

and demonstration of anti-ADV antibodyfrom the glomerular eluates. Sections of thekidneys from 10 ADV-infected mink, whichwere collected 4 months or more after infectionand used as the sources of glomeruli prepara-

tions, were examined for the presence of hostIgG and complement in the glomeruli. All thesections showed strong positive staining withFITC-conjugated RAIgG and RAC3. Fluores-cence showed lumpy and granular patternsalong the glomerular capillary walls (Fig. 10).Normal kidney sections showed no fluorescencewith FITC-conjugated RAIgG and RAC3.

caused the agglutination of

The acid eluates of the isolated glomerulifrom ADV-infected mink kidney showed anti-body activity against ADV antigen but noreaction with control antigen or with normalkidney suspension (Table 2, Fig. 8). Whennormal kidney sections were incubated with theacid eluates from AD glomeruli at room temper-ature for 40 min, then washed and stained withFITC-conjugated RAIgG, the kidney sectionsfailed to show fluorescence. Neither the PBSeluates of the glomeruli from ADV-infectedmink nor the acid nor PBS eluates from normalglomeruli had anti-ADV antibody. Immuno-electrophoresis of the glomerular eluate fromADV-infected mink showed precipitin lines inthe gamma globulin region corresponding toIgG, and in the beta and alpha globulin regions,which may represent mink complement compo-nents (Fig. 7 and 9).

DISCUSSIONSeveral investigators have reported that AD

in mink has some of the characteristic featuresof autoimmune states. The hypothesis that ADis an autoimmune disease is based on thereports of a direct positive Coombs test (19, 20),demonstration of anti-gamma globulin factor(21), and circulating deoxyribonucleic acid(DNA) and anti-DNA antibody (1). Anti-gamma globulin factors, which were generally oflow titer, were found in 68% of 32 normal minksera, compared with 72% of 42 mink infectedwith AD (21). A high concentration of DNA wasdetected in sera of mink both before and afterADV infection (1). The incidence and quantityof "nuclear" antigens and anti-nuclear anti-body detected by immunofluorescence were

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FIG. 6. Immunoelectroosmophoresis of acid eluate from erythrocytes of AD-infected mink.FIG. 7. Immunoelectrophoresis of acid eluate from erythrocytes and from glomeruli of AD-infected mink.FIG. 8. Immunoelectroosmophoresis of acid eluate from glomeruli of AD-infected mink.FIG. 9. Immunoelectrophoresis of acid eluate from glomeruli of AD-infected mink. Abbreviations: A, ADV

antigen prepared- from AD-infected mink tissue; N, negative (control) antigen prepared from normal minktissues; S, mink anti-ADV serum; E, acid eluate from erythrocyte of AD-infected mink; R, normal minkerythrocyte stromata; G, acid eluate from glomeruli ofAD infected mink; K, normal mink kidney suspension;RAS, rabbit anti-mink serum.

greater in sera from mink after ADV infectionthan in sera taken from mink before ADVinfection. A pre-AD serum pool showed nuclearfluorescence with human white blood cell nucleiat dilutions up to 1: 16, whereas the titer ofthe post-AD serum pool was 1: 64. However, the

presence of nuclear antigens and anti-nuclearantibodies did not correlate with the degree ofhypergammaglobulinemia. The significance ofanti-gamma globulin factors and anti-DNA-antibody as pathogenic factors in AD is un-

known at present.

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CHO AND INGRAM

TABLE 2. Antibody to Aleutian disease virus antigenin eluates from erythrocytes and glomeruli of normal

and Aleutian disease-infected minka

Disease Eluate Eluted at:status from: pH 3 pH 7.4

Normal Erythrocytes _Glomeruli _

Infected Erythrocytes +Glomeruli +

a None of the eluates reacted with antigens pre-pared from normal mink tissues, normal mink eryth-rocytes, or normal mink kidney.

FIG. 10. Immunofluorescent reaction of glomerulifrom kidney of a mink infected with Aleutian diseasevirus; unfixed frozen section stained with FITC-con-jugated rabbit anti-mink IgG. IgG is present in thecapillary walls and mesangium and forms a granularpattern.

