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 Avian
Cellular
Immunology

E ditor

Jagdev M. Sharma, B.V.Sc., Ph.D.

Benjamin S. Pomeroy Chair in Avian Health
Department of Veterinary Pathobiology
College of Veterinary Medicine
University of Minnesota
St. Paul, Minnesota

C R C Press
Taylor 8i Francis Group
Boca Raton London New York

C R C Press is an im print of the

Taylor & Francis Group, an in fo rm a business
 Library of Congress Cataloging-In-Publication Data

Avian cellular inununology/editor, Jagdev M. Sharma

p. cm.
Includes bibliographical references.
Includes index.
ISBN 0-8493-6833-2
1. Chickens—Immunology. 2. Birds— Immunology. 3. Cellular
immunity. I. Sharma, lagdev, M ., 1941-
[DNLM: 1. Bird Disease—immunology. 2. Birds—immunology.
3. Immunity, Cellular. SF 99S A9S6S]
SP995.A94 1990
636.5'0896'079—dc20
DNLM/DLC 90-2494
for Library of Congress CIP

This book represents information obtained from authentic and highly regarded sources. Reprinted material is
quoted with permission, and sources are indicated. A wide variety of references are listed. Every reasonable effort
has been made to give reliable data and information, but the author and the publisher caiuiot assume responsibility
for the validity of all materials or for the consequences o f their use.

All rights reserved. This book, or any parts thereof, may not be reproduced in any form without written consent
from the publisher.

Direct all inquiries to CRC Press, Inc., 2000 Corporate Blvd., N .W ., Boca Raton, Florida 33431.

® 1991 by CRC Press, Inc.

International Standard Book Number 0-8493-6833-2

Library of Congress Card Number 90-2494

To Sylvia, Dave, Susan
My Cheering Squad
 PREFA CE

The knowledge on avian cellular immunity has expanded rapidly within the last few
years. It is now well accepted that cell-mediated immunity plays an important role in defense
against neoplastic and nonneoplastic diseases of avian species. New data continue to ac­
cumulate as reproducible in vitro systems to assay immunity are adopted by an increasing
number of laboratories. The advent of hybridoma technology has generated useful mono­
clonal antibody reagents that react against surface markers of avian lymphocytes. The use
of these reagents has contributed significantly to new knowledge on the ontogeny and
functional diversity of chicken lymphocytes. It has become apparent that surface markers
and effector functions of lymphocytes known in the mammalian species are well conserved
in birds. Some of the genes coding for lymphocyte surface marker proteins share sequence
homology between mammals and birds.
Much of the work in avian inununity has been done with the chicken. Other members
of the avian species have not been examined in adequate detail, and it remains to be seen
if interesting comparative differences exist in the immune mechanisms within the species.
The objective in this volume was to bring together the information on various aspects
of avian cellular immunity that now exists scattered in diverse journals. I am very hopeful
that this objective has been adequately met because I had the good fortune of having the
leading authorities in avian cellular immunity contribute chapters in their respective areas
of expertise. It has been a real pleasure to collaborate with these individuals, and this
experience has renewed my appreciation for the sense of cooperation that exists among my
professional colleagues. I wish to express my heartfelt thanks to the contributors, to my
friends and colleagues who didn’t write chapters for this volume but helped me in other
ways, and to the secretarial staff of my department at the University of Minnesota. A special
word of thanks to Pamela Farr, Rosemary Finnegan, and Sean Harrington, who helped in
word processing and literature search for the chapters I have coauthored.
This book comes on the heels of several publications based on proceedings of symposia
on avian immunity and a recent two-volume CRC book. Avian Immunology: Basis and
Practice, edited by Auli and Paavo Toivanen. Each of these publications has unique qualities.
The Toivanens’ excellent book, in particular, is a pioneering effort in compiling extensive
emerging knowledge on diverse aspects of avian immunology. The present volume, in
comparison, has a narrow focus on cellular immunity.
I believe this book will be of value to the students of avian cellular irrununity involved
in all areas of endeavor, i.e., students, teachers, research scientists, and industry scientists.
In addition, the book may also serve as a resource for comparative irrununologists.

J . M. Sharm a
St. Paul, Minnesota
May 1990
 T H E E D IT O R

Jagdev M. Sharm a, B. V. Sc., P h.D ., holds the Benjamin S. Pomeroy Endowed Chair
in Avian Health in the College of Veterinary Medicine, University of Minnesota, St. Paul.
E)r. Sharma received undergraduate training in Punjab, India and obtained his B.V.Sc.
degree in 1961 from the College of Veterinary Science, Haryana Agricultural University,
Hissar, India. He obtained his M.S. and Ph.D. degrees in 1964 and 1967, respectively,
from the Department of Epidemiology and Preventive Medicine, College of Veterinary
Medicine, University of California, Davis. In 1971, after completing postdoctoral work at
Washington State University, Pullman, he was appointed Veterinary Medical Officer at the
Regional Poultry Research Laboratory of the U.S. Department of Agriculture in East Lansing,
Michigan. He remained in Michigan until his move in 1988 to accept the Endowed Profes­
sorship at the University of Minnesota. In 1976, he was appointed Clinical Assistant Professor
in the Department of Veterinary Pathology of the Michigan State University, and promoted
to Clinical Professor in 1982.
Dr. Sharma is a member of a number of professional societies, including the International
Association of Comparative Research on Leukemia and Related Diseases, the World Vet­
erinary Poultry Association, the American Veterinary Medical Association, the American
Association of Avian Pathologists, the Conference of Research Workers in Animal Diseases,
Sigma Xi, and Phi Zeta. He has been the recipient of a number of awards and citations,
including the Upjohn Achievement Award conferred by the American Association of Avian
Pathologists for distinguished research contributions in avian medicine.
Dr. Sharma has presented over SO invited lectures at national and international meetings.
He has published over 130 research papers, the majority of which are in the area of avian
immunology. His current major research interests include the avian cellular immune mech­
anisms and the role of cellular immunity in neoplastic and nonneoplastic viral diseases.
 C O N T R IB U T O R S

Stephen E. Bkwm, Ph.D. M ark H. K aplan, B.Sc.

Professor of Cytogenetics Graduate Assistant
Department of Poultry and Avian Department of Immunology and
Sciences Microbiology
Cornell University Wayne State University School of
Ithaca, New York Medicine
Detroit, Michigan
Chen-lo H. Chen, Ph.D.
Research Associate Professor Jill M. Lahti, Ph.D
Department of Microbiology and Postdoctoral Fellow
Immunology Department of Biochemistry
Division of Developmental and Clinical University of Alabama at Birmingham
Immunology Birmingham, Alabama
University of Alabama at Birmingham
Birmingham, Alabama Hyun S. Lillehoj, Ph.D.
Immunologist
Protozoan Diseases Laboratory
David S. Chi, Ph.D.
Livestock and Poultry Institute
Professor
Agricultural Research Service
Department of Internal Medicine
U.S. Department of Agriculture
East Tennessee State University
Beltsville, Maryland
Johnson City, Tennessee

H aruo M atsuda, D .V .M ., Ph.D.

Max D. Cooper, M.D.
Associate Professor
Professor
Department of Microbiology and Hygiene
Departments of Pediatrics, Medicine,
Faculty of Applied Biological Science
Microbiology, and Pathology
Hiroshima University
Howard Hughes Medical Institute
Higashi-Hiroshima, Japan
University of Alabama at Birmingham
Birmingham, Alabama
Takeshi M ikami, D .V .M ., Ph.D.
Professor
Rodney R. Dietert, Ph.D. Department of Veterinary Microbiology
Professor of Immunogenetics Faculty of Agriculture
Department of Poultry and Avian University of Tokyo
Sciences Tokyo,Japan
Cornell University
Ithaca, New York Jam es M. Picket, B.A.
Graduate Fellow
Karen A. Golemboski, Ph.D. Department of Micrdriology and
Research Associate Immunology
Department of Poultry and Avian Division of Developmental and Clinical
Sciences Immunology
Cornell University University of Alabama at Birmingham
Ithaca, New York Birmingham, Alabama
Stephen J . Prowse, Ph.D.
Principal Research Scientist
Division of Animal Health
CSIRO
Parkville, Victoria
Australia
Pascale Quéré, D .V .M ., Ph.D.
Chargé de Rechercher
Station de Pathologie Aviaire
Institut National de la Recherche
Agronomique
Tours, France
M uquarrah A. Qureshi, Ph.D.
Assistant Professor
Department of Poultry Science
North Carolina State University
Raleigh, North Carolina
Noel R. Rose, M .D ., Ph.D.
Professor and Chairman
Department of Immunology and
Infectious Diseases
School of Hygiene and Public Health
The Johns Hopkins University
Baltimore, Maryland
Karel A. Schat, D .V .M ., Ph.D.
Professor
Department of Avian and Aquatic Animal
Medicine
New York State College of Veterinary
Medicine
Cornell University
Ithaca, New York
Jagdev M. Sharm a, B.V.Sc., Ph.D.
Professor
Benjamin S. Pomeroy Chair
Department of Veterinary Pathobiology
College of Veterinary Medicine
University of Minnesota
St. Paul, Mirmesota
Roy S. Sundick, Ph.D.
Associate Professor
Department of Immunology and
Microbiology
Wayne State University School of
Medicine
Detroit, Michigan
Constantine H. Tempelis, Ph.D.
Professor of Inununology
Department of Biomedical and
Environmental Health Sciences
University of California
Berkeley, California
G. Jeanette Thorbecke, M .D ., Ph.D.
Professor
Department of Pathology
New York University Medical Center
New York, New York
Paavo Toivanen, M.D.
Professor
Department of Medical Microbiology
Turku University
Turku, Finland
Timo Verom aa, M .D ., Ph.D.
Research Associate
Department of Medical Microbiology
Turku University
Turku, Finland
Jennifer J . York, Ph.D.
Experimental Scientist
Division of Animal Health
CSIRO
Parkville, Victoria
Australia
 TABLE O F CONTENTS

Chapter 1
Surface Markers on Avian Immune C e lls ...........................................................
Chen-lo H. Chen, Jam es M. Pickel, Jill M. Lahti, and Max D. Cooper

Chapter 2
Cellular Cooperation in Immune Response and Role of MHC............................................ 23
Timo Verom aa and Paavo Toivanen

Chapter 3
T-Cell Immunity: Mechanisms and SolubleM ediators...........................................................35
K. A. Schat

Chapter 4
Natural Immune Functions.........................................................................................................51
J . M. Sharm a and K. A. Schat

Chapter 5
The Avian Macrophage in Cellular Im m unity.........................................................................71
R. R. Dietert, K. A. Golemboski, S. E. Bloom, and M. A. Qureshi

Chapter 6
Cellular Inunune Suppression................................................................................................... 97
David S. Chi, Pascale Quéré, and G. Jeanette Thorbecke

Chapter 7
Cellular Immune Tolerance....................................................................................................... 119
Constantine H. Tempelis

Chapter 8
Features of Cellular Immune Systems in Avian Species other than Chickens................. 133
H aruo M atsuda and Takeshi Mikami

Chapter 9
Role of Cellular Inununity in Neoplastic and Nonneoplastic Viral Diseases...................139
J . M. Sharm a, S. J . Prowse, and J . J . York

Chapter 10
Cell-Mediated Immunity in Parasitic and Bacterial D iseases............................................ 155
Hyun S. Lillehoj

Chapter 11
Autoimmune Diseases.............................................................................................................. 183
M ark H. Kaplan, Roy S. Sundick, and Noel R. Rose

Index. ,199
 Chapter 1

S U R F A C E M A R K E R S O N A V IA N IM M U N E C E L L S

Chen-lo H. Chen, Janies M. Picket, Jill M. Lahti, and Max D. Cooper

TABLE O F CONTENTS

I. Introduction........................................................................................................................ 2

II. Major HistocompatibilityComplex Antigens..................................................................2

A. B-F Antigens......................................................................................................... 2
B. B-L Antigens......................................................................................................... 3

III. Lymphocyte Antigens....................................................................................................... 4

A. T-Cell A ntigens.................................................................................................... 4
1. C D 2 ...........................................................................................................4
2. C T l............................................................................................................ 5
3. CDS ...........................................................................................................5
4. CD4 and CD 8 .......................................................................................... 6
5. T-Cell Receptors...................................................................................... 6
a. Ontogeny and Tissue Localization of CD3/TCR
Proteins ........................................................................................ 6
b. Biochemical Characterization of the Chicken T-
Cell Receptor Proteins................................................................ 7
c. Lineage Relationships of the Cells Expressing
the Different T-Cell Receptor Isotypes....................................7
d. Functional Capability of Different T-Cell
Receptor Isotypes....................................................................... 8
e. A Third Lineage of Lymphocytes in C hickens...................... 8
6. Interleukin 2 R eceptor............................................................................ 9
7. Other T-Cell A ntigens............................................................................ 9
B. B-Cell A ntigens....................................................................................................9
1. Immunoglobulins.....................................................................................9
2. Nonimmunoglobulin M arkers...............................................................11
a. Bursal Lymphocyte Antigens................................................... 11
i. CB2..................................................................................11
ii. HNK-1............................................................................ 11
b. B-Cell Antigens......................................................................... 11
i. B u -1 ................................................................................12
ii. B u -2 ................................................................................12
iii. CB3................................................................................. 12
iv. Other B-Cell Antigens................................................. 13
c. Antigens of B-Cell Subpopulations.........................................13
d. Antigens of Nonlymphoid Bursal Elements........................... 13
3. Developmental Expression of B-Cell Antigens.................................. 14

IV. Leukocyte Common Antigens and MyeloidAntigens.................................................14

V. Conclusion........................................................................................................................ 15
2 Avian Cellular Immunology

Acknowledgments.......................................................................................................................16

References. 17

I . IN T R O D U C T IO N

Characterization of cell-surface differentiation antigens has been fundamental to under­

standing lymphocyte development. While most of the available information has been derived
from studies in manunals, studies in birds first revealed the separate developmental pathways
of T and B cells,' their hemopoietic stem-cell origin^, and the seeding of the epithelial
thymus by waves of blood-borne precursor cells.^ Recent comparative analysis of T- and
B-cell development using antibodies reactive with avian lymphocyte surface markers has
revealed that the basic antigen recognition molecules are highly conserved in birds and
mammals. The avian major histocompatibility complex (MHC) class 1 and II antigens, T-
cell receptors, accessory T-cell molecules, and the inununoglobulins have been characterized.
Several of the genes encoding these proteins have been cloned and shown to have sequence
homology to their manunalian counterparts.
The phylogenetic distance between birds and mice has made it relatively easy to produce
mouse monoclonal antibodies (mAb) to highly conserved cell-surface proteins on chicken
lymphocytes. Novel antigens have thus been identifled, many of which may well be found
in manunals. This chapter outlines the information on the tissue distribution, structure,
function, and genes of the immunologically important surface molecules that have been
identified to date on chicken leukocytes.

