Fell Pony Syndrome: Characterization of DevelopmentalHematopoiesis Failure and Associated Gene Expression Profiles

Rebecca L. Tallmadge,a Tracy Stokol,b Mary Jean Gould-Earley,c Ed Earley,c Erica J. Secor,a Mary Beth Matychak,a andM. Julia B. Felippea

Departments of Clinical Sciencesa and Population Medicine and Diagnostic Sciences,b College of Veterinary Medicine, Cornell University, Ithaca, New York, USA, andLaurel Highland Farm & Equine Services LLC, Cogan Station, Pennsylvania, USAc

Fell Pony syndrome (FPS) is a fatal immunodeficiency that occurs in foals of the Fell Pony breed. Affected foals present with se-vere anemia, B cell lymphopenia, and opportunistic infections. Our objective was to conduct a prospective study of potentialFPS-affected Fell Pony foals to establish clinical, immunological, and molecular parameters at birth and in the first few weeks oflife. Complete blood counts, peripheral blood lymphocyte phenotyping, and serum immunoglobulin concentrations were deter-mined for 3 FPS-affected foals, 49 unaffected foals, and 6 adult horses. In addition, cytology of bone marrow aspirates was per-formed sequentially in a subset of foals. At birth, the FPS-affected foals were not noticeably ill and had hematocrit and circulat-ing B cell counts comparable to those of unaffected foals; however, over 6 weeks, values for both parameters steadily declined. Abone marrow aspirate from a 3-week-old FPS-affected foal revealed erythroid hyperplasia and concurrent erythroid and myeloiddysplasia, which progressed to a severe erythroid hypoplasia at 5 weeks of life. Immunohistochemical staining confirmed thepaucity of B cells in primary and secondary lymphoid tissues. The mRNA expression of genes involved in B cell development,signaling, and maturation was investigated using qualitative and quantitative reverse transcriptase PCR (RT-PCR). Severalgenes, including CREB1, EP300, MYB, PAX5, and SPI1/PU.1, were sequenced from FPS-affected and unaffected foals. Our studypresents evidence of fetal erythrocyte and B cell hematopoiesis with rapid postnatal development of anemia and B lymphopeniain FPS-affected foals. The transition between fetal/neonatal and adult-like hematopoiesis may be an important aspect of thepathogenesis of FPS.

Fell Pony syndrome (FPS) is a fatal disorder of young foals ofthe Fell Pony breed. FPS-affected foals are born healthy but

rapidly fail to thrive and experience severe anemia and lymphope-nia within several weeks of life. Pedigree analysis of the breedsuggests that FPS may have an autosomal recessive inheritance,and a recent genome-wide association study identified a mutationassociated with the syndrome on chromosome ECA26 (22). Themechanistic implications of this mutation have not been resolved.In the absence of effective therapies, careful breeding manage-ment is necessary to decrease FPS incidence. This is of particularimportance because the Fell Pony breed is already consideredthreatened due to the small population size globally (Rare BreedsSurvival Trust). In 2006, our laboratory reported the first case ofFell Pony syndrome in the United States, along with others in theCzech Republic and the Netherlands (6, 23, 28). Most recently, acase of the same syndrome was reported in a Dales pony, a breedthat also originated in England (21).

Fell Pony syndrome was initially described in the United King-dom in 1998 (60). The fatal illness became clinically apparent infoals within 1 month, and death occurred generally by 3 months oflife. Clinical and postmortem examinations revealed a syndromecomprised of anemia, immunodeficiency, and peripheral gangli-onopathy. Further findings included small numbers of late ery-throid precursors in the bone marrow, small thymi, a lack of sec-ondary lymphoid follicles or germinal centers, and the absence ofplasma cells. Opportunistic infections caused by cryptosporidiaand adenovirus were observed and suggested an immunodefi-ciency condition. The FPS-affected foals were both male and fe-male, no environmental cause could be identified, and the find-ings described were not consistent with other known causes ofanemia in foals. The authors concluded that a genetic defect was

responsible for this syndrome. A second clinical report with sim-ilar findings followed (57). Bell and coworkers (3) described thatFPS-affected foals had fewer total lymphocytes than healthy foals,and the distribution of CD4� and CD8� T lymphocytes appearednormal. The expression of major histocompatibility complex class II(MHC class II) molecule was decreased in peripheral blood lympho-cytes in comparison to that in healthy foals. The B lymphopenia wasconfirmed in 2 subsequent studies (67, 68). A functional consequenceof B lymphopenia is the inability to produce immunoglobulins (Igs);however, several studies did not find decreased serum IgG levels inFPS-affected foals, most likely due to the presence of maternal colos-tral antibodies (57, 60, 68). Nevertheless, enzyme-linked immu-nosorbent assay (ELISA)-based quantification revealed significantreductions in serum IgM concentrations (a parameter not con-founded by maternally derived antibodies) and the absence of a pri-mary humoral immune response in FPS-affected foals (67).

During fetal life, B cells are produced in the yolk sac, liver, andbone marrow at different stages of gestation (46, 63). In mice, ithas been demonstrated that the fetal liver is the major hematopoi-etic organ during embryonic development until birth, when thebone marrow becomes the major blood cell-producing organ(12). In contrast, in fetal pigs, B cells are detected in the bone

Received 20 April 2012 Accepted 9 May 2012

Published ahead of print 16 May 2012

Address correspondence to M. Julia B. Felippe, mbf6@cornell.edu.

Supplemental material for the article may be found at http://cvi.asm.org/.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/CVI.00237-12

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marrow and spleen approximately 2 weeks after their appearancein the liver, by day 45 of the 114-day gestation (62). Studies in ourlaboratory have demonstrated that B lymphopoiesis and Ig generecombination are active during equine fetal life, as with B lym-phopoiesis in fetal pigs (66). At 90 to 120 days (approximatelyone-third of the 330- to 340-day gestation), genes essential for Bcell development, including CD20, CD21, CD22, CD27, CD40,CD40LG, PTPRC/B220, CD79A, CD79B, RAG-2, IGHM, IGHD,IGHA, and IGHG isotypes and the lambda light-chain IGLC gene,are expressed in the liver, bone marrow, and spleen.

B lymphocytes and erythrocytes are the most severely affectedcell populations in ponies with FPS, and both undergo criticaldevelopmental stages in the bone marrow. The condition may becaused by independent or common genetic abnormalities thataffect both cell lines. Our hypothesis is that FPS-affected foals losethe expression or function of a gene essential to hematopoiesis. Toour knowledge, there have been no reports of humans with a sim-ilar clinical course of disease. Mouse models that resemble thecondition in FPS-affected Fell Pony foals include SPI1/PU.1 andEP300KIX mutants. SPI1/PU.1 mutants die during late gestation orshortly after birth with impaired erythroblast maturation and alack of B lymphocytes (1, 36, 49, 61). However, macrophage, neu-trophil, and T cell lineages are also affected in SPI1/PU.1 mutants.Mice with mutations in the EP300KIX domain that diminish bind-ing to CREB1 and MYB proteins exhibit anemia, B cell deficiency,thymic hypoplasia, megakaryocytosis, and thrombocytosis (30).

