INFECTION AND IMMUNITY, Aug. 1973, p. 272-277Copyright © 1973 American Society for Microbiology

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

Hypogammaglobulinemia and ThymicHypoplasia in Horses: a Primary Combined

Immunodeficiency DisorderT. C. McGUIRE AND MARINEL J. POPPIE

Department of Veterinary Pathology, Washington State University, Pullman, Washington 99163

Received for publication 31 January 1973

A severe combined immunodeficiency disorder was demonstrated in twoArabian foals which were full siblings. The defect in the B-lymphocyte systemwas shown by hypogammaglobulinemia, lymphopenia, and absence of germinalcenters. The almost total absence of thymic tissue in one foal and the lack ofthymic dependent lymphocytes in the spleens of both foals demonstrate aT-lymphocyte defect. In a retrospective study of total available Arabian foalcases, 4 of 15 had evidence of immunodeficiency.

The types of immunodeficiencies of man areextremely varied and classification is difficult.When the disorders are viewed from their effecton the lymphoid system, three major groupsemerge (10, 12). Some, such as Bruton-typeinfantile X-linked agammaglobulinemia (3), in-volve a defect in the B-lymphocyte systemwhich leads to a deficiency of immunoglobulinsynthesis, whereas cell-mediated immunity isnormal. Other immunodeficiencies, includingthymic hypoplasia (DiGeorge's Syndrome) (7),have normal immunoglobulin levels but lack aT-lymphocyte system, resulting in defectivecell-mediated immunity. Disorders in the thirdcategory involve defects in both antibody pro-duction and cell-mediated immunity and areclassified as severe combined immunodeficien-cies (10). Autosomal recessive type (Swiss typeagammaglobulinemia) (15) is an example ofthis type of disorder.Few instances of primary defects of the im-

mune system are described in domestic andlaboratory animals. The "nude" mouse strain iscongenitally athymic with alterations in cell-mediated immunity (8, 22, 24). Nude mice havenormal immunoglobulin levels except for im-munoglobulin (Ig)A but have markedly de-pressed responses to thymic dependent anti-gens (5). These mice are an extremely usefulmodel for studying immunodeficiencies as wellas a convenient source of athymic experimentalanimals. A selective immunoglobulin (IgG2) de-ficiency in cattle with susceptibility to infec-tion has been reported (21).This report describes a disorder in Arabian

foals with characteristics of a primary combinedimmunodeficiency disorder. An abstract of thispaper has been published (T. C. McGuire andM. J. Poppie, 1973, Fed. Proc. 32:821). Failureto properly identify the disorder in the originalcase caused the study to be almost entirelyretrospective.

MATERIALS AND METHODSAnimals and sera. The initial case (foal A) was

a female Arabian foal that died at 7 weeks of age after3 weeks of progressive pneumonia. The second case(foal B) was a full sibling to foal A which was born 1year later. This male foal developed a progressivepneumonia at 9 weeks of age. Other foal tissue andhistories were acquired from the files of the Depart-ment of Veterinary Pathology at Washington StateUniversity.

Sera were obtained from foals A and B, their damand sire, and from seven apparently normal Arabianfoals varying from 5 to 8 months of age.

Hematology. Hemoglobin, packed cell volume,total and differential white blood counts, and serumprotein were determined by routine methods.Immunoglobulin quantitation. Serum immuno-

globulin levels were determined by single radialimmunodiffusion (17). Equine immunoglobulinG, IgG(T), IgM, IgA, and aggregating immunoglobu-lin (AI) were isolated by techniques that have beenpreviously described in detail (20). The purity of theisolated immunoglobulins was evaluated by im-munodiffusion and immunoelectrophoresis. These pu-rified immunoglobulins were used as references afterprotein concentrations were determined by the Lowrymethod (18). Specific antisera to IgG, IgG(T), IgM,IgA, and Al were prepared in rabbits and wereevaluated and absorbed as previously described (20).The appropriate dilution of each antiserum was

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determined for single radial immunodiffusion and theamount of each immunoglobulin in pooled normalhorse serum; it was used as a reference in subsequentdeterminations of immunoglobulin levels in horsesera.