I I . M A J O R H IS T O C O M P A T IB IL IT Y C O M P L E X A N T IG E N S

Chicken MHC was originally described as a blood-group B locus.'* The availability of

the first recombinant B h^lotype^ made it possible to propose a three-locus MHC model.
These loci control the synthesis of at least three classes of antigens called B-F, B-L, and
B-G.‘ B-F and B-L antigens correspond to the mammalian MHC class I and II antigens,
resp ectiv ely T he B-G antigen, found only on erythrocytes, erythroid progenitor cells,^ and
thrombocytes,* has no known mammalian counterpart and has been named a MHC class IV
antigen.^ Only B-F and B-L antigens which are involved in the immune response, histo­
compatibility reactions, and disease resistance"-w ill be reviewed here.

A. B-F ANTIGENS
B-F antigens are the chicken MHC class I gene products found on erythrocytes and
leukocytes. Alloantisera and mAb against B-F antigens are available for haplotype deter­
mination and biochemical characterization"-'* and have been reviewed recently.’-**-'* Most
of the available mAb recognize monomorphic determinants of these highly polymorphic
antigens, although mAb which react with B-F determinants shared by only a few B haplotypes
have also been obtained.'* Two mAb that recognized B-F determinants, F21-2 (anti-a chain)’
and F21-21 (anti-^ 2-^ucroglobulin),'* cross-react with turkey cells, but not with Xenopus,
rainbow trout, or human cells. Similar to their mammalian homologues, B-F antigens are
composed of a membrane-bound glycosylated polymcuphic heavy chain (a) of relative
molecular mass (M,) 40,000 to 45,000 noncovalently associated with the invariant light
chain Oj-microglobulin) of M, 14,500. Partial amino-acid sequence analysis has revealed
considerable homology between chicken B-F molecules and mammalian class I antigens.
B-F antigens are expressed on almost all cells in the central lymphoid organs of chickens.
In the thymus, cortical cells are stained less strongly than medullary cells,"'*^ suggesting
that the expression of class I antigens may increase during lymphocyte maturation. The
presence of B-F antigens has not been demonstrated in brain or muscle."
Expression of the B-F antigens has been examined most thoroughly using the mAb INN-
CH-428.'’’ This antibody has been reported to recognize B-F antigens in a hemagglutination
assay, although it has not been shown to immunoprecipitate class I proteins. Using this
antibody, it has been demonstrated that expression of B-F antigens begins relatively late in
ontogeny. INN-CH-428 reactive molecules are not detected on red blood cells (RBC),
peripheral blood leukocytes (PBL), or the cells from bursa and thymus on either embryonic
day 18 (E18) or day 1 after hatching. The frequency of B-F^ cells increases rapidly after
hatching with almost 100% of the RBC, PBL, bursa and spleen cells expressing these antigens
at 2 weeks of age. Medullary cells of the thymus were positive, while those in the cortical
area were negative in these studies. In contrast to these results, expression of B-F antigens
has been observed much earlier during ontogeny using other B-F specific m Ab.'” *^ Further
studies are thus needed to resolve this interesting discrepancy.
The sequence and structure of the B-F genes have been partially resolved. Two B-F
transcripts have been sequenced. One was isolated by hybridizing tissue-specific comple­
mentary deoxyribonucleic acid (cDNA) probes to cosmid clones containing the B-L p
genes,^ indicating that these two loci are linked. The other was isolated by screening an
expression vector cDNA library with an antisera prepared against purified B-F molecules.*
The deduced B-F protein sequences fiom the two transcripts exhibit 84% similarity in the
a , domain, 84% in the Uj domain, 96% in the a , domain, and 100% in the transmembrane
domain.^' The amino acid sequence of B-F molecules showed 35 to 45% homology to
mammalian class 1 antigens: maximal in the Uj domain (40 to 52%), intermediate in the a ,
(32 to 47%) and cytoplasmic (27 to 45%) domains, and lower in the a , (32 to 36%) and
transmembrane (24 to 38%) domains.^ Although the divergence of B-F from the manunalian
MHC class I antigens is striking, many of the amino acids directly involved in the secondary
and tertiary structure are conserved in the chicken protein. Among the residues involved in
the binding of ^2 microglobulin (P 2*m)> four out of nine are substituted in the a , domain,
one out of nine in the U2 domain, and seven out of ten in the domain. Recently, ^j'n i
has been purified from chicken plasma and the N-terminal amino acid sequence determined.
A mixture of the corresponding oligonucleotide probes was used to isolate chicken Pj^ni
cDNA clones. The amino acid sequence of the chicken Pj"™ showed 45 to 55% homology
to its mammalian counterparts,* suggesting that residues involved in the binding of both
chains have diverged together. It is interesting to note, however, that human can
exchange with P 2-m of the MHC class I molecules from chickens.“

B. B-L ANTIGENS
B-L antigens have been defined by alloantisera and mAb as the avian homologues of
the mammalian class II antigens. The available mAb have been reviewed previ­
ously.’-'*-'* Most of the antibodies recognize monomorphic determinants, as mouse mAb
against polymorphic determinants of avian class II molecules are difficult to obtain. Im­
munoprécipitation and gel electrophoresis studies have revealed that the B-L antigen is a
glycoprotein consisting of a single nonpolymoiphic a chain of M, 32,(XX) and one of the
two polymorphic p chains of M, 27,000.**-“ In addition, the B-L heterodimer has been
found to be associated with several basic invariant molecules in the cytoplasm but not on
the cell surface.“
 Avian Cellular Immunology

TABLE 1
Chicken T-Cell Antigens

% Cells expressing surface

M olecular mass
Antigen mAb (isotypc) Thymus Blood Spleen (kD>) Ref.

CTl CTl (7 ,); C T la (7 ,) > 90 <1 <1 65 33

CD2 2— 4; 2— 102 < 95 60 60 40 29
CD3 C T 3(7.) 65—75 75—85 65—75 20,19,17 43,48
CD4 CT4 (7 .) 80—85 35— 45 15— 20 64 40
CTLA I (7 j); CTLA 6 (7 ,) 59 22 16 65 38
2 — 6 ; 2— 35 80 40 20 64 29
CDS CTLA5; CTLA 8 (7 ,) 73 52 45 65,45 38
3—58 90 65 70 56 29
CDS CT 8 (7 .) 8 0 —85 10— 2 0 45—55 63 (34 dimer) 40
CTLA3,4, and 9 (7 ,) 43 18 36 6 6 (33— ^35 dimer) 38
TCRl TCRl (7 ,) 5— 15 15—25 15—25 90 (50,40 dimer) 43,48
TCR2 mAb 6 (7 i) 45— 55 45— 55 35— 10 90 (50,40 dimer) 47—49
TCR3 TCR3 (7 .) 5— 15 10— 15 10— 15 8 8 (48,40 dimer) 48,58
CD25 (IL-2R) INN-CH-16 (|ji) <1 <1 <1 48—50 67

B-L antigens are expressed on B cells, cells of the monocyte-macrophage lineage,

mitogen-stimulated T cells, and Marek’s disease virus-induced T-cell tumors.^*’ They have
also been demonstrated on endothelial cells in bursal capillary blood vessels.^ B-L antigens
appear early during ontogeny, flrst being detectable on E l2 bursa cells and on E13 mac­
rophages and thymic dendritic cells.“ “
Using a probe encoding a human HLA-DQ^ cDNA, the chicken B-L 3 genes have been
cloned.“ “ Five class II p genes defining two related isotypic families have been mapped
to the B complex which is located on the 16th microchromosome. Exons encoding the
^ 2• ^ d transmembrane domains of a B-L chain have been shown to have 63, 66, and 62%
homology with the HLA-DQ^ sequence, respectively. There are highly conserved regions
in each domain which may play an important role in MHC class II function. However, many
unique amino acid substitutions in the chicken ^ chain have also been detected, especially
in the domain which is involved in antigen and T-cell recognition. The presence of B­
L a genes in B-L ^ cosmids was investigated by chromosome walking.“ Although several
genes were localized, none of the identified genes have the transcriptional characteristics of
a MHC class II a-chain gene, indicating that the B-L a gene(s) may not be associated with
the B-L P genes. In contrast, the class II a-chain genes are always found closely linked to
the p-chain gene in the HLA and H-2 complexes in mammals.^'

I I I . L Y M P H O C Y T E A N T IG E N S

A. T-CELL ANTIGENS
The characterization of avian T-cell development and fimction has lagged behind that
of mammals partly because of the absence of antibodies that identify T-cell surface antigens.
However, research in the last S years has begun to elucidate the T-cell differentiation pathway
in chickens. These studies have revealed striking similarities between chicken T-cell surface
molecules and those of their mammalian counterparts. The chicken T-cell system has also
revealed interesting distinctions. The data available on T-cell surface antigens in chickens
will be summarized here (Table 1), with particular emphasis on the similarities and differences
between chickens and mammals.

1. CD2
The first antigen to be recognized on the surface of avian thymocytes during development
is the avian homologue of CD2. This antigen is identified by mAb 2-4 and 2-102.” The
CD2 molecule is expressed by thymocytes beginning around day 11 of embryonic life. CD2
expression is retained throughout adult life on both thymocytes and mature peripheral T
cells, with the exception of *y8 T cells which express the CD2 antigen only in the thymus.” '^
The chicken CD2 antigen is a M, 40,000 single-chain protein.” Its cytoplasmic domain
appears to exhibit considerable homology with the mammalian CD2 antigen, as a rabbit
antiserum to a synthetic peptide from the cytoplasmic region of the human CD2 molecule
is capable of immunoprecipitating the chicken CD2 homologue. The rabbit antibody also
removes all of the CD2 reactive protein from chick thymic lysates in preclearance experi­
ments.”
The mammalian CD2 molecule is expressed very early in ontogeny, in humans at the
pro-T stage.^ Due to the early appearance of this molecule, the CD2 antigen has been
suggested to participate in T-cell growth and differentiation intrathymically. Mammalian
studies have shown that C 02 is a cell-adhesion molecule that binds to LFA-3 molecules
which are expressed by thymic epithelial cells and other cell types.’’ CD2 has also been
shown to be involved in an alternate pathway of T-cell activation.” This alternate pathway
appears to exist in the chicken as suboptimal doses of anti-CD2, and PM A (phorbol myristate
acetate) can activate both embryonic and adult thymocytes and peripheral a p T cells without
involvement of the T-cell receptor (TCR) complex.” The early appearance of the CD2
antigen in conjunction with studies demonstrating the presence of an alternate activation
pathway in chickens suggests that the avian CD2 analogue is functionally equivalent to its
mammalian counterpart.

2. C T l
One of the next antigens to appear during chick embryonic development is the CTl
antigen.” This M, 65,000 molecule, recognized by the CTl and C T la mAb, is first detectable
on thymocytes on E ll to 12. By E15, CTl is expressed by 97% of cortical thymocytes.
The same molecule may also be recognized by the X.14” and T D l” antibodies, although
the reported molecular weights of these antigens differ slightly. Another mAb, T,oA*, reacts
with a M, 65,000 protein that has a similar ontogenetic pattern of expression in the thymus.”
In contrast to C T l, however, this antigen is expressed by a subset of peripheral blood
lymphocytes and splenocytes.
The function of the CTl antigen is unknown although its tissue expression and ontogeny
are similar to the mammalian CDl antigen.” The CTl molecule may not be the avian
homologue of CDl as the protein immunoprecipitated by the CTl antibody has a M, 65,000
as opposed to a M, 40,000 of the CDl antigen. In addition, the human CDl antigen appears
to be associated with which has not yet been identified in CTl immunoprecipitates
from chicken thymocytes. However, the recent characterization of a M, 65,000 form of the
human CDl antigen using the mAB PHM3” leaves open the possibility that the CTl antigen
could be a chicken CDl homologue.