In most FPS-affected foals, the syndrome is not apparent atbirth but progresses rapidly, and anemia and B cell lymphopeniaare already present by the time of diagnosis, limiting the amountof information that can be obtained from absent cells. The sentinelcase identified at Cornell University Hospital for Animals led tothis prospective study of Fell Pony foals born on the same farm(23). An FPS-affected half-sibling of the index case was diagnosed,providing a unique opportunity to assess the progression of FPSwith longitudinal and postmortem samples for hematological,immunological, and molecular gene expression studies. For thefirst time, we were able to measure normal concentrations of Bcells and erythrocytes in the peripheral blood of FPS-affected foalsat birth and to document the progressive and rapid loss of bothcell types within the first few weeks of life. In addition, evaluationof bone marrow aspirates alluded to the importance of the transi-tion period between fetal/neonatal and adult-like differentiationof erythrocytes and B cells and the manifestation of disease. Ourstudies add novel clinical and molecular information about theprogressive depletion of B cells and erythrocytes in FPS-affectedfoals.

MATERIALS AND METHODSBlood and tissue samples. Samples were collected prospectively fromtwo Fell Pony herds in the United States from FPS-affected (n � 3) andhealthy unaffected (n � 49) Fell Pony foals at birth and up to the timeof euthanasia for FPS-affected foals or at 5 months of life for healthyfoals. The initial FPS-affected foal (FPS1) was submitted for euthana-sia at 39 days of life (23), and a second FPS-affected foal identified inthis study (FPS2) died at 51 days of life. Several years after this studywas initiated, a third FPS-affected foal was identified (FPS3), whichwas submitted for euthanasia at 79 days of life. Peripheral blood wascollected via jugular venipuncture into Vacutainer tubes containingheparin, EDTA, or no additives for lymphocyte phenotyping, hemo-gram assessment, or serum immunoglobulin concentration determi-nation, respectively. Peripheral blood lymphocytes were isolated from

heparinized venous samples using methods described previously (19).The isolated cells were processed for immunophenotyping using flowcytometry, and the excess were snap-frozen for gene expression anal-yses. Serum was separated from blood cells using centrifugation(3,000 � g for 15 min) and stored at �20°C. Not all analyses wereperformed on each sample, and some foals were sampled repeatedlyover time.

Bone marrow aspirates and core biopsy specimens were obtained fromthe wing of the ileum from 2 FPS-affected Fell Pony foals and 2 unaffectedFell Pony foals. Bone marrow, spleen, and mesenteric lymph node sam-ples were collected from FPS1 and FPS2 immediately after euthanasia andsnap-frozen in liquid nitrogen or preserved in Tissue-Tek OCT com-pound (optimal cutting temperature medium; SakurFinetek U.S.A., Inc.,Torrance, CA) and stored at �80°C until use. No unaffected, healthy FellPony foal was submitted to euthanasia during the study period; this breedis under danger of extinction, and individuals have high economic value.Therefore, archived tissue samples from 2 age-matched healthy non-FellPony breed foals (Warmblood, at 4 and 8 weeks of life) were used ascontrols. These experiments were approved by the Cornell UniversityCenter for Animal Resources and Education and Institutional AnimalCare and Use Committee for the use of live vertebrates in research.

Hemogram analysis and bone marrow aspirate cytology. EDTA-an-ticoagulated peripheral blood was processed with an automated hematol-ogy analyzer (ADVIA 120 hematology analyzer; Bayer Corporation, Tar-rytown, NJ) in the Clinical Pathology Laboratory at Cornell University.The analyzer provides total leukocyte, erythrocyte, and platelet countsand measurements of erythrocyte and platelet size (mean corpuscularvolume and mean platelet volume, respectively) and erythrocyte hemo-globin content (mean corpuscular hemoglobin concentration). A manualleukocyte differential count (100 leukocytes) and assessment of cell mor-phological features were performed on Wright’s-stained peripheral bloodsmears.

Smears of the bone marrow were prepared immediately after aspira-tion and stained with Wright’s stain before examination by one clinicalpathologist (T. Stokol). Assessment included estimates of cellularity andmegakaryocyte number, calculation of a myeloid cell-to-erythroid cellratio based on a 200 differential cell count, calculation of percentages oflymphocytes and plasma cells, and examination of morphological featuresof hematopoietic cells.

Flow cytometric analysis of lymphocyte surface markers. Isolatedperipheral blood lymphocytes were tested for cell surface molecule expressionusing murine monoclonal antibodies for equine T and B cells and anti-canineparvovirus antibody as a negative control (CD3 [Equine Leukocyte AntigenWorkshop (ELAW) II 98], CD4 [ELAW I 72], CD5 [ELAW I 53], CD8[ELAW I 12], CD19-like [ELAW II 73], IgM [ELAW II 23], and MHC class II[ELAW II 43]) (17, 38, 44, 55). After cell washes and labeling with a fluores-cein isothiocyanate (FITC)-conjugated secondary antimurine antibody(Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), leukocytesubpopulations were displayed in a dot plot and gated according to size basedon forward light scatter (FSC) and granularity based on 90-degree side lightscatter (SSC). Cells compatible with lymphocytes based on size and granular-ity were gated for analyses of percentages of positive cells.

Serum immunoglobulin concentrations. Postsuckle serum IgM andIgG concentrations were determined using commercially available radialimmunodiffusion kits for horses (VMRD, Pullman, WA) per the manu-facturer’s instructions. A standard curve was generated with the knownconcentrations of purified equine immunoglobulins provided in the kitand their respective precipitate diameters. The concentrations of IgM orIgG in each serum sample were determined by comparing the individualprecipitate diameters to that of the standard curve. Serum samples werediluted when values were greater than the upper limit of the standardcurve.

Immunohistochemistry of lymphoid tissues. Five-micrometer tissuesections were cut with a cryotome and placed on glass slides. The sectionswere fixed for 10 min in acetone at 4°C. Immunohistochemical labeling

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was performed in a humidity chamber at room temperature, as previouslydescribed (18). Tris-buffered saline (TBS) was used for washes. Blockingsteps included separate 15-min incubations with (i) 0.3% hydrogen per-oxide (Fisher Scientific, Fair Lawn, NJ) in 0.1% sodium azide (Sigma, St.Louis, MO) and TBS and (ii) 10% normal goat serum in TBS. The tissueswere incubated for 2 h with the following primary monoclonal antibodies:irrelevant monoclonal antibody mouse anti-canine parvovirus, mouseanti-horse B cells (IgM and CD19-like), and mouse anti-horse T cells(CD4, CD8) (38, 44, 55). The secondary antibody, a goat-anti mouse IgGconjugated to horseradish peroxidase (Jackson ImmunoResearch Labo-ratories, Inc.), was incubated for 30 min. Substrate solution was preparedfrom concentrated acetate buffer, 3-amino-9-ethylcarbazole (AEC) chro-mogen, and hydrogen peroxide (AEC staining kit; Sigma). Counterstain-ing was performed using hematoxylin for 1 min (Fisher Scientific).

RT-PCR, subtractive hybridization, and sequencing. RNA was iso-lated from snap-frozen bone marrow and spleen samples following ho-mogenization with a QIAshredder (Qiagen, Valencia, CA) as directed bythe RNeasy kit instructions (Qiagen). One microgram of RNA was treatedwith DNase I (Invitrogen, Carlsbad, CA). cDNA synthesis reaction mix-tures contained 1� Moloney murine leukemia virus (M-MuLV) reversetranscriptase (RT) buffer, 5.5 mM MgCl2, 0.5 mM deoxynucleosidetriphosphates (dNTPs), 2.5 �M oligo(dT) (Applied Biosystems, FosterCity, CA), 0.4 U RNasin RNase inhibitor (Promega, Madison, WI), and 1U Moloney murine leukemia virus reverse transcriptase (MuLV RT; Ap-plied Biosystems); control samples did not receive M-MuLV RT. Ampli-fication reaction mixtures contained 1� PCR buffer, 1.5 mM MgCl2, 0.25mM dNTPs, 0.6 �M forward and reverse primers (Integrated DNA Tech-nologies, Coralville, IA), and 2 U Taq polymerase (Invitrogen).