RESULTSClinical and hematologic findings. Foal A

(female) developed a clinically evident pneu-monia at 4 weeks of age. Bacteriologic examina-tion of nasal exudate revealed the presence ofEscherichia coli, Staphylococcus aureus, andStreptococcus pyogenes. Three weeks of exten-sive antibiotic and supportive therapy wereineffective and the foal died. The most notablehematologic finding (Table 1) was the presenceof only 162 to 580 lymphocytes per mm3,whereas the normal range is 2,000 to 6,000lymphocytes per mm3.

Foal B (male) developed a cough with puru-lent nasal discharge which required therapy at 9weeks of age. The total white blood cell count(WBC) on initial examination was 3,000 permm3 with only 30 lymphocytes per mm3 (Table1). Three days later, the total WBC count was12,000 per mm3. A differential count was notdone at this time. Despite antibiotic therapy,the foal failed to recover. After 3 weeks ofprogressive pneumonia, the owners requestedeuthanasia.

Histopathologic observations. The lungs offoal A were diffusely involved with broncho-pneumonia which was superimposed on a well-developed subacute interstitial pneumonia.Bronchial and bronchiolar epithelium had pro-liferated and epithelial debris was present in the

TABLE 1. Results of hematologic examination

Foal A Foal B,Findings 4 weeks 7 weeks 10 weeks

old old old

Hemoglobin (g%) 15.5 14.6 NDaPacked cell volume 41 38 38

(%)Protein (g%) 6.2 6.5 NDWBC (count/mm3) 11,600 8,100 3,OOObNeutrophils 9,048 6,561 2,888

(segmented/mm3)Neutrophils 698 405 0(band/mm3)

Eosinophils/mm3 1,276 0 60Monocytes/mm3 0 972 0Lymphocytes/mm3 580 162 30

a ND, Not done.b Three days after this white blood cell count, the

total was 12,000. A differential count was not made atthis time.

lumina. Near the luminal surface of the epi-thelium there were large basophilic intranuclearinclusion bodies. These inclusions were typicalof those caused by adenoviruses and have beendescribed in adenoviral pneumonias of youngArabian foals (16, 19, 28).The only lymphoid tissue available for micro-

scope examination from foal A was the spleen.Alterations of the organ were striking: there wasan almost complete absence of lymphocytes.Neither periarteriolar lymphocytic sheaths norgerminal centers were present. Plasma cells andlymphocytes were almost totally absent in otherareas of the spleen.The lung alterations in foal B were very simi-

lar to foal A including the presence of adeno-virus-like inclusion bodies. In the pancreas,there were ductal epithelial proliferation andnecrosis with inclusion bodies similar to thosein the lung. The acini surrounding these ductswere disorganized and necrotic with beginningfibrosis. Most islets of Langerhans were intact.

Germinal centers as well as the surroundingmantle zone of lymphocytes were absent inseveral lymph nodes from foal B (Fig. 1). Theselymph nodes were also deficient in lymphocytesand plasma cells in the diffuse cortex andmedulla. The cell types populating the nodeswere difficult to identify; at least some had theappearance of lymphocytes, whereas many wereelongated and similar to fibroblasts. The arteri-oles of the spleen were surrounded by fibroblas-tic-type cells instead of a lymphocytic sheath(Fig. 2). Most germinal centers were absentbut a few partial ones were detectable. The redpulp contained very few lymphocytes andplasma cells.

FIG. 1. Lymph node cortex demonstrating absenceof germinal centers.

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The most dramatic morphologic changes infoal B were observed in the thymus. The organhad a yellow color and appeared fatty butretained lobular architecture and was subnor-mal in size. Seen through a microscope, over90% of the thymus was adipose tissue. Islandsof lymphoid cells were interspersed in the fatand partially formed Hassal's corpuscles couldbe found in the lymphoid islands (Fig. 3).Immunoglobulin levels. Single radial im-

munodiffusion did not detect IgM, IgA, or Al in

wor; f =̂ t4

6*0~~~~~

* r. *4g;t~_- S

'I~~~~~~~~~~~~~~~~~I

FIG. 2. Spleen of immunodeficient foal with anabsence of thymic dependent periarteriolar lym-phocytic sheaths.