Fluorescein-labeled normal- and ADV-infected mink sera did not react with cells ofnormal mink tissues (15, 18). Anti-ADV anti-body titers, determined by the immunofluores-cence test, were extremely high, and at 60 daysafter ADV infection the geometric mean anti-body titer reached 100,000 (18). The antibodytiters were correlated with the degree of hyper-gammaglobulinemia (13, 18; Cho and Ingram,Proc. Can. Fed. Biol. Soc. 15:299, 1972; Cho andIngram, Can. J. Comp. Med., in press). Porteret al. (16) failed to demonstrate the systemiclupus erythematosus (SLE) cell phenomenon oranti-nuclear factor in ADV infections of mink.

In the present investigation, erythrocytesfrom ADV-infected mink were shown to haveIgG and complement components on their sur-faces (Table 1, Fig. 7). The immunoglobulins

eluted from the erythrocytes of ADV-infectedmink had anti-ADV antibody activity, but noantibody against the surface antigen of normalmink red blood cells.

Erythrocytes from ADV-infected mink gave astronger reaction with RAC3 than with RAIgG,and erythrocytes from some mink reacted withRAC3 but not RAIgG (Table 1). By means of acomplement-fixing antibody consumption test,Gilliland et al. (5) reported that previouslyundetectable red cell-bound IgG could be de-tected and quantified from the erythrocytes ofpatients with SLE. These red cells showedpositive direct antiglobulin tests with anti-com-plement serum but negative reactions usinganti-IgG serum. The amount of IgG fixed on thesurface of these erythrocytes was below the levelrequired for detection by the direct antiglobulintest. These workers have been able to eluteanti-erythrocytic IgG from the reactive redblood cells. The mechanisms responsible for thecoating of the erythrocytes with anti-ADV anti-body and complement components are not clearat the present time. However, the erythrocytesapparently are coated with a complex of thevirus, the antibody and complement and acidelution results in the separation of the antibodyand complement. The virus antigen was notdemonstrated in the eluates.The renal glomerular lesions in ADV-infected

mink resemble those in man with SLE (12) andin NZB mice (8). It has been reported thatgamma globulin and complement were depos-ited in the glomeruli of ADV-infected mink, andthe possible source of the immune complexesdeposited in glomeruli were ADV-antibody-complement complexes (7, 15) or circulatingDNA-anti-DNA antibodies (7) as was reportedin SLE (12). However, the acid eluate fromglomeruli of ADV-infected mink did not reactwith the nuclei of normal mink kidney cells(15). Porter and co-workers (18) eluted anti-ADV antibody, as determined by the immuno-fluorescence test, from ADV-infected mink kid-ney and concluded that the pathogenesis of theglomerulonephritis of AD is related to theformation of viral antigen-antibody-comple-ment complexes which lodge in glomerularcapillaries. In the present investigation, serumproteins eluted from isolated glomeruli of ADV-infected mink kidney contained antibodyagainst the ADV antigen as assayed by IEOP.These results supported the earlier finding ofPorter and co-workers (18).The present investigations strongly suggested

that the positive direct Coombs reaction andglomerulonephritis in AD were due to the per-sistence of the virus and formation and deposi-

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VOL. 8, 1973 PATHOGENESIS OF ALEU

tion of ADV antigen-antibody-complementcomplexes on the surfaces of erythrocytes and inglomerular capillaries.

ACKNOWLEDGMENT

This work was supported by grant MA 2001 from theMedical Research Council of Canada and by the OntarioMinistry of Agriculture and Food.

LITERATURE CITED

1. Barnett, E. V., R. C. Williams, Jr., A. J. Kenyon, and J.B. Henson. 1969. Nuclear antigens and antinuclearantibodies in mink sera. Immunology 16:241-253.

2. Cho, H. J., and D. G. Ingram. 1972. Antigen and antibodyin Aleutian disease in mink. I. Precipitation reaction byagar-gel electrophoresis. J. Immunol. 108:555-557.

3. Cho, H. J., and D. G. Ingram. 1972. An accurate andrapid test for detecting Aleutian disease infected mink.Fur Trade J. Can. 50:6-7.