Based on the literature, we composed a list of genes that, if mutatedor dysregulated, might play a role in the lack of B cell and erythrocytedevelopment in foals with FPS. RT-PCR assays were designed for thefollowing genes, which were sorted into four categories based on func-tion: (i) genes known to be important for supporting hematopoiesis inthe bone marrow, including IL-7 and its receptor (IL7R), FLT3 and itsligand (FLT3LG), KIT and its ligand (KITLG), and PTPN2, (ii) genesimperative for B lymphocyte development, including SPI1/PU.1,EBF1, TCF3/E2A, PTPRC/B220, PAX5, CD19, and IGHM, (iii) genescritical for B lymphocyte function and signaling, including IGHG,CD79A, CD79B, BTK, POU2F1, POU2F2, POU2AF1, JAK1, JAK3,STAT5A, and TRAF2, and (iv) other genes identified by subtractivehybridization and literature searches, including hemoglobin genes,EP300, CREBBP, CREB1, and MYB (2, 5, 9, 11, 14, 15, 24, 25, 29, 30,32–35, 37, 40, 41, 45, 48, 50, 52, 54, 56, 58, 59, 61, 71–77). RT-PCRamplification of the �-actin (ACTB) gene was included as a cDNAquality control.

Primers were designed from equine sequence databases (EST andgenomic trace archives) or consensus sequences (see Table S1 in the sup-plemental material; the assembled genome sequence was not available atthat time). Thermal cycling parameters were 95°C for 5 min, 35 cycles of95°C for 60 s, 58°C for 60 s, and 72°C for 30 s, and a final extension of 72°Cfor 10 min. Amplification products were run on 1% agarose gels andstained with ethidium bromide for visualization.

Quantitative real-time reverse transcriptase PCR was performed with

50 nanograms of RNA, 500 nM primer, and iScript one-step RT-PCR withSYBR green mix (Bio-Rad, Hercules, CA) in a CFX96 real-time PCR de-tection system (Bio-Rad). Reactions were performed in triplicate. Cyclingparameters were as follows: 1 cycle of 50°C for 10 min, 1 cycle of 95°C for5 min, and 40 cycles of 95°C for 10 s and then 60°C for 30 s, followed bymelt curve analysis. SYBR primers spanning intron/exon boundaries weredesigned with Beacon Designer 7.91 software (Premier Biosoft Interna-tional, Palo Alto, CA) and were synthesized by Eurofins MWG Operon(Huntsville, AL) (see Table S1 in the supplemental material).

For absolute quantitation, RNA standard curves were prepared foreach gene. Each target gene was amplified from healthy adult bone mar-row cDNA (primers are listed in Table S1 in the supplemental material)(iProof polymerase; Bio-Rad), cloned (pJET1.2 vector; Fermentas, GlenBurnie, MD), and sequenced at the Cornell University Life Sciences CoreLaboratories Center Genomics Facility (Ithaca, NY). RNA transcriptionwas performed on linearized plasmid DNA (Fermentas), purified (ZymoResearch Corporation, Irvine, CA), and quantified (NanoDrop). Tenfoldserial dilutions were made for the standard curve. SYBR primers werevalidated on the RNA standard curve. Data were analyzed with CFX Man-ager software (Bio-Rad) to determine numbers of mRNA transcripts. Dueto the small sample size, no statistical comparisons were performed.

Subtractive hybridization was performed as directed by the instruc-tions of a PCR-Select cDNA subtraction kit (Clontech, Mountain View,CA) in bone marrow samples from FPS2 and a Warmblood control foal.

To obtain full-length transcripts for sequencing of selected genes,gene-specific cDNA synthesis was performed with the Superscript IIIfirst-strand synthesis system for RT-PCR (Invitrogen) and amplificationwas performed with iProof high-fidelity DNA polymerase (Bio-Rad).PCR products were purified and cloned, and at least 3 clones per gene weresequenced. Sequences were analyzed with VectorNTI (Invitrogen) soft-ware.

Nucleotide sequence accession numbers. Accession numbers ofgenes submitted to GenBank are listed in Table 1.

RESULTS

To assess the temporal pattern of anemia, hematocrits were deter-mined prospectively and sequentially from Fell Pony foals. Atbirth, two FPS-affected foals had hematocrits below or within thelow range of the unaffected foals, and all 3 FPS-affected foals be-came severely anemic by 3 to 6 weeks of life. Data are not availablefor one of the affected foals at birth (Fig. 1).

Total lymphocyte counts were also monitored in FPS-affectedand unaffected Fell Pony foals to determine the presence of lym-phopenia. At birth, FPS-affected foals had total lymphocytecounts comparable to those of age-matched healthy control foals(Fig. 2). The lymphocyte counts of two of the foals failed to in-crease with age. One affected foal had lymphocyte counts compa-rable to those of unaffected foals at 6 weeks of life.

We assessed the percentage of peripheral blood lymphocytesexpressing the cell surface markers for B cells (CD19-like andIgM), T cells (CD3, CD4, CD5, and CD8), and the MHC class II

TABLE 1 Genes of interest sequenced from FPS-affected Fell Pony foals

GeneSequencelength (bp)a Regions sequencedb Sequence difference(s)

GenBankaccession no.

CREB1 1,412 Exon 1 through 3= UTR None JQ044378EP300 6,533 5= UTR to last exon 1 A to G and 1 T to C synonymous substitution JN979561MYB 2,150 Exon 2 through 3= UTR None JQ070070PAX5 1,173 5= UTR through 3= UTR 1 C to T synonymous substitution JQ044379SPI1/PU.1 820 5= UTR through last exon None JN979559a Nucleotide sequence length excluding primers.b UTR, untranslated region.

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molecule in all lymphocytes using flow cytometry. FPS2 and FPS3foals were born with a peripheral blood B cell subpopulation dis-tribution (CD19-like� IgM�) comparable to those of unaffectedfoals and adult Fell Pony horses. However, a B cell lymphopeniadeveloped rapidly with age in all 3 FPS-affected foals. The presenceof B lymphopenia is a key feature of the FPS phenotype (67, 68).The expression of MHC class II on lymphocytes in FPS2 and FPS3was comparable to that of unaffected foals in the time points mea-sured, whereas FPS1 had low expression by 6 weeks of life. Thelatter observation was not due to poor reagent recognition forindividual polymorphic molecules because monocytes from thesame blood samples of FPS-affected foals labeled positively withappropriate mean fluorescence intensity (data not shown). Thedistribution of CD3�, CD4�, CD5�, and CD8� T cell subpopu-lations in FPS-affected foals was comparable to that of unaffectedfoals (Fig. 3).

FPS-affected foals failed to increase in serum IgM concentra-tions with age, in contrast to unaffected Fell Pony foals (Fig. 4).The postsuckle serum IgM concentration in FPS2 in the first dayof life was comparable to that in unaffected foals but decreasedwithin the first month of life. Conversely, serum IgG concentra-tions remained above protective levels in FPS1 and FPS2 untileuthanasia (1,600 mg/dl for both).