the sera from foals A and B (Table 2). IgG andIgG(T) were present in these sera but at levelsbelow the normal range for Arabian foals (Table2). Immunoglobulin levels for the dam and sireof the foals were within the normal range.Other foals. After the defect was verified in

these two foals, the files of the WashingtonState University Department of VeterinaryPathology were searched for similar cases. Eventhough the data was incomplete, at least 4 ofthe 15 Arabian foal cases available had evidenceof immunodeficiency (Table 3). Spleens fromthree foals lacked germinal centers and periar-teriolar lymphocytic sheaths (foals 1, 2, and 3 inTable 3). Hematologic results from two of thesedemonstrated a lymphocytopenia. The casehistory from a fourth foal revealed lymphocy-topenia and hypogammaglobulinemia (foal 4in Table 3). No lymphoid tissue was available

TABLE 2. Immunoglobulin levels in immunodeficientand normal Arabian foals

Immunoglobulin Foal A Foal B Normal foal range

IgG 1.50a 2.80 3.46-8.20IgG(T) 0.31 0.44 3.00-6.40IgM <0.03 <0.03 0.24-0.46IgA <0.02 <0.02 0.34-0.88Al <0.03 <0.03 <0.03-0.15

a Values are expressed as milligrams per milliliterand were determined by single radial immunodiffu-sion.

* < <> 4P

FIG. 3. Thymus from an immunodeficient foal. Most of the organ was fat tissue but a few islands of cellsresembling lymphocytes were present. Insert shows Hassal's corpuscle in lymphoid island of thymus.

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TABLE 3. Summary of data from Arabian foals with probable immunodeficiency

Foal no.Determinations

1 ~~~23 4

Age 12 weeks olda 12 weeks old 2 weeks old 10 weeks oldSex M F M MDuration of clinical disease 4 weeks 4 weeks 1 week 3 weeksLymphocytopenia (< 1,000/mm3) Yes Yes NAb YesAbsence of B- and T-lymphocytesc Yes Yes Yes NAAdenovirus pneumonia Yes Yes Yes YesSecondary bacterial pneumonia Yes Yes No YesHypogammaglobulinemia NA NA NA Yes

a Age at time of death.b NA, Not available.c Absence of B- and T-lymphocytes was demonstrated by a complete lack of splenic germinal centers and

periarteriolar lymphocytic sheaths.

from this case. This information suggests thatimmunodeficiency may be common in Arabianfoals. These four cases plus three others (nosera, leukocyte counts, or lymphoid tissue wereavailable from the last three cases) had an in-terstitial pneumonia with basophilic intranu-clear epithelial inclusion bodies apparently dueto adenovirus infection. This was further com-plicated by a purulent bronchopneumoniaevidently caused by bacteria. The remainingeight Arabian foal cases had no evidence ofimmunodeficiency or adenovirus infections andhad died of a variety of causes. An examinationof tissue from 15 non-Arabian foals with pneu-monia ranging in age from 3 days to 3 monthsof age did not reveal evidence of immunodefi-ciency or adenovirus infection.

DISCUSSIONThat a defect is present in the B-lymphocyte

system of the foals described is clearly shown bythe hypogammaglobulinemia, lymphopenia,and absence of germinal centers in spleens andlymph nodes. The absence of IgM providesconsiderable support for this conclusion. Previ-ous studies in our laboratory (T. C. McGuireand T. B. Crawford, 1973, Amer. J. Vet. Res.,in press) and others (14) demonstrated thatpre-suckle foal serum contains IgM, suggestingthe foal has the ability to synthesize this im-munoglobulin prior to birth. The lack of IgMand IgA with only low levels of IgG and IgG(T)in the serum of 2- and 3-month-old foals is firmevidence. of a defect in immunoglobulin synthe-S1S.The absence of thymus and thymic depend-

ent lymphoid tissue (periarteriolar lymphocyticsheaths) (4, 23) provides evidence of a T-lym-phocyte defect. However, functional studies willbe necessary to definitively validate a disorderin cell-mediated immunity.