3. CDS
An avian homologue of the CD5 antigen also appears on approximately E l 1 to 12. The
CD5 antigen is identified by three different monoclonal antibodies: 3-58, CTLA5, and
CTLA 8.” “ This antigen is expressed on the majority of chick thymocytes and peripheral
T lymphocytes, but its expression by the various T-cell subsets has not yet been reported.
Incubation of chick splenocytes with anti-CD5 antibodies eliminates concanavalin A-ie-
sponsive lymphocytes.’* As in mammals,” the CD5 antigen is also expressed by a sub­
population of bursal lymphocytes and B cells (see Section IIl.B). Monoclonal antibodies
CTLA5 and CTLA 8 immunoprecipitate glycoproteins of M, 65,000 and 45,000, while 3­
58 immunoprecipitates a M, 56,000 protein. These values are similar to the mouse CD5
protein, which has a M, 67,000 and is associated with other, smaller proteins.”
6 Avian Cellular Immunology

4. CD4 and CD 8
Expression of the avian homologues of both CD4 and CDS begins in the thymus on
E12 to 13.'*° Initially, the CDS antigen is expressed by a small subpopulation of chick
thymocytes before the CD4 antigen is expressed in detectable levels. The expression of both
CD4 and CDS then increases rapidly, with approximately 95% of the thymocytes expressing
both by E17. After hatching, expression of these molecules is seen on S5 to 95% of the
thymocytes. As in nuunmals, most cortical thymocytes express both CD4 and CDS, whereas
mature thymocytes expressing either the CD4 or CDS antigens ate present in the thymic
medulla and in the peripheral blood and spleen.*°
Monoclonal antibodies reactive with the CDS molecule have identified a M, 63,000
protein under notueducing conditions from thymocytes and T-cell membranes.*** Under
reducing conditions, a single protein band of M, 34,000 has been observed, suggesting that
the CDS antigen is a disulfide-linked dimer. Another CDS mAb also immunoprecipitates a
protein of M, 65,000, in addition to the M, 33,000 to 35,000 dimer, under reducing con­
ditions.^* Whether this represents a CDS-associated molecule or nonreduced material remains
unclear. The chicken CD4 antigen has been identified by mAb as a single chain polypeptide
of M, 64,000 from thymocytes, splenocytes, and peripheral T cells.'*“ Three mAb reactive
with CD4 molecules have been reported.” “ *“
The CDS molecule in mammals is involved in signal transduction and TCR recognition
probably by facilitating the interaction of T cells with class I MHC on antigen-presenting
cells. The CDS molecule also marks a sublineage of T cells with cytotoxic capabilities.*' *^
CDS appears to function similarly in chickens and serves to delineate cytotoxic T cells.“ *“
As in manunals,**’*^ the CD4*’ T cells in the chicken have helper capabilities, mediated
at least in part by lymphokine activity.” “ *“ Avian CD4 mAb inhibit T-cell proliferative
responses,” “ *“ and this effect is synergistic with that of anti-class 11 MHC antibodies.”

5. T-Cell Receptors
a. Ontogeny and Tissue Localization o f CD3ITCR Proteins
The CD3/TCR complex appears on thymocytes soon after the CD4 and CDS molecules.**
Elements of the CD3 complex, the invariant portion of the TCR, are ftrst observed in the
cytoplasm on E9, 3 d prior to CD3 expression on the cell surface.** As in mammals, the
avian CD3 complex is always expressed on the cell surface in conjunction with the poly­
morphic T-cell receptor molecules.** ** The CD3 complex can be recognized by the CT3
mAb.** Three mutually exclusive sublineages of CT3*' cells are recognized by the TCRl,*“
TCR2,** and TCR3 mAb.** The first TCR isotype to appear in development is the *y8 TCR
(TCRl).** *“ This TCR isotype appears on the surface of E12 thymocytes in conjunction
with the CD3 complex. Relatively high levels of yS TCR molecules ( > 1 X 10*) are present
on the TCRl*^ cells from the onset of their expression.*“ The TCRl population expands
rapidly to reach a peak of approximately 30% of the thymocyte pool on E15. The proportion
of TCRl *■ thymocytes declines thereafter to approximately 10%. TCRl *■cells rapidly trav­
erse the thymus and can be seen in the spleen on E15.** In adult birds, approximately 20
to 50% of the peripheral blood lymphocytes and splenocytes express the TCRl isotype.** *'
While TCRl thymocytes rarely express either CD4 or CD 8, most of the TCRl *■ cells in
both the spleen and intestine express the CD 8 molecule.*“-**
The vast majority of chicken thymocytes eventually express the TCR2 (ap ) isotype.** *“
Expression of the TCR2 isotype begins on E15, 2 d after 78 *■ cells are first observed, thus
resembling the pattern observed in mammals.** ** The frequency of TCR2* cells increases
gradually until hatching when the majority of the thymocytes express this receptor isotype.
In contrast to the TCRl *■cells, the initial level of TCR2 receptors is very low (approximately
600 per cell) .*“ The density of TCR2 molecules then increases as these cells mature within
the thymus. TCR2*' cells in the thymic cortex express both CD4 and CD 8, whereas mature
TCR2^ cells in the thymic medulla, peripheral blood, spleen, and intestine express either
CD4 or CD 8.**-^’ A difference in thymic transit time is also seen for TC R l* and TCRZ"^
cells, with the former passing through the cortex within approximately 1 d as compared
with a 3 to 4 d transient time for the latter.^ All of these findings suggest differences in
the process of thymic education for the two cell lineages.
The tissue localization of the two TCR isotypes also differs. TCRl"^ splenocytes are
preferentially located in the splenic sinusoidal regions and in the intestinal epithelium in
both birds and mammals.” **” Whereas TCRl cells appear in the spleen by E15, TCR2"^
cells begin to enter the spleen on E l9, where they are localized to the periarteriolar sheath
regions. They reach the lamina propia of the intestine beginning on E20.”
One of the more interesting differences between the gallinaceous birds and mammals is
the existence of a third sublineage of T cells.” These express a CD3-associated heterodimer
that is not recognized by either the TCRl or TCR2 mAb but recognized by the TCR3 mAb.”
The TCR3"^ cells first appear in the thymus on E l7, and their frequency increases slowly
until the time of hatching when approximately 9% of the thymocytes express the TCR3
receptor."** Like the TCR2 population of thymocytes, the numbers of TCR3 molecules per
cell increase gradually as these cells mature.'*' TCR3* cells in the thymic cortex express
both CD4 and CD 8. In the medullary region, a small percentage of these cells express either
the CD4 or the CD 8 molecules.'*' The TCR3* cells begin to seed to the periphery after
hatching. In young adults, approximately 12 to 1S% of the peripheral T cells express this
receptor, 80% of these cells express CD4, and 20% express CD 8.** ** The TCR3* cells are
preferentially localized in the periarteriolar sheaths in the spleen, as are the TCR2* cells.
In contrast, TCR3* cells are rarely found in the chicken intestine.'*'

b. Biochemical Characterization o f the Chicken T-Cell Receptor Proteins

The CT3 mAb immunoprecipitates a complex of at least three proteins from T cells
with M, of 20,000, 19,000, and 17,000.** At least two of these proteins contain N-linked
oligosaccharides. When the T-cell surface molecules are solubilized with a mild detergent,
the TC R l, TCR2, and TCR3 heterodimers can be coprecipitated with the CT3 antibody.**
Each heterodimer can also be immunoprecipitated with the TCRl,** TCR2,*® or TCR3**
mAb. Both TCRl and TCR2 molecules are M, 90,000 heterodimers composed of M, 50,000
and 40,000 disulfide-linked glycoproteins.*’ Removal of the N-linked oligosaccharides re­
veals core proteins of M, 36,000 and 33,000 from TCRl and of M, 34,000 and 29,000 from
TCR2.*’ The TCR3 mAb immunoprecipitates a slightly smaller (M, 88,000) heterodimer
composed of M, 48,000 and 40,000 polypeptides from T-cell lysates.** Removal of the N-
linked sugar from the TCR3 molecules yields core proteins of M, 31,000 and 34,000, clearly
differentiating this TCR from TCRl.** The relationship between the TCR2 and TCR3 T-
cell sublineages is more difficult to discern. Both TCR2 and TCR3 contain a core protein
of M, 34,000, and in addition, the M, 50,000/48,000 peptides have similar isoelectric points
(pi 6.2 to 7.6) as do the 40,000 chains (pi 5.7 to 7.0).'** Deglycosylation of the isolated
M, 40,0(X) chains from both TCR2 and TCR3 immunoprecipitates yields M, 34,000 core
proteins. Peptide mapping data suggest that these two proteins are highly similar, but not
the same.**

c. Lineage Relationships o f the Cells Expressing the Different T-Cell Receptor Isotypes
The lineage relationships between the T C R l, TCR2, and TCR3 subpopulations have
been examined in embryonic antibody-suppression experiments.** *’-'*' The results support
the conclusion that all three subpopulations represent different T-cell sublineages. Injection
of the TCRl mAb selectively inhibits development of TC R l* cells without affecting de­
velopment of either the TCR2* or TCR3* cells.*’ Combining the TCRl mAb injection with
neonatal thymectomy results in a dramatic permanent depletion of TCRl * cells, suggesting
8 Avian Cellular Immunology

that this lineage of cells has a limited expansion capacity and requires prolonged seeding to
fill the peripheral compartment. Embryonic injections of TCR2 and TCR3 mAb do not affect
the development of the TCRl* cells.*' ** '^' Injection of the TCR2 mAb appears to abort
the maturational process, as cells expressing high levels of TCR2 are not observed in treated
birds. If combined with thymectomy, repeated TCR2 mAb injections dramatically diminish
the number of TCR2* cells.*' “ This reduction of TCR2* cells is compensated by a dramatic
increase in the nequency of TCRS*' cells. Reciprocal results are obtained using the TCR3
mAb, suggesting that the TCR2* and TCR3* cells represent distinct sublineages.
The waves of precursor cells for these three populations of T cells have been examined
using the chicken-quail chimera system."" Transplantation of chicken E9 thymus containing
the first wave of chicken stem cells into E3 quail embryos"' resulted in the development of
all three sublineages of chicken T ceils, indicating that the first wave of precursor cells gives
rise to all three sublineages of chicken T cells. Similarly, the second wave of thymic
precursors also gives rise to these cell lineages. The results of these studies indicate that the
thymus is the only site in which cells expressing TCR/CD3 complexes are generated."'

d. Functional Capability o f Different T-Cell Receptor Isotypes

Functional studies on the different T-cell lineages are still limited. Preliminary studies
indicate that both the TCR2''' and TCR3''^ cells can elicit a graft vs. host response, but the
frequencies of alloreactive cells differ in the two populations of T cells.'"' This suggests
that the repertoire of the TCR2 and TCR3 cell lineages differs. The TCRl * cells frequently
coexpress the CDS antigen in the spleen and intestine, suggesting that they recognize antigen
in association with class I and/or class l-like molecules which have been postulated to be
involved in the recognition process in mammals."^ Consistent with this view, the TCRl*
cells are impotent in the graft vs. host assay"* that depends on class II alloreactivity, whereas
TC R l* cells from class I alloantigen-stimulated chickens exhibit specific T-cell cytotox­
icity.'"* This can be inhibited by either anti-CD3 or anti-CD 8 antibodies. Cross-linkage of
the TCR1/CD3 complex with antibody has also been shown to elict the cytotoxic capabilities
of TCRl * cells.'"" TCRl */CD 8* cells may also be capable of suppressor activity."" These
functional clues, in association with the preferential tissue localization patterns of TCRl*
cells, suggest that these cells may play an important role in host defense. The large number
of y 8 cells relative to the mammal, moreover, makes the chicken an excellent model system
to examine the function of the TCRl lineage.
The TCR2* cell population appears to contain cells with both helper and cytotoxic
capabilities. TCR2* cells exhibit proliferative responses to both conconavalin A and allo­
geneic cells. These cells produce lymphokines and are capable of cytotoxic responses to
allogeneic target cells."*
While the in vitro responses of TCR3* cells have not been examined, TCR2-suppressed
chickens, in which the percentage of TCR3* cells is increased, are capable of mounting
antibody responses to sheep red blood cells,'"" suggesting that the TCR3* cells are capable
of helper functions as well as graft vs. host potential.

e. A Third Lineage o f Lymphocytes in Chickens

In the course of studies of T-cell development, we identified another lymphocyte lineage.
These cells are characterized by cytoplasmic expression of a CD3 epitope even though they
lack cell surface CD3/TCR."* "" These cells often express CDS on their surface. We have
named these cells TCRO because they express some of the characteristic markers of T cells,
but lack the capacity to express the T-cell receptor complex on their surface. They appear
initially in the E8 spleen, prior to the appearance of cells expressing the CD3/TCR complex
in the thymus. In studies of chick-quail chimeras, we have shown that TCRO cells do not
have the capacity to acquire cytoplasmic or surface expression of either immunoglobulin or
TCR proteins. In adults, TCRO cells are found in the spleen (1% of the CT3* cells), intestine
(30 to 40% of the CT3‘*^ cells), thymic medulla, and bursa.“
The origin of TCRO cells has been examined in the chicken-quail chimera system.“
These studies indicate that TCRO cells are derived from splenic precursors prior to the
migration of the first wave on stem cells to the thymus. Transplantation of E6 chicken spleen
into E3 quail embryos resulted in the normal development and localization of chick TCRO
cells in the spleen, thymic medulla, bursa, and intestine, indicating that TCRO cells represent
a separate lineage of lymphoid cells.