Sequential bone marrow aspirates were performed at 3, 5, and8 weeks of life in FPS2, whereas a single marrow sample was col-lected from FPS3 at 6 weeks of life. Bone marrow aspirates werealso collected from 6 unaffected Fell Pony foals between 1 and 6weeks of life, with sequential aspirations at 2 and 6 weeks beingperformed for 1 foal. Bone marrow cytology results varied withtime of sampling in the FPS-affected foals. When FPS1 was se-verely anemic, with a hematocrit of 13% at 3 weeks of life, therewas an erythroid hyperplasia, with left-shifted maturation (in-creased proportion of immature precursors and relatively fewstages seen beyond basophilic rubricytes) and concurrent dyspla-sia (characterized by multinucleation, abnormal nuclear shapes,syncytial formation, hypochromic nuclei, and apoptosis) (Fig. 5).

Phagocytosis of erythroid progenitors by macrophages was ob-served. Mild dysplasia in myeloid cells (hypersegmented neutro-phils) was also evident. The morphological diagnosis was ineffec-tive erythropoiesis with bilineage dysplasia. These morphologicalabnormalities in erythroid and myeloid progenitors were not ob-served in aspirates taken from unaffected Fell Pony foals. Sequen-tial bone marrow aspirates revealed that the ineffective erythro-poiesis progressed in the foal to a severe erythroid hypoplasia at 5weeks of life (myeloid cell-to-erythroid cell ratio of 7.1 to 99:1;range in 5- to 6-week-old unaffected foals of 0.4 to 1.1:1), withonly early-stage erythroid progenitors (prorubricytes and baso-philic rubricytes, some of which were binucleate) being observed.Neutrophil hypersegmentation was still present. Similar resultsfor erythroid and myeloid progenitors were obtained with mar-row collected from FPS3 at 6 weeks of life. A bone marrow aspirateand a section of bone marrow taken at necropsy at 8 weeks of lifefrom FPS2 revealed a hypoplastic marrow consisting of myeloidprecursors and erythrophagocytic histiocytes, with no erythroidprecursors identified, compatible with a pure red cell aplasia.

To characterize the distribution of lymphocyte subpopulationsand organization of lymphoid tissues, samples collected at nec-ropsy from FPS1 and FPS2 were assayed using immunohisto-chemical staining (Fig. 6). In contrast to what was observed withthe control samples from age-matched healthy Warmblood foals,two equine B cell markers (CD19-like and IgM) revealed the ab-sence of B cells in the bone marrow and lymph nodes of FPS-affected foals and a paucity of B cells in the spleen, with the ab-sence of germinal centers. The distributions of T cells (CD4 andCD8 markers) were similar in the lymph nodes and spleens ofFPS-affected and healthy foals; however, T cells were absent fromthe bone marrow of FPS1.

The fact that B cells were absent in the bone marrow led us totest for the mRNA expression of genes known to be involved in Bcell hematopoiesis and development; RT-PCR was initially used toscreen for absent gene expression. To account for differences dueto developmental stages, FPS1 and FPS2 lymphoid tissue samples

FIG 2 Sequential total lymphocyte counts of FPS-affected and unaffected FellPony foals from birth to 18 weeks of life. At birth, FPS-affected foals (n � 3)had total lymphocyte counts comparable to those of age-matched healthycontrol foals (n � 29). The lymphocyte counts of two of the affected foals failedto increase with age. One affected foal had lymphocyte counts comparable tothose of unaffected foals at 6 weeks of life. Foals were sampled sequentially,although not all foals were sampled at each time point.

FIG 1 Sequential hematocrits of FPS-affected and unaffected Fell Pony foalsfrom birth to 18 weeks of life. At birth, FPS-affected foals (n � 2; data are notavailable for FPS3) had hematocrits below or within the low range of theunaffected foals (n � 29), and all 3 FPS-affected foals became severely anemicby 4 weeks of life. Foals were sampled sequentially, although not all foals weresampled at each time point.

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FIG 3 Sequential flow cytometric assessment of peripheral blood lymphocyte subpopulation distributions in FPS-affected and unaffected Fell Pony foals frombirth up to 21 weeks of life and in adult Fell Pony horses. Lymphocyte subpopulations are expressed as percentages of cells positive for the selected marker withinthe defined lymphocyte gate in flow cytometric dot plot analyses. FPS-affected foals (n � 2; data are not available for FPS1) were born with a peripheral blood Bcell subpopulation distribution (CD19-like� IgM�) comparable to those of unaffected foals (n � 46) and adult Fell Pony horses (n � 6). However, a B celllymphopenia developed rapidly with age in all 3 FPS-affected foals. The expression of MHC class II on lymphocytes in 2 FPS-affected foals was comparable to thatof unaffected foals in the time points measured, whereas FPS1 had low expression by 6 weeks of life. The distribution of CD3�, CD4�, CD5�, and CD8� T cellsubpopulations in all FPS-affected foals was comparable to that of unaffected foals. Foals were sampled sequentially, although not all foals were sampled at eachtime point.

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along with age-matched healthy Warmblood control sampleswere assayed by RT-PCR. mRNA expression was detected in bothFPS-affected and healthy Warmblood foals for IL-7 and its recep-tor (IL7R), FLT3 and its ligand (FLT3LG), KIT and its ligand(KITLG), PTPN2, SPI1/PU.1, TCF3/E2A, PTPRC/B220, CD19,and IGHM (Fig. 7). No mRNA for EBF1 was detected in the spleenof FPS2, but transcripts were detected in the bone marrow of FPS1and FPS2. Remarkably, PAX5 mRNA expression was not detectedin either the bone marrow or spleens of FPS1 and FPS2, and thusthe entire coding sequence of PAX5 was determined from an age-matched healthy Warmblood foal and FPS2. Only one nucleotidedifference was identified in the PAX5 sequence from FPS2 (Table1); yet, this polymorphism did not cause an amino acid change.Since this work was completed, the annotated equine genome se-quence has become available. The predicted equine PAX5 mRNAsequence is identical to the sequence that we determined from thehealthy Warmblood foal. Although mRNA expression was de-tected, the coding sequence of SPI1 was specifically determinedbecause SPI1/PU.1 mutants have impaired erythroblast matura-tion and lack B lymphocytes (61). No sequence differences wereidentified between an FPS-affected and a healthy Warmblood foal(Table 1). The sequence predicted from the horse genome pub-lished more recently is also identical to the sequences that weobtained.

The mRNA expression of genes essential to B lymphocyte func-tion and signaling, IGHG, CD79A, CD79B, BTK, POU2F1,POU2F2, POU2AF1, JAK1, JAK3, STAT5A, and TRAF2, was de-tected in bone marrow and spleen from FPS1, FPS2, and thehealthy Warmblood foals.

To identify differentially expressed genes in an unbiasedmanner, subtractive hybridization was undertaken using sam-ples from FPS2 and an age-matched healthy Warmblood foal.Five genes that differed in mRNA expression levels were de-tected with this method: Ig heavy and light chain, hemoglobinalpha (HBA), hemoglobin beta (HBB), and eukaryotic elonga-tion translation factor 1 alpha 1 (EEF1A1). The expression of

HBA, HBB, and EEF1A1 was further investigated by RT-PCRand detected in FPS1, FPS2, and healthy Warmblood foal bonemarrow or spleen tissues (Fig. 7).