The primary lesion in the lungs of both foalswas an interstitial pneumonia apparentlycaused by an adenovirus infection. This wascomplicated by a secondary bacterial-inducedbronchopenumonia. Adenovirus inclusionbodies and interstitial pneumonia were presentin 7 of 15 other Arabian foal cases in a retrospec-tive study. Four of these seven foals had evi-dence of an immunodeficiency disorder. Otherinvestigators have described fatal adenoviralinfections in Arabian foals with apparently nocases occurring in other breeds (16, 19, 28).Lymphocytopenia and lymphoid atrophy weredescribed as consistent findings (19). We feelthat, in the Arabian foal cases we studied andpossibly in those reported by others (16, 19, 28),an immunodeficiency disorder was primary andimpaired the ability of the foals to combatadenovirus and other infections. This conclu-sion is based on several considerations. (i) Ourstudies indicate a high prevalence of im-munodeficiency in the Arabian breed. Six of the17 Arabian foal cases available had evidence ofimmunodeficiency, whereas none were found inother breeds. (ii) The presence of immunodefi-ciency in full siblings suggests a genetic origin.(iii) The severity of the involvement of both theB-lymphocyte system (lymphocytopenia, hypo-gammaglobulinemia and absence of germinalcenters) and the T-lymphocyte system (thymichypoplasia and absence of thymic dependentlymphocytic sheaths in the periarteriolar areasof the spleen) is similar to that described insevere primary combined immunodeficienciesof man (10, 12, 15).

Certain viruses are known to cause im-munosuppression and some even cause thymicatrophy. The most notable of these are murine(2, 9) and feline (1, 25) leukemia viruses whichcause marked thymic atrophy but the effects onthe thymic dependent areas and germinal cen-

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ters of the spleen and lymph node are not nearlyas marked as described in the foals. The sever-ity of lymphoid changes induced by other vi-ruses (13, 27, 28) do not approach those we havedescribed in Arabian foals. Furthermore, wehave been unable to find any significant lymph-oid lesions described in adenovirus infections ofother species. (iv) Precipitating antibody to agroup-specific adenovirus antigen has beendetected in 43 of 178 horse sera from unknownbreeds indicating that adenoviruses are com-mon among horses. It seems likely that theseinfections are only serious when some type ofimmunodeficiency occurs. Fatal pulmonary in-fections due to adenovirus have been reported inhuman patients with severe combined im-munodeficiences (11).

If a genetic basis for the disorder is assumed,certain important deductions can be made. Asex-linked mode of inheritance seems unlikelysince one of the foals was female and the othermale. Further, the disorder is apparently suble-thal. The dam and sire of the foals had normalimmunoglobulin levels and were without histo-ries of recurrent infections. These factors sug-gest the immunodeficiency was inherited as anautosomal recessive trait.

Defects in the T- and B-lymphocyte systemsand a possible autosomal recessive mode ofinheritance suggest that this condition in horsesis very similar to the Swiss type of severecombined immunodeficiency in man. This con-dition in horses represents the only currentlyknown example of primary combined im-munodeficiency in any animal other than man.A breeding program is being conducted withdams and sires of affected foals. Subsequentstudies with this defect in horses should provideimportant information concerning the basis ofthe defect and T- and B-lymphocyte functionand provide a model for the evaluation of thera-peutic procedures which may be applicable toman.

ACKNOWLEDGMENTSThis investigation was supported in part by Public Health

Service grant RR-00515 from the National Institutes ofHealth and the Agricultural Research Service, U. S. Depart-ment of Agriculture.

The assistance and cooperation of Keith Banks, GeraldHegreberg, Barrie Grant, Dan Dahl, and Bob Saunders wasappreciated.

LITERATURE CITED1. Anderson, L. J., W. F. H. Jarrett, 0. Jarrett, and H. M.

Laird. 1971. Feline leukemia-virus infection of kittens:mortality associated with atrophy of the thymus andlymphoid depletion. J. Nat. Cancer Inst. 47:807-816.

2. Arnesen, K. 1958. Preleukemic and early leukemicchanges in the thymus of mice. Acta. Pathol. Micro-biol. Scand. 43:350-364.

3. Bruton, 0. C. 1952. Agammaglobulinemia. Pediatrics9:722-728.

4. Craddock, C. G., R. Longmire, and R. I. McMillian. 1971.Lymphocytes and the immune response. N. Engl. J.Med. 285:378-384.

5. Crewther, P., and N. L. Warner. 1972. Serum immuno-globulins and antibodies in congenitally athymic(Nude) mice. Aust. J. Exp. Biol. Med. Sci. 50:625-635.