4. Eklund, C. M., W. J. Hadlow, R. C. Kennedy, C. C.Boyle, and T. A. Jackson. 1968. Aleutian disease ofmink: properties of the etiologic agent and the hostresponses. J. Infect. Dis. 118:510-526.

5. Gilliland, B. D., J. P. Leddy, and J. H. Vaughan. 1970.The detection of cell-bound antibody on complement-coated human red cells. J. Clin. Invest. 49:898-906.

6. Greenspon, S. A., and C. A. Krakower. 1950. Directevidence for the antigenicity of the glomeruli in theproduction of nephrotoxic serums. Arch. Pathol.49:291-297.

7. Henson, J. B., J. R. Gorham, G. A. Padgett, and W. C.Davis. 1969. Pathogenesis of the glomerular lesions inAleutian disease of mink. Immunofluorescent studies.Arch. Pathol. 87:21-28.

8. Hicks, J. D., and F. M. Burnet. 1966. Renal lesions in the"autoimmune" mouse strains NZB and Fl NZB x

NZW. J. Pathol. Bacteriol. 91:467-477.9. Karstad, L. 1967. Aleutian disease. A slowly progressive

viral infection of mink. Curr. Top. Microbiol. Immu-nol. 40:1-21.

10. Kenyon, A. J., J. E. Gander, C. Lopez, and R. A. Good.

JTIAN DISEASE OF MINK 271

1973. Isolation of Aleutian mink disease virus byaffinity chromatography. Science 1:187-189.

11. Kochwa, S., and R. E. Rosenfield. 1964. Immunochemi-cal studies of the Rh system. I. Isolation and character-ization of antibodies. J. Immunol. 92:682-692.

12. Koffler, D., P. H. Schur, and H. G. Kunkel. 1967.Immunological studies concerning the nephritis ofsystemic lupus erythematosus. J. Exp. Med.126:607-624.

13. McGuire, T. C., T. B. Crawford, J. B. Henson, and J. R.Gorham. 1971. Aleutian disease of mink: detection oflarge quantities of complement-fixing antibody to viralantigen. J. Immunol. 107:1481-1482.

14. Mardiney, M. R., and H. J. Miiller-Eberhard. 1965.Mouse ,Blc-globulin: characterization in the comple-ment reaction. J. Immunol. 94:877-882.

15. Pan, I. C., K. S. Tsai, and L. Karstad. 1970. Glomerulo-nephritis in Aleutian disease of mink: histological andimmunofluorescent studies. J. Pathol. 101:119-127.

16. Porter, D. D., F. J. Dixon, and A. E. Larsen. 1965.Metabolism and function of gammaglobulin in Aleu-tian disease of mink. J. Exp. Med. 121:889-900.

17. Porter, D. D., and A. E. Larsen. 1967. Aleutian disease ofmink: infectious virus-antibody complexes in the se-rum. Proc. Soc. Exp. Biol. Med. 126:680-682.

18. Porter, D. D., A. E. Larsen, and H. G. Porter. 1969. Thepathogenesis of Aleutian disease of mink. I. In vivoviral replication and the host antibody response to viralantigen. J. Exp. Med. 130:575-589.

19. Saison, R., L. Karstad, and T. J. Pridham. 1966. Viralplasmacytosis (Aleutian disease) in mink. VI. Thedevelopment of positive Coombs tests in experimentalinfections. Can. J. Comp. Med. 30:151-156.

20. Saison, R., and L. Karstad. 1968. Evidence of an autoim-mune reaction in viral plasmacytosis (Aleutian disease)of mink as demonstrated by the Coombs test. Proc.11th Congr. Int. Soc. Blood Transf., Sydney, Biblio-theca Haematol., 29. Part 2, pp. 486-493. Karger,Basel, Switzerland.

21. Williams, R. C., Jr., J. D. Russel, and A. J. Kenyon. 1966.Anti-gammaglobulin factors and immunofluorescentstudies in normal mink and mink with Aleutian dis-ease. Amer. J. Vet. Res. 27:1447-1454.

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