Pursuing a candidate gene approach based on published liter-ature, we investigated the expression of genes involved in the phe-notype of mice with mutations in the EP300 KIX domain (EP300,CREBBP, CREB1, and MYB) (Fig. 7). mRNA expression was de-tected for EP300 and CREBBP genes in FPS1, FPS2, and healthyWarmblood foals using RT-PCR. The mRNA expression ofCREB1 was weak in the bone marrow of all foals tested and wasabsent (FPS1) or weak (FPS2) in the spleens of affected foals incomparison to healthy foals. MYB mRNA expression appeared tobe absent from FPS1 and FPS2 tissues, unlike in the bone marrowand spleens of healthy foals. Due to the importance of the EP300KIX domain in erythropoiesis and B cell lymphopoiesis (30), wenext sequenced the EP300 transcript from FPS2 and a healthyWarmblood foal, although primers flanking the entire coding se-quence could not be designed (lacking the last 1,130 bases of the7,263-nucleotide coding sequence). Two synonymous substitu-tions were identified between FPS-affected and healthy foals (Ta-ble 1), but the amino acid sequence of the KIX domain obtainedfrom these foals was identical to the human EP300 KIX domainsequence and to the predicted equine EP300 sequence.

Quantitative RT-PCR was undertaken for the three genes withsubjective differences in mRNA expression identified by standardRT-PCR: PAX5, CREB1, and MYB (Fig. 8). PAX5 mRNA tran-script numbers were low in the bone marrow of FPS1 and FPS2(272 and 615 copies, respectively), in contrast to transcript num-bers in age-matched healthy Warmblood foals (16,967 and 3,030copies). A similar pattern of PAX5 mRNA expression was ob-served in the spleen. The numbers of CREB1 transcripts detectedin FPS1 and FPS2 were comparable to the numbers in age-matched healthy Warmblood control foal tissues. Transcriptnumbers of MYB were low in FPS2 bone marrow, in contrast only

A B

C D

FIG 5 Photomicrographs of Wright’s-stained smears of a bone marrow aspi-rate collected from an FPS-affected Fell Pony foal at 3 weeks of life. Earlyerythroid progenitors (prorubricytes, basophilic rubricytes) dominate, withevidence of erythroid and myeloid dysplasia. (A) Abnormal nuclear shapes(lobulation, blebbing) in metarubricytes; (B) multinucleation or syncytial for-mation in a basophilic rubricyte (arrow); (C) binucleate prorubricyte; (D)binucleate prorubricyte (arrow), abnormal nuclear shape in a metarubricyte(arrowhead), and hypersegmented neutrophil. Scale bar � 10 �m.

FIG 4 Sequential serum IgM concentrations in FPS-affected and unaffectedFell Pony foals from birth (postsuckle) to 18 weeks of life and in adult Fell Ponyhorses. No increase in IgM concentrations with age was measured in post-suckle serum samples of FPS-affected foals (n � 3), unlike in unaffected FellPony foals (n � 35). Results for 2 adult Fell Pony horses overlap. Foals weresampled sequentially, although not all foals were sampled at each time point.

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to the transcript numbers in FPS1 (2.1 � 108 versus 4.5 � 1010

copies) and a healthy Warmblood control foal (1.1 � 1010 versus2.3 � 1010 copies) tissues. We also sequenced sections of theCREB1 and MYB genes in FPS1 and FPS2 spleen tissues and anage-matched healthy Warmblood foal. No sequence differences fromthe healthy foal sequence or the GenBank predictions were identifiedin the coding sequence of either CREB1 or MYB (Table 1).

DISCUSSION

This study describes the first prospective clinical and molecularinvestigation of Fell Pony syndrome from birth to euthanasia,including sequential collection of bone marrow samples for cyto-logic analysis of FPS-affected and unaffected Fell Pony foals. Thenovel findings about the temporal distribution of events that leadto anemia and B cell lymphopenia described in this study includethe following: (i) FPS-affected foals may be born with erythroidprecursors in the bone marrow and hematocrits and peripheralblood B cell distributions equivalent to those of healthy unaffectedfoals, suggesting a productive hematopoiesis during fetal life, (ii)they exhibit perinatal development of a progressive B lymphope-nia and anemia, (iii) there is bone marrow cytologic evidence ofinitial erythroid hyperplasia with dysplasia, followed by a rapidand progressive hypoplasia and aplasia, along with anemia, and(iv) they have concomitant mild myeloid dysplasia. Despite theevidence that B cells seem to be produced during fetal life, B cellswere not found in the bone marrow in postmortem samples, andthis absence was accompanied by low PAX5 gene expression in thebone marrow. All together, our data suggest a limited but detect-able B cell and red cell poiesis during fetal life that is not sustainedafter birth in FPS-affected foals.

The prospective aspect of our study was essential for revealingthe morphological abnormalities of erythroid and myeloid pre-cursors, which were likely missed by the previous studies due tothe timing of sample analysis and technique used (histology versuscytology) (28, 57, 60). We hypothesize that the timing of diseaseonset may be significant, since the disease manifests during the

transitioning period from fetal/neonatal to foal stages; perhapsbone marrow function is more critical in that period than duringfetal life. Indeed, there remains an important question about theimportance of the liver versus bone marrow as hematopoietic or-gans during the fetal life of FPS-affected individuals; one mayspeculate that the red cells and B cells detected in the peripheralblood of FPS-affected equine neonates may have been developedin the fetal liver, with or without limited bone marrow contribu-tions. All together, the erythroid hypoplasia/dysplasia/aplasia, Bcell lymphopenia/depletion, and myeloid dysplasia suggest a com-mon underlying defect affecting hematopoietic stem cell lineagedifferentiation rather than three separate abnormalities in ery-throid, lymphoid, and myeloid cells. Cytologic examination ofbone marrow aspirates from additional FPS-affected foals taken inthe first week of life would help to further elucidate the cell differ-entiation defect. Similar morphological abnormalities in erythro-poiesis are observed in other congenital hematopoietic disordersin humans and animals, including congenital dyserythropoieticanemia and Fanconi syndrome (8, 26). However, these syndromeslack the concurrent B cell lymphopenia.

The inability of FPS-affected foals to produce IgM in the crit-ical phase of primary pathogen exposure indicates an intrinsichumoral dysfunction. Equine colostrum is a poor source of IgMand has a half-life of 5 to 8 days; therefore, maternally derived IgMis depleted in foals by 4 weeks of life (39). In the absence of endog-enous production, serum IgM concentrations decrease over time,as we observed in the FPS-affected but not in the unaffected FellPony foals. The equine fetus produces IgM during gestation andmay be born with serum IgM concentrations of 10 to 25 mg/dl(66). Of note, FPS1 postsuckling serum IgM concentrations closeto birth were within the range observed for healthy Fell Pony foals,likely due to colostral transfer. Without analysis of presuckle sam-ples, we cannot rule out the possibility that affected foals also havea failure of fetal IgM production. In contrast to IgM, IgG has a longhalf-life of 23 to 30 days; thus, the normal serum IgG concentra-

FIG 6 Immunohistochemical staining for equine-specific lymphocyte markers for B cells (CD19-like and IgM) and T cells (CD4 and CD8) in lymphoid tissuesfrom FPS-affected Fell Pony foals and a healthy Warmblood foal. Lymphoid tissues were collected at necropsy and stained for lymphocyte markers usingimmunohistochemistry. FPS1 was submitted to euthanasia at the age of 5 weeks and FPS2 at 7 weeks; tissues from a 4-week-old Warmblood foal (shown) andan 8-week-old foal (not shown) were used as controls. No B cells were detected in the bone marrow and lymph nodes of FPS-affected foals. Splenic germinalcenters revealed a paucity of B cells in FPS-affected foals, in contrast to results for the age-matched healthy foal spleen. The distributions of T cells in the lymphnode and spleen are similar in all foals; however, T cells were not present in the bone marrow of FPS1.