6. Interleukin 2 Receptor
The chicken homologue of the interleukin 2 (IL-2) receptor, recognized by the mAb
INN-CH-16,“ “ is expressed only on activated T cells with >1% of the unstimulated
splenocytes being reactive. The antibody stains less than 1% of the thymocytes and PBL.
In immunized chickens, cells expressing the IL-2 receptor are primarily localized in the
periarteriolar lymphatic sheath regions.
The IL-2 receptor mAb immunoprecipitates a M, 48,000 to 50,000 protein under reducing
conditions. This antibody has the ability to block mitogen-induced T-cell proliferation and
to reduce IL-2 absorption by T lymphoblasts from conditioned media. The kinetics of the
expression of IL-2 receptors are similar in birds and mammals.*’ **

7. O ther T-Cell Antigens

Several other T-cell surface antigens have been identified by mAb, but these antigens
have not been characterized extensively and their functions are unknown. Three such antigens
have been reported by Mazella and co-workers.“ One of these has a of 40,000 and is
expressed by 95% of the thymocytes, but not by peripheral T cells or other splenocytes.
This antigen is recognized by the TA3, T B l, and TB 6 mAb. While the ontogenetic pattern
of expression of this molecule has not been reported, its biochemical characteristics and
tissue distribution are reminiscent of the mammalian GDI antigen. Two additional antibodies
reported by this group react with both thymocytes and subsets of peripheral T cells. One of
these, TC4, identifies a cell-surface protein of M, 110,000 expressed by 90% of the medullary
thymocytes, 70 to 80% of the peripheral blood lymphocytes, and 60 to 70% of the splen­
ocytes. The other antibody, T A l, recognizes a 16,000 protein expressed by 40% of the
thymocytes (60 to 70% of the medullary subpopulation), 30 to 35% of the peripheral blood
lymphocytes, and 70 to 80% of the splenocytes. Two other anti-thymocyte antibodies, INN-
CH/T51 and INN-CH/T53, have been reported.™ These antibodies identify antigens of M,
186,000 and 135,000, respectively. No other information on function, ontogeny, and tissue
distribution has been reported.

B. B-CELL ANTIGENS
With the exception of immunoglobulins and a CD5 equivalent, the chicken B-lymphocyte
antigens identified by mAb have no known mammalian homologues. Mouse mAb have
identified chicken molecules that are antigenically divergent though their functional roles
may prove to be conserved in vertebrates. These novel antigens thus may provide an op­
portunity to describe aspects of B-cell maturation and function that have not yet been revealed
in mammalian systems. The patterns of expression and structural features of these proteins
offer clues to their functional roles in B-cell development.

1. Immunoglobulins
Immunoglobulin (Ig) is the most conspicuous and well-understood protein on the sur­
face of B lymphocytes. As in mammals, these antibody molecules define avian B cells as
a function of their state of differentiation. Functional analogues of mammalian IgM, IgG,
10 Avian Cellular Immunology

and IgA have been described,^' and the genes encoding chicken IgM and IgG have been
cloned.” ™ Mouse mAh which react with chicken Ig have been produced,” -” and the
structural features of the Ig proteins have been reviewed elsewhere.™
Chicken IgM, a pentameric molecule with a total mass of M, 880,000 to 890,000, is
composed of subunits of heavy and light chains of 63,000 to 70,000 and M, 22,000 to
24.000, respectively.™ Virtually all bursal lymphocytes and the majority of circulating B
lymphocytes express IgM on their surfaces.™
Chicken IgG is structurally distinct from the mammalian analogue, and some have
proposed that a unique appellation, IgY, be used to acknowledge these differences.^ Because
the chicken IgG molecule contains an additional constant domain™ when compared to the
M, 150,000 mammalian molecule, its weight is correspondingly higher at M, 165,000 to
180.000. In adult spleen, 6% of the lymphocytes are surface IgG *, while fewer cells express
IgG in blood (1.6%) and in bursa (0.2%).” In the bursa, some IgG"^ cells also express IgM,
evincing the sequential expression of IgM and then IgG by maturing B lymphocytes.” -™
IgA is expressed by fewer cells than IgG in spleen (0.6%), blood (0.2%), and bursa
(0.1%).” Soluble IgA concentrations are increased in bile and in secretions from mucosal
membranes.^' A secretory component, which binds to chicken IgA, can be detected as a
free protein in agammaglobulinémie birds, further suggesting the conservation of IgA func­
tion in avian species.” -“
An avian IgD candidate with a heavy chain of higher molecular weight (M, 81,000)
than that of p. chain has been identified. An anti-light-chain mAb immunoprecipitates the
putative IgD molecules from B-cell lysates cleared of IgM, IgG, and IgA by inununopre-
cipitation with isotype specific mAb.” In addition, light chains were detected on the surface
of cells from which IgM had been removed by modulation.” However, IgD-specific anti­
bodies have not been produced, and an avian 8-chain constant region gene has not been
identified.
Monoclonal antibodies have also been produced against Ig idiotypic epitopes. The Cld-
1, CId-2, and CId-3 antibodies recognize 0.3%, 0.2%, and 1% of the B cells in the bursa
and 1%, 0.2%, and 1% of circulating B lymphocytes, respectively.“ -“ These mAb were
produced against chicken antibodies to V-acetylglucosamine or p-aminobenzoic acid. The
specificity of these mAb for variable determinants was demonstrated by immunochemical
analysis. Lymphocytes expressing Ig with CId-1 and CId-2 idiotypes were increased four­
fold and sixfold in birds which were immunized with the original antigens.“
While these anti-idiotypic mAb react with small populations of B cells, another mAb
against the variable region of heavy chains, CV h- 1, binds up to one third of adult B cells
and plasma cells expressing IgM, IgG, and IgA.“
The cloning and sequencing of immunoglobulin genes encoding X light chains (probably
the only light chain isotype in chickens) and heavy chains have recently been accomplished.
These studies have shown that the mechanism for the generation of a repertoire of antigen­
binding immunoglobulins is quite different from the mammalian system.^^™-“ ~“ The X and
heavy chain loci both have a single functional variable region gene segment which is
rearranged to (joining) or J„ and D„ (diversity) gene segments.“ -“ Untranslatable pseu­
dogenes (25 ijfVx and at least 45 contribute nucleotide sequence diversity to the
intact and translated variable regions by a mechanism of copy replacement (gene conversion).
However, the resulting immunoglobulin proteins are structurally similar to mammalian im­
munoglobulins; variation in the coding Vh and genes is confined to the complementarity
determining regions (CDR).™-“
Allotypic markers of chicken IgM and IgG, defined by antisera, have also been useful
in studies of B-cell development. Allelic exclusion was originally described in studies that
took advantage of IgM allotype-specific antisera.“ The dynamics of the peripheral B-cell
population have also been examined by using immunoglobulin allotypic markers.“ In a
 11

TABLE 2
Chicken B-Cell Antigens

% Cells cxprcssiiig surface

M olecular mass
AnUgen m Ab (isotype) Bursa Blood Spleen (kDa) Ref.

Bu-Ia L-22 (-yj 90—95 1 0 -2 0 15—25 70 34

21-lA (7,) 94
Bu-lb 5 -1 1 0 2 (7 .) 85—90 2— 18 15— 27 70 94
Bu-2 Hy30 (7.) 98 17 35 66 98
CBl A-21 (7.) 97 17 18 110(55 dimer) 13
CB2 M2C2 (p ) 86 2 3 80— 125 13
CB3 3 - 1 6 (7.) 62— 83 20 26 50 13,99
CB4 5 - 1 5 (7.) 98 20 28 39,53,107 13
CBS 7— 19 (fi) 96 16 23 167 13
CB7 7— I0a2 (p) SI 1 2 148
CBS 32c4 (p) 90 15 10 148
CB9 65a3 (7 J 98 5 7 120 (65 dimer) 148
CBIO 65bS (7.) 97 5 10 220 148
CB II 3 U 6 ( m.) 26 15 21 37 148

review. Benedict and Berestecky^” have defined 11 allotypic markers for chicken
immunoglobulin.

2. Noninununoglobulin M arkers
Unlike the avian immunoglobulins, TCR and other T-cell antigens, a majority of B-cell
antigens in chickens are novel, and their functional roles have not been defined. These
antigens have been identified by mouse mAb, and their structures and patterns of expression
are described (Table 2). One group of such mAb is designated the CB series because they
react with chicken B cells. These are numbered in the order of their identification.

a. Bursal Lymphocyte Antigens

During the period of proliferation and clonal diversification that occurs in the bursa, B
cells may express adhesion molecules, receptors for growth or differentiation factors, or
enzymes. Two antigens expressed by all bursal lymphocytes, but not by peripheral B cells,
are identified by the CB2 and HNK-1 mAb.

I. CB2
CB2, a cell-surface molecule of M, 80,000 to 12S,0(X), is expressed by all bursal
lymphocytes. The level of expression varies widely on individual cells, with some being
only dimly positive while others are more than 100 times brighter in immunofluorescence
assays. The level of CB2 expression is not related to cell size or to the precise location of
the lymphocytes within the bursal follicles.

II. HNK-I
The HNK-1 mAb*' binds a myelin-associated glycoprotein on human neural and natural
killer (NK) cells.’^ It also binds chicken neurons, cortical thymocytes, and bursal lympho­
cytes. The HNK-1 antigen is not expressed by peripheral lymphocytes in the chicken.'^ The
antigen on bursal cells has a M, of 110,(XX), the same molecular mass as the myelin-associated
protein on neural celis,^^ suggesting that HNK-1 reactive molecules may play a role in
governing the developmental patterns of cells in both the nervous and immune systems.

b. B-Cell Antigens
Of the markers listed in Table 2, several are expressed by virtually all B cells. These
12 Avian Cellular Immunology

include Bu-1, Bu-2, C B l, CB3, CB4, and CBS. Others (CB7, CBS, CB9, CBIO, and C B ll)
are detected by mAb only on subpopulations of B cells.

I. Bu-l
One of the best-studied B-cell maricers is Bu-1. This M, 70,000 antigen was detected
first by alloantisera created by Gilmour and co-woiicers.” Xenoantisera and mouse mAb” **
were subsequently made against this cell-surface alloantigen. In young chickens, Bu-1 is
expressed by virtually all bursal lymphocytes, as well as the peripheral B cells (Table 2),
but is not found on plasma cells.^ In the liver, macrophage-like cells express Bu-1, but
these cells are not phagocytic, and cultured macrophages are not stained with a Bu-1 specific
mAb.’^
Allelic differences between Bu-1 molecules are recognized by alloantisera, and two
alleles have been defined by mAb. The L22” and 21-1A4** mAb recognize B u-la allelic
forms, while 511G2®^ recognizes B u-lb molecules. While not genetically linked to the MHC,
Bu-1 alleles appear to influence the level of expression of MHC class II (B-L) genes.^
When these allelic differences were used to study B-cell development in the bursa,’^ the
origin of precursors that colonize each follicle could be distinguished in chimeras of different
Bu-1 allotypes. By scoring the number of follicles containing Bu-la alone, Bu-lb alone, or
mixtures of the two allotypes, it was possible to estimate the average number of precursors
in each follicle at three or fewer.”