6. Darbyshire, J. H., and H. G. Pereira. 1964. An adenovirusprecipitating antibody present in some sera of differentanimal species and its association with bovine respira-tory disease. Nature (London) 201:895-897.

7. DiGeorge, A. M. 1965. Congenital absence of the thymusand its immunologic consequences: concurrence withcongenital hypoparathyroidism, p. 116-121. In D. Berg-sma and R. A. Good (ed.), Immunologic deficiencydiseases in man. Birth defects original article series.National Foundation Press, New York.

8. Flanagan, S. P. 1966. 'Nude', a new hairless gene withpleiotropic effects in the mouse. Genet. Res. 8:295-298.

9. Frey-Wettstein, M., and E. F. Hays. 1970. Immuneresponse in preleukemic mice. Infect. Immunity2:398-403.

10. Fudenberg, H. H., R. A. Good, W. H. Hitzig, H. G.Kunkel, I. M. Roitt, F. S. Rosen, D. S. Rowe, M.Seligmann, and J. R. Soothill. 1971. Primary immunedeficiencies: report of a WHO committee. Pediatrics47:927-945.

11. Fulginiti, V. A. 1969. Immunologic deficiency states.Basic review of mechanisms of immunity. Clin. Pediat.8:216-224.

12. Good, R. A. 1972. Recent studies on the immunodeficien-cies of man. Amer. J. Pathol. 69:484-490.

13. Hanaoka, M., S. Suzuki, and J. Hotchin. 1969. Thymus-dependent lymphocytes: destruction by lymphocyticchoriomeningitis virus. Science 163:1216-1219.

14. Helms, C. M., and P. Z. Allen. 1971. A comparativeimmunological examination of some immunoglobulinsof several equine species. Comp. Biochem. Physiol.38B:439-449.

15. Hitzig, W. H. 1968. Swiss type of agammaglobulinemia,p. 82-87. In D. Bergsma and R. A. Good (ed.),Immunologic deficiency diseases in man. Birth defectsoriginal article series. National Foundation Press, NewYork.

16. Johnston, K. G., and D. R. Hutchins. 1967. Suspectedadenovirus bronchitis in Arabian foals. Aust. Vet. J.43:600.

17. Kohler, P. F., and H. J. Muller-Eberhard. 1967. Immuno-chemical quantitation of the third, fourth and fifthcomponents of human complement: concentrations inthe serum of healthy adults. J. Immunol. 99:1211-1216.

18. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-269.

19. McChesney, A. E., J. J. England, J. L. Adcock, L. L.Stackhouse, and T. L. Chow. 1970. Adenoviral infec-tion in suckling Arabian foals. Pathol. Vet. 7:547-565.

20. McGuire, T. C., and T. B. Crawford. 1972. Identificationand quantitation of equine serum and secretory immu-noglobulin A. Infect. Immunity 6:610-615.

21. Nansen, P. 1972. Selective immunoglobulin deficiency incattle and susceptibility to infection. Acta Pathol.Microbiol. Scand. 80:49-54.

22. Pantelouris, E. M. 1968. Absence of a thymus in a mousemutant. Nature (London) 217:370-371.

23. Parrott, J. M., M. A. B. deSoussa, and J. East. 1966.Thymus dependent areas in the lymphoid organs ofneonatally thymectomized mice. J. Exp. Med.123:191-204.

24. Pennycuik, P. R. 1971. Unresponsiveness of nude mice toskin allografts. Transplantation 11:417-418.

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25. Perryman, L. E., E. A. Hoover, and D. S. Yohn. 1972. 27. Snodgrass, M. J., S. D. Lowere, and H. G. Hanna. 1972.Immunologic reactivity of the cat: immunosuppression Changes induced by lactic dehydrogenase virus inin experimental feline leukemia. J. Nat. Caneer Inst. thymus and thymus-dependent areas of lymphatic49:1357-1365. tissue. J. Immunol. 108:877-892.

26. Rohovsky, M. W., and R. A. Griesemer. 1967. Experimen- 28. Todd, J. D. 1969. Comments on rhinoviruses and parain-tal feline infectious enteritis in the germ free cat. fluenza viruses of horses. J. Amer. Vet. Med. Ass.Pathol. Vet. 4:391-410. 155:387-390.

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