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tions measured in older affected foals at the time of death or eu-thanasia were likely due to the persistence of circulating maternalantibodies (39). It is likely that serum IgG concentrations wouldhave progressively decreased in affected foals had they survivedlonger.

Absolute lymphopenia in FPS-affected foals likely reflects Bcell lymphopenia in peripheral blood and primary and secondarylymphoid tissues. It also may be a consequence of T cell lym-phopenia or dysfunction. The presence of T cell lymphopenia issupported by our data showing a lack of increases in total lympho-cyte counts with age in 2 FPS-affected foals and the absence of Tcells in the bone marrow of 1 FPS-affected foal. Also, affected foalstypically have small thymi and suffer from opportunistic infec-tions with cryptosporidia and adenovirus, despite normal (colos-trum-derived) serum IgG concentrations (23). Murine studieshave demonstrated that T cells are important in resisting primary

and secondary Cryptosporidium muris infection and in clearingthe pathogen (27, 47). CD4� and CD8� T cells are also imperativein the control of adenovirus infection (20, 64, 65). Our study didnot document a failure in the expression of MHC class II mole-cules in lymphocytes of FPS-affected foals, reported in other stud-ies; yet, such finding would further support abnormal lymphocytedevelopment (3, 23, 43). Further studies focused on T cell differ-entiation and function in FPS-affected foals are necessary to con-firm the involvement of yet another cell lineage in this syndrome,the T cells.

Our RT-PCR survey assessed the mRNA expression of genesinvolved in hematopoiesis, B lymphocyte development, function,and signaling and genes with potential involvement in FPS, basedon the literature. Results revealed that most of these genes, withthe exception of PAX5 and, potentially, MYB, were expressed inFPS-affected and healthy foals. PAX5 is essential for B lineage

FIG 7 mRNA expression of selected B cell and erythrocyte developmental genes in the bone marrow and spleens of FPS-affected Fell Pony foals and age-matchedhealthy Warmblood foals. The mRNA expression of most investigated genes is not different in the bone marrow of FPS-affected foals (FPS1, FPS2) from that inhealthy horse samples (H; n � 2), except for CREB1, MYB, and PAX5, which showed decreased mRNA expression in both tissues. RT-PCR amplification of the�-actin (ACTB) gene was included as a cDNA quality control.

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commitment and development, as well as in suppressing alterna-tive cell fates in the bone marrow; the expression of PAX5 is alsoessential for B cell survival peripheral tissues (51, 53). PAX5mRNA expression measured by standard and quantitative RT-PCR was low in both the bone marrow and spleens of FPS-affectedfoals but not in control foals; yet, downstream PAX5-dependentCD19 and CD79A genes were measured in FPS-affected foals us-ing standard RT-PCR (16, 54, 70). We hypothesize that the de-tected CD19 and CD79A mRNA expression results from the per-sistence of a limited number of B cells developed during fetal life.MYB expression is required for definitive erythropoiesis duringfetal life, and it dictates both T cell numbers and early T cell dif-ferentiation events (13, 59, 69). Our results are inconclusive for anabnormal expression of MYB in FPS-affected foals, and additionalsamples would be necessary to determine its role in FPS.

To date, it is difficult to reconcile our findings, the progressionof FPS, or the manifestation of postnatal anemia and B lymphope-nia with the nonsynonymous SLC5A3 mutation reported to be

predictive of disease status (22). The SLC5A3 P446L substitutionwas proposed to affect substrate binding, which would presum-ably result in the loss of SLC5A3 function (22). However, thephenotype of SLC5A3 null mice includes prenatal skeletal devel-opment defects, postnatal bone formation defects, and deathshortly after birth due to hypoventilation (4, 10). If indeed theSLC5A3 mutant is not functional, it may be consistent with theperipheral ganglionopathy observed in FPS-affected foals andthe requirement for myo-inositol for the development of periph-eral nerves (7). Human genome-wide association studies haveidentified associations between SLC5A3 variations and the risk ofcoronary artery disease and early-onset myocardial infarction (31,42). No impairments in cardiac development or function havebeen reported in FPS-affected foals. It is noteworthy that the tissuedistribution of SLC5A3 is uncertain and that a causal relationshipbetween the SLC5A3 mutation and Fell Pony syndrome has notbeen established. Therefore, it remains possible that other muta-tions in that genomic region may be responsible for the phenotypeof Fell Pony syndrome, which warrants continued studies of can-didate genes.

In summary, our study brings evidence of fetal erythrocyte andB cell hematopoiesis, with rapid postnatal development of anemiaand B lymphopenia, in FPS-affected foals. Sequential cytologicanalyses of bone marrow aspirates collected close to birth and inthe first few weeks of life provided valuable information about therapid progression of the syndrome, allowed us to observe a failurein hematopoiesis, introduced the finding of myeloid dysplasia,and highlighted the importance of early testing before clinicalsigns are fully manifested. Most essential B cell differentiationgenes and genes associated with erythropoiesis are expressed inaffected foals, and a potential common hematopoietic genetic de-fect is still under investigation. Further, this primary immunode-ficiency is a unique natural model for studying mammalian hema-topoiesis and developmental regulation in the perinatal period.The transition between fetal/neonatal and adult-like hematopoi-esis may be an important aspect of the pathogenesis of FPS.

ACKNOWLEDGMENT

We thank the staff in the Clinical Pathology Laboratory for excellent tech-nical assistance.

REFERENCES1. Back J, Dierich A, Bronn C, Kastner P, Chan S. 2004. PU.1 determines

the self-renewal capacity of erythroid progenitor cells. Blood 103:3615–3623.

2. Bedell MA, Cleveland LS, O’Sullivan TN, Copeland NG, Jenkins NA.1996. Deletion and interallelic complementation analysis of Steel mutantmice. Genetics 142:935–944.

3. Bell SC, Savidge C, Taylor P, Knottenbelt DC, Carter SD. 2001. Animmunodeficiency in Fell ponies: a preliminary study into cellular re-sponses. Equine Vet. J. 33:687– 692.

4. Berry GT, et al. 2003. Loss of murine Na�/myo-inositol cotransporterleads to brain myo-inositol depletion and central apnea. J. Biol. Chem.278:18297–18302.

5. Bordon A, et al. 2008. Enforced expression of the transcriptional coacti-vator OBF1 impairs B cell differentiation at the earliest stage of develop-ment. PLoS One 3:e4007. doi:10.1371/journal.pone.0004007.

6. Butler CM, Westermann CM, Koeman JP, Sloet van Oldruitenborgh-Oosterbaan MM. 2006. The Fell pony immunodeficiency syndrome alsooccurs in the Netherlands: a review and six cases. Tijdschr. Diergeneeskd.131:114 –118.

7. Chau JF, Lee MK, Law JW, Chung SK, Chung SS. 2005. Sodium/myo-inositol cotransporter-1 is essential for the development andfunction of the peripheral nerves. FASEB J. 19:1887–1889.