a. Bu-2
The Bu-2 antigen, a protein of M, 66,(XX), is expressed by bursal and peripheral B cells.
The level of expression on B cells is high, but low levels of Bu-2 are also detected on
macrophages and dendritic cells in the thymic medulla and in splenic periellipsoidal cuffs.^
Though of similar molecular weight and tissue distribution, Bu-2 was shown to be distinct
from the Bu-1 antigen by sequential immunoprecipitation, modulation studies, and two-
color immunofluorescence of bone marrow where Bu-2 expression exceeds that of B u-l.’*

ill. CB3
The CB3 antigen is a glycoprotein of M, 50,(XX) that is associated with the P j micro­
globulin (P 2*tn, M, 14,(XX)) on the surface of bursal and peripheral B lymphocytes.” Both
CB3 and Pj'in are immunoprecipitated by mAb against one or the other protein. The anti-
Pj-m antibody precipitates class I (B-F) heavy chains of M, 45,(XX) as well as the CB3 heavy
chain from B-cell lysates. The ^ 2-m associated with either class I or CB3 has an identical
pi and molecular mass. In addition to the difference in molecular weight, the pi of CB3
(6.5 to 8.5) and the class I heavy chain (< 5 to 6) are quite distinct.” Partial trypsin digestion
of these heavy chains releases peptides of different sizes, indicating that the distribution of
the basic amino acids is different in the two molecules. Although the CB3 molecule differs
from class I molecules in both physical characteristics and tissue distribution, the association
of CB3 with ^ 2-In suggests structural similarity to other ^ 2-In-associated class I-like proteins.
P 2-tnicroglobulin-associated proteins in mammals, C D l, Qa, and TL may resemble class
I molecules in their ability to present antigen. TL and Qa (voteins can be recognized by
T C R r T cells ('y8+ ).'“ '°' CDl is recognized by a TCR2"^ (ap ) CTL line which is
CD4” C D 8~.‘“ The hypothetical functions of class I molecules include the presentation of
specific antigens (possibly carbohydrates) to T C R l’’ cells.“ '®’ The presentation of these
antigens may occur in specific locations, such as the skin in the case of C D l''' Langerhans
cells.“ '®’ A similar situation may exist in the intestinal epithelium of chickens where TCRl ^
cells are preferentially localized.“ If CB3 functions as a class I-like molecule, B lymphocytes
could present antigens to T C R l''' cells in association with CB3 proteins, in addition to class
I- and Il-mediated recognition.
 13

iv. Other B-Cell Antigens

C B l, CB4, and CBS'^ are also expressed in peripheral B cells, as well as in the bursa.
The tissue distribution and molecular masses of these antigens are summarized in Table 2.
The CBl antigen, 110,000, was originally detected only on bursal lymphocytes and
B-cell tumors. Recent evidence indicates that the C B 1 mAb also reacts with normal peripheral
B cells. In the bursa, the CBl antigen is a disulfide-linked dimer composed of SS,000
subunits whose pi are slightly different.'^’ The CB4 antigen is a three chain structure with
noncovalently associated subunits of M, 107,000, 53,000, and 39,000. The CBS antigen,
a protein of M, 167,000, is expressed on plasma cells as well as B cells.

c. Antigens o f B-Cell Subpopulations

Subsets of B cells have been described in mammals.” '®* '“ In the chicken, CBS, CB9,
and CB 10 antigens are expressed on the majority of bursal lymphocytes, but only on a subset
of peripheral B cells. CBS is detected on approximately 60% of B lymphocytes in the blood
and about 90% of IgM** lymphoblasts and plasma cells. In contrast, both CB9 and CBIO
are expressed only by a small population of peripheral B lymphocytes. While CB9 is a
single polypeptide of M, 220,000, CBIO is a 120,000 dimer consisting of two disulfide-
linked 65,000 chains. The CBS molecule has not been characterized.
The C B l l antigen, although detected on the majority of peripheral B lymphocytes, is
expressed only by a subset of bursal cells. In young chicks, CB7 is expressed on 51% of
bursal cells and CBl 1 on 26%.'** The distribution of these antigens in the bursa is striking.
CB7*^ cells are located in the medulla, while CBl 1 cells are limited to the cortex and the
surrounding interfoliicular tissue. The mutually exclusive expression of these antigens by
two physically and maturationally distinct populations of cells suggests different roles for
these antigens in precise stages of differentiation. They also provide a convenient marker
with which to distinguish these differentiation stages in single cell suspensions. C B l l is an
antigen of 37,000 while the molecular weight of CB7 has not been determined.
The CD5 mAb (Section III. A) recognizes a subpopulation of B lymphocytes. In the
bursas of 8-week-old chickens, less than 10% of the lymphocytes bind these antibodies, but
by 3 months betwenn 30 and 50% of bursal cells are stained.” Between 55 and 65% of
peripheral B cells express CD5.” -^* Whether these CD5*' B cells represent an independent
lineage with a specific tissue distribution, as has been proposed in manunals,” has not been
explored.

d. Antigens o f Nonlymphoid Bursal Elements

Interactions of developing bursal lymphocytes with nonlymphoid cells in the bursa are
likely to be important, but these are not yet understood. One approach toward gaining the
relevant information is to make monoclonal antibodies against cell-surface molecules on
nonlymphoid bursal cells that may be involved in their interaction with developing B cells.
Two mAb, BEP-1 and BEP-2, mark antigens on bursal epithelial cells.'®* BEP-1 appears
on all epithelial cells in the bursa: plical epithelium, basement membrane-associated epi­
thelium, cortico-medullary epithelium, and, sparsely, on the follical-associated epithelium
(FAE). BEP-2 is a cytoplasmic antigen that is secreted by plical and medullary epithelium
in bursa, thymus, and goblet cells in the intestine.
The B.2 mAb recognizes another nonlymphoid antigen that is expressed by Ig~ cells
in the bursa.'®’ B.2 also binds the basement membrane, the cortico-medullary epithelium,
FAE (but not the rest of the plical membrane), and a mesh work of reticular cells in the
medulla."” Most phagocytic cells in the bursa express B.2 as do scattered cells in the spleen
and thymus. High endothelial venules and skeletal and smooth muscle are also reactive with
this antibody.
The B.3 mAb reacts with cells in the medulla region of bursal follicles, as well as thymic
14 Avian Cellular Immunology

and intestinal epithelium.“” Other antigens have been identified with antisera raised against
bursal cells: bursa reticular fiber antigen (CBRFA) and gut-associated mucin antigen
(CGAMA).“” These maricers may provide the ligands which mediate the communication of
B lymphocytes with the extracellular environment during the critical bursal phase of de­
velopment.

3. Developmental Expression of B-Cell Antigens

The bursa is colonized by lymphocyte precursors only between E7 and £14."° IgM is
first evident both in the cytoplasm and on the surface of the bursal lymphocytes around
£12 76.ni Immunoglobulin gene rearrangements can be detected between £10 and £12,"*
but the accumulation of somatic changes in the transcribed and gene segments
continues beyond the third week after hatching.**-“ '“ -"* Immunoglobulin gene rearrange­
ment, modification, and translation can thus be used as hallmarks for the different stages
in B-cell maturation, and the expression of other B-cell antigens can be placed within this
ftameworit. As the percentage of IgM*^ cells increases in the embryonic bursa, there is a
concordant increase in the percentages of cells that express most of the other B-cell antigens.
The level of their expression appears to be higher than IgM. An exception is the CB2 antigen
which is expressed by only a subpopulation of IgM*^ cells until late in ontogeny when all
of the IgM"^ cells become CB 2 "^."*
An intriguing recent fínding suggests that Ig gene rearrangement may also occur in
extra-bursal cells present in the £10 to 12 spleen, liver, and bone marrow."* Because these
rearrangements are very rare, it is not yet clear whether these are precursors of B cells or
if they are emigrants that have already traversed the bursal microenvironment."* Several of
the mAb that mark B cells in adults also react with Ig~ cells in embryonic spleen. B u-1,'"
319-D3,"*-'“ CB3, CB4, CB 8, CB9, CBIO, and CBll'**-'“ are expressed by embryonic
spleen cells that do not express immunoglobulin. The ftequency of cells expressing these
antigens decreases just before hatching when the frequency of Ig*^ cells in the periphery
begins to increase.

IV . L E U K O C Y T E C O M M O N A N T IG E N S A N D M Y E L O ID
A N T IG E N S

Leukocyte cotiunon antigens (LCA; CD4S; and T200) are expressed by virtually all
chicken leukocytes, but not by nonhemopoietic tissues. The LCA glycoproteins expressed
on different lymphocyte populations in mammals vary in apparent molecular weight from
180,000 to 220,000 as a function o f their differentiation status and lineage."* The size
heterogeneity is due to differential mRNA splicing and glycosylation status."* Certain LCA
epitopes are lineage-restricted, and mAb against these can be used to separate ftinctiorudly
important subsets of lymphoid cells. ' The large cytoplasrrtic domain of this highly conserved
molecule (~ 700 amino acids) contains multiple serine phosphorylation sites. Amino acid
sequence analysis has recently revealed striking homology between a major protein tyrosine
phosphatase (PTPase IB) and the tandem C-terminal homologous domains of LCA.'*° LCA
is now thought to function in specific signal transduction pathways via protein tyrosine
déphosphorylation. '*'
LCA molecules have also been identifred on the cells of avian species by mAb, most
of which are chicken specific. The L-17 mAb reacts with all myeloid and lymphoid cells
in blood and lymphoid organs, but not with erythrocytes or thrombocytes.** The L-17 antigens
have molecular weights of approximately 180,000 on thymocytes and 190,000 and 210,000
on bursal lymphocytes. Other antibodies reactive with similar molecules include the CL-
1'** and CLA3 m Ab.'“ A lineage-restricted mAb, CLA-1, which reacts with T cells, early
B cells, and myeloid cells,'* precipitates a glycoprotein composed of noncovalently linked
 15

chains of M, 180,000 and 195,000. The specificity of this LCA mAh appears to be similar
to the BCIO mAb which recognizes human leukocytes.'“ Few studies have addressed the
structure and function of the avian LCA.
Other nonlineage restricted mAb which may identify LCA or other leukocyte molecules
have also been reported, but the antigens have not been well characterized. The 56-3-C
mAb reacts with cell lines of myeloid and lymphoid origin, normal thymus and bursa cells,
and erythroblasts. HIS-C7 reacts with a membrane-bound determinant present on T and
B lymphocytes, granulocytes, monocytes, and macrophages.'“ The 17-B-2 mAb, which
identifies a M, 30,000 protein,'“ and L-43, which detects a molecule of M, 23,000,'* are
clearly different from the LCA mAb, but are similar to CAMPATH-1 which identifies a M,
23,000 to 30,000 glycoprotein expressed on most lymphoid cells and monocytes in hu­
m ans.'“ Another mAb, MB-1, reacts with quail, but not chicken, hemopoietic and endoth­
elial cells.*“ '“ The differential species-specific reactivity of these mAb is useful to study
stem cell differentiation in quail-chick chimeras.
Another family of structurally related leukocyte glycoproteins in mammals includes
molecules that mediate cellular adhesion in virtually all phases of the inunune response.
These adhesion molecules share a common ^ unit (M, 95,000) that associates noncovalently
with a series of different a chains of molecular weights ranging from 150,000 to 180,000.'“ '“
None of the avian antigens have been clearly characterized as homologues of these adhesion
proteins. We have produced a mAb, CLA-2, that reacts with most of the leukocytes and
precipitates a heterodimer consisting of M, 180,000 and 100,000 chains.'*® CLA-2 may thus
be a candidate for a chicken adhesion molecule. Several mAb reactive with monocytes/
macrophages have been generated. Four such antibodies react predominantly with immature
myeloid cells and detect glycoproteins of M, 100,000 to 190,000. '“ CVI-ChNL-68. 1 reacts
with monocytes, macrophages, and interdigitating cells.'“ K -1 reacts with adherent mono­
cytes from spleen and peripheral blood and a virus-transformed monocytic cell line. '^'
Several mAb against human myeloid cells react with NK cells. NK cells represent a
small fraction of peripheral blood mononuclear cells that are phenotypically and functionally
heterogenous.'^^ Both CD3~ and CDS*" lymphocytic clones with NK activity have been
defined.*’* NK cells in birds have been defined only by their spontaneously killing of tumor
cells in v i t r o . Recently, the K-14 and K-24 mAb were reported to stain 7 to 10% of
the splenocytes, 8 to 10% of blood leukocytes, and < 2% of the thymus and bursal lym­
phocytes. Pretreatment of spleen cells with either the K-14 or K-24 mAb abrogated NK-
cell activity, suggesting that these mAb react with molecules expressed on NK cells.'*' The
biochemical nature of these molecules is presently unknown.