FIG 8 Quantitative RT-PCR of selected genes in the bone marrow and spleensof FPS-affected Fell Pony foals and healthy Warmblood foals. The expressionof PAX5 mRNA transcripts is decreased in both tissues from FPS-affected foals(FPS1, FPS2) compared to the healthy horses (H; n � 2). Statistical compari-son was not performed due to the small sample size. Bars represent the meansplus standard deviations of results for each sample, tested in triplicate.

Tallmadge et al.

1062 cvi.asm.org Clinical and Vaccine Immunology

on May 24, 2021 by guest

http://cvi.asm.org/

Dow

nloaded from

8. Cioc AM, Wagner JE, MacMillan ML, DeFor T, Hirsch B. 2010. Diag-nosis of myelodysplastic syndrome among a cohort of 119 patients withfanconi anemia: morphologic and cytogenetic characteristics. Am. J. Clin.Pathol. 133:92–100.

9. Corcoran LM, Koentgen F, Dietrich W, Veale M, Humbert PO. 2004.All known in vivo functions of the Oct-2 transcription factor require theC-terminal protein domain. J. Immunol. 172:2962–2969.

10. Dai Z, et al. 2011. Sodium/myo-inositol cotransporter 1 and myo-inositol are essential for osteogenesis and bone formation. J. Bone Miner.Res. 26:582–590.

11. Dolence JJ, Gwin K, Frank E, Medina KL. 2011. Threshold levels ofFlt3-ligand are required for the generation and survival of lymphoid pro-genitors and B-cell precursors. Eur. J. Immunol. 41:324 –334.

12. Douagi I, Vieira P, Cumano A. 2002. Lymphocyte commitment duringembryonic development, in the mouse. Semin. Immunol. 14:361–369.

13. Emambokus N, et al. 2003. Progression through key stages of haemopoi-esis is dependent on distinct threshold levels of c-Myb. EMBO J. 22:4478 –4488.

14. Engel P, et al. 1995. Abnormal B lymphocyte development, activation,and differentiation in mice that lack or overexpress the CD19 signal trans-duction molecule. Immunity 3:39 –50.

15. Eynon EE, Kuida K, Flavell RA. 1996. Disruption of cytokine signaling inlymphoid development: unique contributions of the common cytokinegamma chain and the Jak3 kinase. J. Interferon Cytokine Res. 16:677– 684.

16. Fitzsimmons D, et al. 1996. Pax-5 (BSAP) recruits Ets proto-oncogenefamily proteins to form functional ternary complexes on a B-cell-specificpromoter. Genes Dev. 10:2198 –2211.

17. Flaminio MJ, et al. 2007. The effect of CpG-ODN on antigen presentingcells of the foal. J. Immune Based Ther. Vaccines 5:1.

18. Flaminio MJ, LaCombe V, Kohn CW, Antczak DF. 2002. Commonvariable immunodeficiency in a horse. J. Am. Vet. Med. Assoc. 221:1296 –1302, 1267.

19. Flaminio MJ, et al. 2000. Characterization of peripheral blood and pul-monary leukocyte function in healthy foals. Vet. Immunol. Immuno-pathol. 73:267–285.

20. Flomenberg P, Piaskowski V, Truitt RL, Casper JT. 1995. Characteriza-tion of human proliferative T cell responses to adenovirus. J. Infect. Dis.171:1090 –1096.

21. Fox-Clipsham L, et al. 2009. Immunodeficiency/anaemia syndrome in aDales pony. Vet. Rec. 165:289 –290.

22. Fox-Clipsham LY, et al. 2011. Identification of a mutation associatedwith fatal foal immunodeficiency syndrome in the Fell and Dales pony.PLoS Genet. 7:e1002133. doi:10.1371/journal.pgen.1002133.

23. Gardner RB, Hart KA, Stokol T, Divers TJ, Flaminio MJ. 2006. Fell Ponysyndrome in a pony in North America. J. Vet. Intern. Med. 20:198 –203.

24. Hoelbl A, et al. 2006. Clarifying the role of Stat5 in lymphoid develop-ment and Abelson-induced transformation. Blood 107:4898 – 4906.

25. Huntington ND, et al. 2006. CD45 links the B cell receptor with cellsurvival and is required for the persistence of germinal centers. Nat. Im-munol. 7:190 –198.

26. Iolascon A, Russo R, Delaunay J. 2011. Congenital dyserythropoieticanemias. Curr. Opin. Hematol. 18:146 –151.

27. Jalovecka M, et al. 2010. Activation of protective cell-mediated immuneresponse in gastric mucosa during Cryptosporidium muris infection andre-infection in immunocompetent mice. Parasitol. Res. 106:1159 –1166.

28. Jelinek F, Faldyna M, Jasurkova-Mikutova G. 2006. Severe combinedimmunodeficiency in a Fell pony foal. J. Vet. Med. A Physiol. Pathol. Clin.Med. 53:69 –73.

29. Jung S, Rajewsky K, Radbruch A. 1993. Shutdown of class switch recom-bination by deletion of a switch region control element. Science 259:984 –987.

30. Kasper LH, et al. 2002. A transcription-factor-binding surface of coacti-vator p300 is required for haematopoiesis. Nature 419:738 –743.

31. Kathiresan S, et al. 2009. Genome-wide association of early-onset myo-cardial infarction with single nucleotide polymorphisms and copy num-ber variants. Nat. Genet. 41:334 –341.

32. Khan WN, et al. 1995. Defective B cell development and function inBtk-deficient mice. Immunity 3:283–299.

33. Khan WN, et al. 1997. Impaired B cell maturation in mice lacking Bru-ton’s tyrosine kinase (Btk) and CD40. Int. Immunol. 9:395– 405.

34. Kikuchi K, Kondo M. 2006. Developmental switch of mouse hematopoi-etic stem cells from fetal to adult type occurs in bone marrow after birth.Proc. Natl. Acad. Sci. U. S. A. 103:17852–17857.

35. Kikuchi K, Lai AY, Hsu CL, Kondo M. 2005. IL-7 receptor signaling isnecessary for stage transition in adult B cell development through up-regulation of EBF. J. Exp. Med. 201:1197–1203.

36. Kim HG, et al. 2004. The ETS family transcription factor PU.1 is neces-sary for the maintenance of fetal liver hematopoietic stem cells. Blood104:3894 –3900.

37. Kitamura D, Rajewsky K. 1992. Targeted disruption of mu chain mem-brane exon causes loss of heavy-chain allelic exclusion. Nature 356:154 –156.

38. Kydd J, et al. 1994. Report of the First International Workshop on EquineLeucocyte Antigens, Cambridge, UK, July 1991. Vet. Immunol. Immuno-pathol. 42:3– 60.

39. Lavoie JP, Spensley MS, Smith BP, Mihalyi J. 1989. Absorption ofbovine colostral immunoglobulins G and M in newborn foals. Am. J. Vet.Res. 50:1598 –1603.

40. Lin H, Grosschedl R. 1995. Failure of B-cell differentiation in mice lack-ing the transcription factor EBF. Nature 376:263–267.

41. Lipovsky K, Bennett LA, Chen J. 2003. B-cell deficiency and reducedB-cell reconstitution in hemoglobin-deficit mice. Exp. Hematol. 31:1230 –1236.

42. Lluis-Ganella C, et al. 2010. Additive effect of multiple genetic variants onthe risk of coronary artery disease. Rev. Esp. Cardiol. 63:925–933.

43. Lunn D, Holmes M, Duffus W. 1993. Equine T-lymphocyte MHC IIexpression: variation with age and subset. Vet. Immunol. Immunopathol.35:225–238.