V . C O N C L U S IO N

The avian model has contributed to our understanding of the embryonic development
of lymphocyte populations in the primary lymphoid organs and their peripheral migration.
Cooperation between T and B cells and the functions of these cells follow many of the same
rules in birds and marrunals. However, study of the relevant cell interactions and structure-
function relationships has been limited by the absence of suitable markers of the avian
lymphocytes. Availability of the mAb and lymphocyte gene probes outlined in this review
may enable the avian model to contribute new insight into the immune system.
The MHC class I and class II antigens, immunoglobulins, T-cell receptors, and their
accessory molecules on chicken lymphocytes are the best characterized surface markers of
avian cells. The biochemical characteristics of the identified molecules are highly conserved
in birds and mammals. The most unusual feature of the chicken MHC is the very small size
of the introns flanking the MHC genes.*® *' “ The B-L pH gene is only 1.7 kb long compared
to 8 kb for HLA-DQ p and up to 20 kb for HLA-DP p genes. The B-F and B-L p genes
16 Avian Cellular Immunology

are also two orders of magnitude closer than are the class I and class II genes in the mammalian
MHC complex. This may account for the absence of B-F/B-L recombinants.
Like the MHC antigens, the basic antigen recognition molecules and the differentiation
pathways defined for mammalian T cells also appear to be conserved in birds. The sequential
development of avian TCRl and TCR2'*’ cells during ontogeny parallels that noted for the
yd and a ^ T cells in mammals. However, the subsequent generation of a third TCR3-
bearing lineage of T cells in birds is an unprecedented finding. Our data suggest that the
M, 40,000 chain of TCR2 and TCR3 represent a shared a chain, while the M, 50,000 chain
of TCR2 and M, 48,000 chain of TCR3 represent distinctive ^ chains. Molecular cloning
of chicken TCR is in progress to determine the genetic basis for the sequential generation
of these two sublineages of T cells.
The avian CD3 antigen has been used to identify a novel population of cytoplasmic
CD3* lymphocytes that lack the TCR/CD3 receptor complex on their surface. These so-
called TCRO cells are abundant in the intestine and may represent a primitive lymphoid cell
that plays an important local role in body defense. In support of this idea, gut-associated
lymphoid tissue is found in all vertebrates and in some invertebrates.'^ A subpopulation of
human lymphocytes capable of specific lysis of allogeneic target cells expresses the CD3 e
protein in the absence of other elements of the TCR/CD3 complex.'” The expression of
CD3 C chain has also been noted in human CD3~ NK c e l l s . I t remains to be determined
whether the avian TCRO cells have NK activity or represent a novel lineage of cells.
Chicken immunoglobulins resemble their mammalian counterparts in both structure and
function. However, mechanisms for generating these molecules, as well as the site of origin
of the cells that produce them, differ from the mammalian model in many respects. The
bursa of Fabricius, which is required for normal avian B-cell development, has no anatomical
equivalent in mammals. Cells with the pre-B phenotype (cytoplasmic p. heavy chain but no
light chain expression), which are the hallmark of B-lymphopoietic sites in mammals, are
not detected in the bursa. The primary role of the bursa is to expand the B-cell repertoire.
Diversity is generated by a somatic mechanism of gene conversion in which a pool of Vh
and Vi, pseudogenes are used as donors of sequence to the productively rearranged variable
gene segment.’^-” At both the light chain and heavy chain gene loci, only a single variable
gene segment is rearranged, and usually this occurs on only one allele. This strict constraint
on rearrangement appears to limit aberrant rearrangements. This is an important safeguard
since any non-fiinctional rearrangements could be corrected by gene conversion events which
occur continuously in the bursa, violating the principle of allelic exclusion.
Unlike immunoglobulins, most of the other B-cell maikers identified so far have no
known mammalian homologues. Studies of the structure, function, and genes encoding these
novel antigens may be valuable in our understanding o f the evolution of the immune system.
The information obtained may also yield insight into the roles that analogous molecules play
in mammals.

ACKNOW LEDGM ENTS

We thank Mrs. E. A. Brookshire for her excellent secretarial assistance. This work has
been supported by grants CA 16673 and CA 13148, awarded by the National Institutes of
Health. MDC is an HHMI investigator.
 17

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Another random document with
no related content on Scribd:
had married a seafaring youth, and borne him one fair child. Her
husband was returning from a distant voyage; had entered the sea of
Solway; his native hills—his own home—rose to his view, and he saw
the light streaming from the little chamber window, where his wife
and his sweet child sat awaiting his return. But it was not written
that they were to meet again in life. She heard the sweep of a
whirlwind, and she heard a shriek, and going to her chamber-door,
she saw the ship sinking, and her husband struggling in the agitated
water. It is needless to lengthen a sorrowful story: she now threw
herself weeping over his grave, and poured out the following wail:—
“He was the fairest among men, yet the sea swept him away: he
was the kindest hearted, yet he was not to remain. What were all
other men compared to him,—his long curling hair, and his sweet
hazel eyes, and his kind and gladsome tongue? He loved me long,
and he won me from many rivals; for who could see his face, and not
love him? who could listen to his speech, and refuse him aught?
When he danced, maids stood round, and thought his feet made
richer music than the instruments. When he sang, the maids and
matrons blessed him; and high-born dames loved the song of my
frank and gentle sailor. But there is no mercy in the ocean for the
sons of men; and there is nought but sorrow for their daughters. Men
go gray-headed to the grave, who, had they trusted the unstable
deeps, would have perished in their prime, and left fatherless babes,
and sorrowing widows. Alas, alas! in lonely night, on this eerie spot,
on thy low and early grave, I pour forth my heart! Who now shall
speak peace to my mind, and open the latch of my little lonely home
with thy kind and anxious hand? Who now shall dandle my sweet
babe on his knee, or love to go with me to kirk and to preaching,—to
talk over our old tales of love and courtship,—of the secret tryst and
the bridal joy!”
And, concluding her melancholy chant, she looked sorrowfully and
steadfastly at the grave, and recommenced anew her wailing and her
tears.
The widow’s grief endured so long that the moon began to make
her approach manifest by shooting up a long and a broad stream of
thin, lucid, and trembling light over the eastern ridge of the
Cumberland hills. She rose from her knees, shed back her moist and
disordered locks, showing a face pale but lovely, while the watery
light of two large dark eyes, of liquid and roving blue, was cast
mournfully on the way homewards, down which she now turned her
steps to be gone. Of what passed in the pastor’s mind at this
moment, tradition, which sometimes mocks, and at other times
deifies, the feelings of men, gives a very unsatisfactory account. He
saw the hour of appointment with his shadowy messenger from the
other world arrive and pass without his appearance; and he was
perhaps persuaded that the pure, and pious, and overflowing grief of
the fair young widow had prevented the intrusion of a form so
ungracious and unholy. As she advanced from the burial-ground, the
pastor of her parish stood mute and sorrowful before her. She passed
him as one not wishing to be noticed, and glided along the path with
a slow step and a downcast eye.
She had reached the side of a little lonely stream, which glided half
seen, half hid, underneath its banks of broom and honeysuckle,
sprinkled at that hour with wild daisies, and spotted with primroses
—when the voice of Ezra reached her ears. She made a full stop, like
one who hears something astounding, and turned round on the
servant of the altar a face radiant with tears, to which her tale of woe,
and the wild and lonely place, added an interest and a beauty.
“Young woman,” he began, “it is unseemly in thee to bewail thy
loss at this lonely hour, and in this dreary spot: the youth was given
to thee, and ye became vain. I remarked the pride of thy looks, and
the gaudiness of thine apparel, even in the house of holiness; he is
taken from thee, perhaps, to punish thy pride. There is less meekness
in thy sorrow than there was reason in thy joy; but be ye not
discomforted.”
Here the weeping lady turned the sidelong glance of her swimming
eyes on Ezra, shed back the locks which usurped a white brow and
snowy temples, and folding her hands over a bosom, the throbbings
of which made the cambric that concealed it undulate like water,
stood still, and drank in his words of comfort and condolence.
Tradition always conducts Ezra and the mariner’s widow to this
seldom frequented place. A hundred and a hundred times have I
mused over the scene in sunlight and moonlight; a hundred and a
hundred times have I hearkened to the wild and variable accounts of
the peasantry, and sought to make bank, and bush, and stream, and
tree assist in unravelling the mystery which must still hang over the
singular and tragic catastrophe. Standing in this romantic place, a
pious man, not over-stricken in years, conversing with a rosy young
widow, a vain and a fair creature, a bank of blossomed flowers beside
them, and the new risen moon scattering her slant and ineffectual
beams on the thick budded branches above them,—such is the
picture which tradition invariably draws, while imagination
endeavours to take up the tender thread of the story, and
imagination must have this licence still. Truth contents herself with
the summary of a few and unsatisfactory particulars. The dawn of
morning came, says Truth, and Ezra had not returned to his manse.
Something evil hath happened, said Imagination, scattering as she
spoke a thousand tales of a thousand hues, many of which still find
credence among the pious people of Galloway.
Josiah, the old and faithful servant of Ezra, arrived in search of his
master at the lonely burial-ground, about the dawn of the morning.
He had become alarmed at his long absence, and his alarm was not
abated by the unholy voices which at midnight sailed round the
manse and kirk, singing, as he imagined, a wild and infernal hymn of
joy and thanksgiving. He traced his steps down the footpath by the
rivulet side till he came to the little primrose bank, and found it
trodden upon and pressed as if two persons had been seated among
the flowers. Here all further traces ceased, and Josiah stood
pondering on the power of evil spirits, and the danger of holding
tryst with Beelzebub or any of the lesser spirits of darkness.
He was soon joined by an old shepherd, who told a tale which
pious men refuse to believe, though they always listen to it. The
bright moonlight had made him imagine it was morning, and he
arose and walked forth to look at his lambs on the distant hill—the
moon had been up for nearly an hour. His way lay near the little
lonely primrose bank, and as he walked along he heard the
whispering of tongues: he deemed it some idle piece of lovemaking,
and he approached to see who they might be. He saw what ought not
to be seen, even the reverend Ezra seated on the bank, and
conversing with a buxom young dame and a strange one. They were
talking wondrous kindly. He observed them for a little space; the
young dame was in widow’s weeds; the mariner’s widow wore the
only weeds, praise be blest, in the parish, but she was a raven to a
swan compared to the quean who conversed with the minister. She
was indeed passing fair, and the longer he looked on her she became
the lovelier—ower lovely for mere flesh and blood. His dog shrunk
back and whimpered, and an owl that chased a bird in the grove
uttered a scream of terror as it beheld her, and forsook its prey. At
length she turned the light of her eyes on himself; Will-o’-the-wisp
was but a proverb to them; they had a glance he should never get the
better of, and he hardly thought his legs carried him home, he flew
with such supernatural speed.
“But, indeed,” added the cautious peasant, “I have some doubts
that the whole was a fiction of the auld enemy, to make me think ill
of the douce man and the godly; and if he be spared to come home,
so I shall tell him. But if Ezra, pious man, is heard of nae mair, I shall
be free to believe that what I heard I heard, and what I saw I saw.
And Josiah, man, I may as weel give you the benefit of my own
opinion. I’ll amaist aver on my Bible, that the minister, a daring man
and a courageous,—ower courageous, I doubt,—has been dared out
to the lonely place by some he, or, maybe, she-fiend—the latter maist
likely; and there he has been overcome by might or temptation, and
now Satan may come atween the stilts of the gospel plough, for the
right hand of Ezra will hold it no longer; or I shouldna wonder,”
added the shepherd, “but that the old dour persecutor Bonshaw has
carried him away on his fiend-steed Geordie Johnstone; conscience!
nought mair likely; and I’ll warrant even now they are ducking him
in the dub of perdition, or picking his banes ahint the hallan o’ hell.”
The whole of this rustic prediction was not fulfilled. In a little deep
wild dell, at the distance of a gunshot, they found Ezra Peden lying
on the ground, uttering words which will be pardoned, since they
were the words of a delirious tongue. He was carried home amid the
sympathy and sorrow of his parishioners; he answered no question,
nor seemed to observe a single face, though the face of many a friend
stood round him. He only raved out words of tenderness and
affection, addressed to some imaginary person at his side; and
concluded by starting up, and raising such an outcry of horror and
amazement, as if the object of his regard had become a demon: seven
strong men could hardly hold him. He died on the third day, after
making a brief disclosure, which may be readily divined from this
hasty and imperfect narrative.
 YOUNG RONALD OF MORAR:
A TRADITIONARY TALE OF THE WESTERN
HIGHLANDS.

Angus Macdonald, a son of Clanranald, having quarrelled with his

neighbour and namesake, the Laird of Morar, he made an irruption
into that district, at the head of a select portion of his followers. One
of his men was celebrated for his dexterity as a marksman; and on
their march he gave a proof of this, by striking the head off the
canna, or moss cotton, with an arrow. This plant is common on
mossy ground in the Highlands; it is as white as the driven snow, and
not half the size of the lily.
Having got possession of the cattle, Angus was driving away the
spreith to his own country; but Dugald of Morar pursued him with a
few servants who happened to be at hand; and, being esteemed a
man of great bravery, Angus had no wish to encounter him. He
ordered the marksman to shoot him with an arrow; but the poor
fellow, being unwilling to injure Dugald, aimed high, and overshot
him. Angus observed this, and expressed his surprise that a man who
could hit the canna yesterday, could not hit Dugald’s broad forehead
that day; and drawing his sword, swore that he would cleave the
marksman’s head should he miss him again. John then reluctantly
drew his bow, and Dugald fell to rise no more.
Angus got into his hands the only son of the dreaded Morar, then
very young; and the treatment which the unfortunate boy received
was calculated to injure his health and shorten his life. A poor girl,
who attended the calves, had pity on him, and at last contrived to
carry him away, wrapped up in a large fleece of wool. Having escaped
from her pursuers, she made her way to the house of Cameron of
Lochiel. Here she and the boy were most hospitably received; and,
according to the custom of the country in those days, they passed a
year and a day without being asked any question. At the end of that
period, Lochiel made inquiry regarding the boy, and the girl candidly
told him her story. He thus discovered that the boy was the son of his
own wife’s sister; but he concealed the whole from his lady, of whose
secrecy he was not very confident. But he treated young Ronald with
great kindness. Lochiel had a son much of the same age; the two boys
frequently quarrelled, and the lady was angry to see her own son
worsted. She at last swore that “the girl and her vagabond must quit
the house next morning.” The generous Lochiel set out with the boy
to Inverness, where he boarded him under a false name, and placed
the woman in the service of a friend in the neighbourhood, that she
might have an eye to his condition.
Ronald received such education as befitted his birth; and when he
grew up to manhood, he paid a visit to Lochiel, his kind benefactor,
in Lochaber, who was so much satisfied with him, that he
determined on giving him his powerful assistance in recovering his
paternal estate, which was then in the possession of Angus.
Lochiel ordered a hundred men to attend himself and Ronald on
this occasion; and they arrived in Morar on a Sunday, when the
usurper and all his people were in church at mass. He congratulated
the young man on the opportunity he now had of avenging his
father’s blood, and destroying all his enemies at once, by burning
them in the church. Ronald humanely objected, that though many of
those persons then in the church were guilty of his father’s death, yet
there were others innocent of that crime; and he declared that if his
estate could not be recovered otherwise, he would rather want it, and
trust to Providence and his own valour. Lochiel did not at all relish
such sentiments, and left Ronald to his fate.
Ronald took refuge in a cavern, and the daughter of Angus, his
only child, frequently passed that way, in looking after her father’s
fold. He sometimes got into conversation with her; and, though but a
child, she became attached to him. He prevailed upon her to get his
shirts washed for him. Her father having accidentally discovered the
linen bleaching, observed the initial letters of Ronald’s name; and
making inquiry into the circumstances, soon suspected that he was at
hand. He attempted to persuade his daughter to decoy Ronald into
his power; but she told the young man all that her father proposed to
her; and he, finding that Angus was still thirsting for his blood,
immediately left the country, and took the girl along with him. With
much difficulty he conveyed her in safety to Inverness, from whence
he procured a passage to France, where he placed her in a convent.
He entered the French army, and was much distinguished for his
bravery; he was thus enabled to support himself, and to defray the
expense of her education. When the young woman was of age, they
were married, and returned to Scotland. Ronald having obtained
strong recommendations to the king, he found means of being
reconciled to Angus, who was then old, and had become very
penitent. He made great professions of friendship and attachment to
Ronald; but his daughter was always doubtful of his sincerity, and it
would appear that she had justly appreciated his disposition. One
night, Ronald having feigned intoxication and retired to rest, the old
barbarian calculated that he would sleep very soundly, and slunk into
his apartment, armed with a dirk, to stab his son-in-law; but the
young man watched the treacherous hypocrite, and put him to death.
Ronald obtained possession of his paternal estate, and, after a long
and prosperous life, became the founder of a very respectable family.
—Lit. Gazette.
 THE BROKEN RING.

By one of the Authors of the “Odd Volume.”

“Hout, lassie,” said the wily Dame Seton to her daughter, “dinna
blear your een wi’ greeting. What would honest Maister Binks say, if
he were to come in the now, and see you looking baith dull and dour?
Dight your een, my bairn, and snood back your hair—I’se warrant
you’ll mak a bonnier bride than ony o’ your sisters.”
“I carena whether I look bonny or no, since Willie winna see me,”
said Mary, while her eyes filled with tears. “Oh, mother, ye have been
ower hasty in this matter; I canna help thinking he will come hame
yet, and make me his wife. It’s borne in on my mind that Willie is no
dead.”
“Put awa such thoughts out o’ your head, lassie,” answered her
mother; “naebody doubts but yoursel that the ship that he sailed in
was whumelled ower in the saut sea—what gars you threep he’s
leeving that gate?”
“Ye ken, mother,” answered Mary, “that when Willie gaed awa on
that wearifu’ voyage, ‘to mak the crown a pound,’ as the auld sang
says, he left a kist o’ his best claes for me to tak care o’; for he said he
would keep a’ his braws for a day that’s no like to come, and that’s
our bridal. Now, ye ken it’s said, that as lang as the moths keep aff
folk’s claes, the owner o’ them is no dead,—so I e’en took a look o’ his
bit things the day, and there’s no a broken thread among them.”
“Ye had little to do to be howking among a dead man’s claes,” said
her mother; “it was a bonny like job for a bride.”
“But I’m no a bride,” answered Mary, sobbing. “How can ye hae
the heart to speak o’t, mother, and the year no out since I broke a
ring wi’ my ain Willie!—Weel hae I keepit my half o’t; and if Willie is
in this world, he’ll hae the other as surely.”
“I trust poor Willie is in a better place,” said the mother, trying to
sigh; “and since it has been ordered sae, ye maun just settle your
mind to take honest Maister Binks; he’s rich, Mary, my dear bairn,
and he’ll let ye want for naething.”
“Riches canna buy true love,” said Mary.
“But they can buy things that will last a hantle longer,” responded
the wily mother; “so, Mary, ye maun tak him, if you would hae me
die in peace. Ye ken I can leave ye but little. The house and bit garden
maun gang to your brother, and his wife will mak him keep a close
hand;—she’ll soon let you see the cauld shouther. Poor relations are
unco little thought o’; so, lassie, as ye would deserve my benison,
dinna keep simmering it and wintering it any longer, but take a gude
offer when it’s made ye.”
“I’ll no hae him till the year is out,” cried Mary. “Wha kens but the
ship may cast up yet?”
“I fancy we’ll hae to gie you your ain gate in this matter,” replied
the dame, “mair especially as it wants but three weeks to the year,
and we’ll need that to hae ye cried in the kirk, and to get a’ your
braws ready.”
“Oh, mother, mother, I wish ye would let me die!” was Mary’s
answer, as she flung herself down on her little bed.
Delighted at having extorted Mary’s consent to the marriage,
Dame Seton quickly conveyed the happy intelligence to her son-in-
law elect, a wealthy burgess of Dunbar; and having invited Annot
Cameron, Mary’s cousin, to visit them, and assist her in cheering the
sorrowful bride, the preparations for the marriage proceeded in due
form.
On the day before that appointed for the wedding, as the cousins
sat together, arranging the simple ornaments of the bridal dress,
poor Mary’s feelings could no longer be restrained, and her tears fell
fast.
“Dear sake, Mary, gie ower greeting,” said Annot; “the bonny white
satin ribbon is wringing wet.”
“Sing her a canty sang to keep up her heart,” said Dame Seton.
 “I canna bide a canty sang the day, for there’s ane rinnin’ in my
head that my poor Willie made ae night as we sat beneath the rowan-
tree outby there, and when we thought we were to gang hand in hand
through this wearifu’ world,” and Mary began to sing in a low voice.
At this moment the door of the dwelling opened, and a tall, dark-
complexioned woman entered, and saying, “My benison on a’ here,”
she seated herself close to the fire, and lighting her pipe, began to
smoke, to the great annoyance of Dame Seton.
“Gudewife,” said she gruffly, “ye’re spoiling the lassie’s gown, and
raising such a reek, so here’s an awmous to ye, and you’ll just gang
your ways, we’re unco thrang the day.”
“Nae doubt,” rejoined the spaewife, “a bridal time is a thrang time,
but it should be a heartsome ane too.”
“And hae ye the ill-manners to say it’s otherwise?” retorted Dame
Seton. “Gang awa wi’ ye, without anither bidding; ye’re making the
lassie’s braws as black as coom.”
“Will ye hae yer fortune spaed, my bonny May?” said the woman,
as she seized Mary’s hand.
“Na, na,” answered Mary, “I ken it but ower weel already.”
“You’ll be married soon, my bonny lassie,” said the sibyl.
“Hech, sirs, that’s piper’s news, I trow,” retorted the dame, with
great contempt; “can ye no tell us something better worth the
hearing?”
“Maybe I can,” answered the spaewife. “What would you think if I
were to tell you that your daughter keeps the half o’ the gold ring she
broke wi’ the winsome sailor lad near her heart by night and by day?”
“Get out o’ my house, ye tinkler!” cried Dame Seton, in wrath; “we
want to hear nae such clavers.”
“Ye wanted news,” retorted the fortune-teller; “and I trow I’ll gie ye
mair than you’ll like to hear. Hark ye, my bonnie lassie, ye’ll be
married soon, but no to Jamie Binks,—here’s an anchor in the palm
of your hand, as plain as a pikestaff.”
“Awa wi’ ye, ye leein’ Egyptian that ye are,” cried Dame Seton, “or
I’ll set the dog on you, and I’ll promise ye he’ll no leave ae dud on
your back to mend another.”
 “I wadna rede ye to middle wi’ me, Dame Seton,” said the fortune-
teller. “And now, having said my say, and wishing ye a blithe bridal,
I’ll just be stepping awa;” and ere another word was spoken, the
gipsy had crossed the threshold.
“I’ll no marry Jamie Binks,” cried Mary, wringing her hands; “send
to him, mother, and tell him sae.”
“The sorrow take the lassie,” said Dame Seton; “would you make
yoursel and your friends a warld wonder, and a’ for the clavers o’ a
leein’ Egyptian,—black be her fa’, that I should ban.”
“Oh, mother, mother!” cried Mary, “how can I gie ae man my
hand, when another has my heart?”
“Troth, lassie,” replied her mother, “a living joe is better than a
dead ane ony day. But whether Willie be dead or living, ye shall be
Jamie Binks’ wife the morn. Sae tak nae thought o’ that ill-deedy
body’s words, but gang ben the house and dry your een, and Annot
will put the last steek in your bonny white gown.”
With a heavy heart Mary saw the day arrive which was to seal her
fate; and while Dame Seton is bustling about, getting everything in
order for the ceremony, which was to be performed in the house, we
shall take the liberty of directing the attention of our readers to the
outside passengers of a stagecoach, advancing from the south, and
rapidly approaching Dunbar. Close behind the coachman was seated
a middle-aged, substantial-looking farmer, with a round, fat, good-
humoured face, and at his side was placed a handsome young sailor,
whose frank and jovial manner, and stirring tale of shipwreck and
captivity, had pleasantly beguiled the way.
“And what’s taking you to Dunbar the day, Mr Johnstone?” asked
the coachman.
“Just a wedding, John,” answered the farmer. “My cousin, Jamie
Binks, is to be married the night.”
“He has been a wee ower lang about it,” said the coachman.
“I’m thinking,” replied the farmer, “it’s no the puir lassie’s fault
that the wedding hasna been put off langer; they say that bonny
Mary has little gude will to her new joe.”
“What Mary is that you are speaking about?” asked the sailor.
 “Oh, just bonny Mary Seton that’s to be married the night,”
answered the farmer.
“Whew!” cried the sailor, giving a long whistle.
“I doubt,” said the farmer, “she’ll be but a waefu’ bride, for the
sough gangs that she hasna forgotten an auld joe; but ye see he was
away, and no likely to come back, and Jamie Binks is weel to pass in
the world, and the mother, they say, just made her life bitter till the
puir lassie was driven to say she would take him. It is no right in the
mother, but folks say she is a dour wife, and had aye an ee to the
siller.”
“Right!” exclaimed the young sailor, “she deserves the cat-o’-nine
tails!”
“Whisht, whisht, laddie,” said the farmer. “Preserve us! where is he
gaun?” he continued, as the youth sprung from the coach and struck
across the fields.
“He’ll be taking the short cut to the town,” answered the
coachman, giving his horses the whip.
The coach whirled rapidly on, and the farmer was soon set down at
Dame Seton’s dwelling, where the whole of the bridal party was
assembled, waiting the arrival of the minister.
“I wish the minister would come,” said Dame Seton.
“We must open the window,” answered Annot, “for Mary is like to
swarf awa.”
This was accordingly done, and as Mary sat close by the window,
and gasping for breath, an unseen hand threw a small package into
her lap.
“Dear sirs, Mary,” said Dame Seton, “open up the bit parcel, bairn;
it will be a present frae your Uncle Sandie; it’s a queer way o’ gieing
it, but he ne’er does things like ony ither body.” The bridal guests
gathered round Mary as she slowly undid fold after fold. “Hech!”
observed Dame Seton, “it maun be something very precious to be in
such sma’ bouk.” The words were scarcely uttered when the half of a
gold ring lay in Mary’s hand.
“Where has this come frae?” exclaimed Mary, wringing her hands.
“Has the dead risen to upbraid me?”
 “No, Mary, but the living has come to claim you,” cried the young
sailor, as he vaulted through the open window, and caught her in his
arms.
“Oh, Willie, Willie, where hae ye been a’ this weary time?”
exclaimed Mary, while the tears fell on her pale cheek.
“That’s a tale for another day,” answered the sailor; “I can think of
nothing but joy while I haud you to my breast, which you will never
leave mair.”
“There will be twa words to that bargain, my joe,” retorted Dame
Seton. “Let go my bairn, and gang awa wi’ ye; she’s trysted to be this
honest man’s wife, and his wife she shall be.”
“Na, na, mistress,” said the bridegroom, “I hae nae broo o’
wedding another man’s joe: since Willie Fleming has her heart, he
may e’en tak her hand for me.”
“Gude save us,” cried the farmer, shaking the young sailor by the
hand, “little did I ken wha I was speaking to on the top of the coach. I
say, guidwife,” he continued, “ye maun just let Willie tak her; nae
gude e’er yet come o’ crossing true love.”
“’Deed, that’s a truth,” was answered by several bonny
bridesmaids. Dame Seton, being deserted by her allies, and finding
the stream running so strongly against her, at length gave an
unwilling consent to the marriage of the lovers, which was celebrated
amidst general rejoicings; and at the request of his bride, Willie, on
his wedding-day, attired himself in the clothes which the moths had
so considerately spared for the happy occasion.
 A PASSAGE OF MY LIFE.