44. Lunn DP, et al. 1998. Report of the Second Equine Leucocyte AntigenWorkshop, Squaw Valley, California, July 1995. Vet. Immunol. Immuno-pathol. 62:101–143.

45. Mackarehtschian K, et al. 1995. Targeted disruption of the flk2/flt3 geneleads to deficiencies in primitive hematopoietic progenitors. Immunity3:147–161.

46. Marcos MA, et al. 1997. Antigenic phenotype and gene expression pat-tern of lymphohemopoietic progenitors during early mouse ontogeny. J.Immunol. 158:2627–2637.

47. McDonald V, Robinson HA, Kelly JP, Bancroft GJ. 1994. Cryptospo-ridium muris in adult mice: adoptive transfer of immunity and protectiveroles of CD4 versus CD8 cells. Infect. Immun. 62:2289 –2294.

48. McKenna HJ, et al. 2000. Mice lacking flt3 ligand have deficient hema-topoiesis affecting hematopoietic progenitor cells, dendritic cells, and nat-ural killer cells. Blood 95:3489 –3497.

49. McKercher SR, et al. 1996. Targeted disruption of the PU.1 gene results inmultiple hematopoietic abnormalities. EMBO J. 15:5647–5658.

50. Meffre E, Nussenzweig MC. 2002. Deletion of immunoglobulin beta indeveloping B cells leads to cell death. Proc. Natl. Acad. Sci. U. S. A. 99:11334 –11339.

51. Michaelson JS, Singh M, Birshtein BK. 1996. B cell lineage-specificactivator protein (BSAP). A player at multiple stages of B cell develop-ment. J. Immunol. 156:2349 –2351.

52. Nielsen PJ, Georgiev O, Lorenz B, Schaffner W. 1996. B lymphocytes areimpaired in mice lacking the transcriptional co-activator Bob1/OCA-B/OBF1. Eur. J. Immunol. 26:3214 –3218.

53. Nutt SL, Morrison AM, Dorfler P, Rolink A, Busslinger M. 1998.Identification of BSAP (Pax-5) target genes in early B-cell development byloss- and gain-of-function experiments. EMBO J. 17:2319 –2333.

54. Nutt SL, Urbanek P, Rolink A, Busslinger M. 1997. Essential functionsof Pax5 (BSAP) in pro-B cell development: difference between fetal andadult B lymphopoiesis and reduced V-to-DJ recombination at the IgHlocus. Genes Dev. 11:476 – 491.

55. Parrish CR, Carmichael LE, Antczak DF. 1982. Antigenic relationshipsbetween canine parvovirus type 2, feline panleukopenia virus and minkenteritis virus using conventional antisera and monoclonal antibodies.Arch. Virol. 72:267–278.

56. Pelanda R, Braun U, Hobeika E, Nussenzweig MC, Reth M. 2002. B cellprogenitors are arrested in maturation but have intact VDJ recombinationin the absence of Ig-alpha and Ig-beta. J. Immunol. 169:865– 872.

57. Richards AJ, Kelly DF, Knottenbelt DC, Cheeseman MT, Dixon JB.2000. Anaemia, diarrhoea and opportunistic infections in Fell ponies.Equine Vet. J. 32:386 –391.

58. Rodig SJ, et al. 1998. Disruption of the Jak1 gene demonstrates obligatoryand nonredundant roles of the Jaks in cytokine-induced biologic re-sponses. Cell 93:373–383.

59. Sandberg ML, et al. 2005. c-Myb and p300 regulate hematopoietic stemcell proliferation and differentiation. Dev. Cell 8:153–166.

Developmental Hematopoiesis Failure in Fell Pony Syndrome

July 2012 Volume 19 Number 7 cvi.asm.org 1063

on May 24, 2021 by guest

http://cvi.asm.org/

Dow

nloaded from

60. Scholes SF, Holliman A, May PD, Holmes MA. 1998. A syndrome ofanaemia, immunodeficiency and peripheral ganglionopathy in Fell ponyfoals. Vet. Rec. 142:128 –134.

61. Scott EW, Simon MC, Anastasi J, Singh H. 1994. Requirement oftranscription factor PU.1 in the development of multiple hematopoieticlineages. Science 265:1573–1577.

62. Sinkora M, Butler JE. 2009. The ontogeny of the porcine immune system.Dev. Comp. Immunol. 33:273–283.

63. Sinkora M, Sinkorova J, Butler JE. 2002. B cell development and VDJrearrangement in the fetal pig. Vet. Immunol. Immunopathol. 87:341–346.

64. Smith CA, Woodruff LS, Kitchingman GR, Rooney CM. 1996. Adeno-virus-pulsed dendritic cells stimulate human virus-specific T-cell re-sponses in vitro. J. Virol. 70:6733– 6740.

65. Souberbielle BE, Russell WC. 1995. Human T cell proliferative responseto polypeptides from adenovirus type 2. J. Infect. Dis. 172:1421–1422.

66. Tallmadge RL, et al. 2009. Expression of essential B cell genes and immu-noglobulin isotypes suggests active development and gene recombinationduring equine gestation. Dev. Comp. Immunol. 33:1027–1038.

67. Thomas GW, Bell SC, Carter SD. 2005. Immunoglobulin and peripheralB-lymphocyte concentrations in Fell pony foal syndrome. Equine Vet. J.37:48 –52.

68. Thomas GW, et al. 2003. Aid to the antemortem diagnosis of Fell ponyfoal syndrome by the analysis of B lymphocytes. Vet. Rec. 152:618 – 621.

69. Tober J, McGrath KE, Palis J. 2008. Primitive erythropoiesis and mega-

karyopoiesis in the yolk sac are independent of c-myb. Blood 111:2636 –2639.

70. Urbanek P, Wang ZQ, Fetka I, Wagner EF, Busslinger M. 1994. Com-plete block of early B cell differentiation and altered patterning of theposterior midbrain in mice lacking Pax5/BSAP. Cell 79:901–912.

71. von Freeden-Jeffry U, et al. 1995. Lymphopenia in interleukin (IL)-7gene-deleted mice identifies IL-7 as a nonredundant cytokine. J. Exp. Med.181:1519 –1526.

72. Wang VE, Schmidt T, Chen J, Sharp PA, Tantin D. 2004. Embryoniclethality, decreased erythropoiesis, and defective octamer-dependent pro-moter activation in Oct-1-deficient mice. Mol. Cell. Biol. 24:1022–1032.

73. Waskow C, Terszowski G, Costa C, Gassmann M, Rodewald HR. 2004.Rescue of lethal c-KitW/W mice by erythropoietin. Blood 104:1688 –1695.

74. Yeh WC, et al. 1997. Early lethality, functional NF-kappaB activation, andincreased sensitivity to TNF-induced cell death in TRAF2-deficient mice.Immunity 7:715–725.

75. You-Ten KE, et al. 1997. Impaired bone marrow microenvironment andimmune function in T cell protein tyrosine phosphatase-deficient mice. J.Exp. Med. 186:683– 693.

76. Zhu JW, Brdicka T, Katsumoto TR, Lin J, Weiss A. 2008. Structurallydistinct phosphatases CD45 and CD148 both regulate B cell and macro-phage immunoreceptor signaling. Immunity 28:183–196.

77. Zhuang Y, Soriano P, Weintraub H. 1994. The helix-loop-helix gene E2Ais required for B cell formation. Cell 79:875– 884.

Tallmadge et al.

1064 cvi.asm.org Clinical and Vaccine Immunology

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