Maiden aunts are very tough. Their very infirmities seem to bring
about a new term of life. They are like old square towers—nobody
knows when they were built, and nobody knows when they will
tumble down. You may unroof them, unfloor them, knock in their
casements, and break down their doors, till the four old black walls
stand, and stand through storm and sunshine year after year, till the
eye, accustomed to contemplate the gradual decay of everything else,
sickens to look at this anomaly in nature. My aunt, dear good soul,
seemed resolved never to die,—at least to outlive her hopeful
nephew. I thought she was to prove as perdurable as a dried
mummy,—she was by this time equally yellow and exsiccated as any
of the daughters of Pharaoh.
I had run myself quite aground. But my extravagances, as well as
my distresses, I had the policy to conceal from my aged relative. She,
honest lady, occasionally had pressed me to accept of some slight
pittances of two or three £50’s at different times, which, after much
difficulty and entreaty, I made a merit of accepting, stoutly asserting
that I only received them to avoid hurting her feelings—that my own
income was amply sufficient for the limited wants of a scholar, or to
any one who could put in practice the rules of wholesome economy;
but this trifle certainly would enable me to purchase a few rather
expensive publications which I could not otherwise have hoped to do,
and which would prove of essential use in furthering the progress of
the two great works I had commenced while at college, and had been
busy with ever since, viz.: “A History of Antediluvian Literature, Arts,
and Sciences,” and, “A Dissertation on the Military Tactics of the
Assyrians,” which I intended should appear along with the last
volume of Valpy’s Greek Dictionary, or the first of Sir James
Mackintosh’s History of Great Britain.
 Fortune at last grew tired of persecuting me; she fairly turned her
wheel, and put me on the brightest spoke. My aunt’s factor called one
day, and let me know that he thought I should make my visits at
Broadcroft more frequent—take a little interest in looking over the
ditching and draining of the estate (short-sighted man, he little knew
how much I had ditched and drained it by anticipation!)—walk
through the woods and plantations, and bestow my opinion as to
thinning them (they were long ago, in my own mind, transferred to
the timber-yard)—apply myself a little to master the details of
business connected with agricultural affairs, such as markets, green
and white crops, manure, &c. &c.; and concluded by telling me that
his son was a remarkably clever lad, knew country matters
exceedingly well, and would be a most valuable acquisition as factor
or land grieve to any gentleman of extensive landed property. The
drift of this communication I perfectly understood. I listened with
the most profound attention, lamented my own ignorance of the
subjects wherein his clever son was so much at home, and wished
only that I had an estate, that I might entrust it to the care of so
intelligent a steward. After dispatching a bottle or two of claret, we
parted mutually pleased.
He had seen my aunt’s will, and, in the fulness of his heart, ran
over the legal jargon which constituted me the owner of Broadcroft,
Lilliesacre, Kittleford, Westerha’, Cozieholm, Harperston, and
Oxgang, with hale parts and pendicles, woods and fishings, mills and
mill-lands, muirs and mosses, rights of pasturage and commonty. I
never heard more delightful music all my days than the hour I spent
hearkening to this old rook cawing over the excellent lands that were
mine in prospective. My aunt’s letters, after this, I found assumed a
querulous tone, and became strongly impregnated with religious
commonplaces—a sure sign to me that she herself was now winding
up her earthly affairs—and generally concluded with some such
sentence as this: “I am in a comfortable frame of spirit, but my
fleshly tabernacle is sorely decayed—great need hath it of a sure prop
in the evening of its days.” These epistles I regularly answered,
seasoning them with scriptural texts as well as I could. Some, to be
sure, had no manner of connection or application whatsoever; but I
did not care for that if they were there. I stuck them thick and
threefold, for I knew my aunt was an indulgent critic, provided she
got plenty of matter. I took the precaution also of paying the postage,
for I learned, with something like satisfaction, that of late she had
become rather parsimonious in her habits. I also heard that she daily
took much comfort in the soul-searching and faith-fortifying
discourses of Mr Samuel Salmasius Sickerscreed, a migratory
preacher of some denomination or other, who had found it
convenient for some months to pitch his tent in the Broadcroft.
Several of my aunt’s letters told me, in no measured terms, her high
opinion of his edifying gifts. With these opinions, as a matter of
course, I warmly coincided. Sheet after sheet now poured in from
Broadcroft. I verily thought all the worthy divines, from the
Reformation downwards, had been put in requisition to batter me to
pieces with choice and ghostly counsel.
This infliction I bore up against with wonderful fortitude, and
repaid with my weightiest metal. To supply the extraordinary drafts
thus made on my stores of devout phraseology, I had to call in my
worthy friend Tom ——. He had been a regularly-bred theologian,
but finding the casque more fitting for his hot head than the
presbyter’s cowl, he now lived in elegant starvation as a dashing
cornet in the —— Dragoons, and a better fellow never breathed. His
assistance was of eminent service: when we exhausted our own
invention, we immediately transcribed the sermon of some forgotten
divine of last century, and sent it thundering off. These we
denominated shells. At this time Tom’s fortune and mine were
hanging on the same pin; we were both up to the chin in debt; we
had stretched our respective personal credits, as far as they would go,
for each other. We were involved in such a beautiful multitude and
labyrinth of mutual obligations, that we could neither count them
nor see our way out of them. In the holy siege of Broadcroft citadel
we therefore joined heart and hand.
In this manner things went on smoothly. My aunt was becoming
daily weaker, seldom left her own bedroom, and permitted no person
to see her save the Rev. S. S. Sickerscreed. Indeed, every letter I
received from my aunt intimated more plainly than its predecessor
that I might make up my mind for a great and sudden change, and
prepare myself for afflictions. As in duty bound, my answers
breathed of sorrow and resignation—lamented the mutability of this
world—its nothingness—the utter vanity of all earthly joys. I really
loved the good old lady; but I was hampered most villanously. I knew
not a spot where I could put the sole of my foot, without some legal
mine blowing me up a shivered rag into the azure firmament,—a fate
a thousand times more picturesque than pleasant. I may therefore be
excused for confessing that I looked upon my aunt’s release from this
world as the dawn of my own deliverance. Yet, even then, I felt
shame when I looked into the chambers of my heart, and found that
every feeling of grief I had there for my aunt’s illness was beautifully
edged with a gleam of satisfaction. The cypresses and yews, and
other mournful trees that threw their pensive shadows around me,
were positively resting above a burning volcano of joy. No; it was not
in human nature for a desperate man like me to exclude from his
contemplation the bills, bonds, moneys, and manors that had
accumulated for years under her thrifty and prudent management.
One morning, while musing in this indescribable state of feeling, a
little ragged boy, besmeared with dust and sweat, whom I recognised
as turnspit and running footman of the establishment at Broadcroft,
thrust a crumpled greasy-like billet in my hand.
“Come awa, laird, come awa, gin ye would like to see your auld
auntie afore she gangs aff a’thegither.”
I started up, threw down the “Sporting Magazine,” and
instinctively snatched up my hat.
“When did it happen, wee Jamie?”
“This morning, nae far’er gane—but come awa; everything’s gaun
tap-salteerie at Braidcraft—sae unexpected by us a’! Has your horse
been fed yet? Dinna put aff, but come awa. We’re a’ dementit ower
the way, and ye’re muckle wanted, and sair missed.”
With this wee Jamie darted away; I roared after him to obtain
further particulars, but wee Jamie shot off like an arrow, only
twisting his head over his shoulder, notwithstanding his trot, he
screamed—
“Gerss maunna grow under my heels, if I care for my lugs. But it’s
a’ by noo, and there’s nae gude in granin’.”
With which sapient remark the kitchen boy got out of hearing, and
soon out of sight.
I now hastily broke the black wax of the billet. The note was
subscribed by Mr S. S. Sickerscreed, and was written in his most
formal small-text hand. He had been a schoolmaster in his youth,
and could write legibly, which no gentleman who regards his caste
should do. The three big S S S were dearer to me than a collar of
knighthood. It required my immediate presence at Broadcroft to talk
over certain serious and impressive matters. So had Mr Samuel
Salmasius Sickerscreed penned his billet, and in the fulness of my
heart I gave the poor man credit for an excess of delicacy more than I
ever noticed had belonged to him before. Poor dear man, he, too, has
lost a valuable friend. Judging of the exquisiteness of my feelings by
the agony of his own, he has kindly delayed the fatal announcement
of my aunt’s demise, till my heart has been prepared to meet the
shock with becoming fortitude. How considerate—how very
compassionate he has been! Worthy man—would I could repay his
kindness with a benifice! Thus did I soliloquise over the dispatch
from Broadcroft; but notwithstanding the tumult which it and its
bearer raised in my bosom, I did not omit communicating to Tom the
unexpected change which a few hours had produced in our destinies,
and charging him at the same time to moderate his transports till I
returned with a confirmation of our hopes.
Then backing my stoutest hunter, and taking a crow’s flight across
the country, I spared not her heaving flanks, nor drew bridle, till I
reached the long, straight, dusky avenue that led to the tall, narrow
slip of a house yclept Broadcroft Place. Here I slackened my pace,
and left my wearied and panting brute to crawl as lazily as she liked
along the avenue. I, too, lengthened my visage to the requisite degree
necessary for the melancholy purpose on which I came. The very
trees had a lugubrious and sepulchral aspect. I took them in fancy to
be so many Sawlies waiting the time for heading the funeral
procession of my lamented aunt. They seemed to mourn for her in
sincere sorrow, and, in fact, walking under their shadows disposed
my mind very much to melancholy. Now a green leaf, now a withered
one, dropped on my beaver as I passed, and in the deep silence that
reigned around me, I could not, despite my constitutional
recklessness, be wholly insensible to the appeals these mute
emblems of man’s mortality made to reflection.
But a pleasanter train of feelings arose when I looked at the stately
trunks of the venerable oaks, their immense girth, and (with a glow
of patriotic virtue, quite common now-a-days) pictured forth to
myself how admirably they were suited to bear Britannia’s thunders
triumphantly across the wave. Yes, every tree of them shall be
devoted to the service of my country. Perish the narrow thought, that
for its own gratification would allow them to vegetate in unprofitable
uselessness, when they can be so beneficially employed for the state.
Every old, druidical-looking oak which my eye scanned was, of
course, devoted to the axe. I already saw the timber yards piled with
Broadcroft oak, and the distant sea my imagination soon whitened
with a fleet of noble barks wholly built of them. Thus did I speculate
till I reached the end of the avenue, where, to my surprise, I found a
travelling post-chaise and four drawn up before the door of the
mansion. This vehicle, an apparition of rare occurrence in so
secluded a part of the country, and at the residence of so retired a
lady as my departed aunt, was literally crushed with trunks, and
boxes, and bags, and packages of one kind or another, strapped
above, behind, and before it.
Being never unfertile in surmises, I immediately guessed that the
equipage I saw must, of necessity, belong to the clerk to the signet,
my aunt’s city lawyer, who had trundled himself into the country
with the whole muniments of my estate, for the mere purpose of
welcoming me, and regulating my deceased relative’s affairs. His
prompt appearance, I attributed, with my usual goodness of heart, to
the kindly foresight of Mr Samuel. I really did not know how I could
sufficiently recompense him for the warm, disinterested, and
valuable services he had rendered in this season of affliction. But my
aunt must have remembered him in her testament. She was ever
grateful. She cannot possibly have overlooked him. As the d—l would
have it, I then asked myself, now, if your aunt has forgotten Mr
Samuel Salmasius Sickerscreed altogether, how will you act? At first,
I said he must have £100 at least; then as I looked on my own
necessities, the uncertainty of rents, the exorbitance of taxes, this
sum speedily subsided into half the amount. And by the time I fairly
reached my aunt’s door, I found my mind reconciling itself to the
handsome duty of presenting Mr Sickerscreed with a snuff-box, value
£2, 10s., a mourning ring worth 30s., a new coat, and ten guineas; in
all, some twenty pieces of gold or thereby.
On alighting, I gave my horse to the servant to walk and cool. John
was old as his late mistress—a very good, foolish, gray-headed
domestic, marvellously fond of the family he served with, and
marvellously fond of conversation. He looked profoundly melancholy
when he took my reins.
“It’ll be a sair dispensation to you, Maister William,” quoth John,
“this morning’s news. Ye wud be wonderfully struck and put about
when ye heard it.”
“It is, indeed,” said I, throwing as much of mournfulness as
possible into the tones of my voice. “Heavy news indeed, and most
unexpected. Great cause have I to grieve. My poor dear aunt to be
thus lost to me for ever!”
“Nae doubt, nae doubt, Maister William, ye maun hae a heavy
heartfu’. We were a’ jalousing as muckle,—that’s me, Souple Rab, and
wee Jamie; however, it’ll no do to be coosten down a’thegither,—a
rainy night may bring a blithe morrow. Every thing is uncertain in
this world but death! But come on, Kate;” and John and my reeking
jade disappeared in the direction towards the stable; John, no doubt,
bursting with impatience till he could communicate to his select
cabinet, Souple Rab and wee Jamie, the awsome and doncie looks of
the young laird.
I was yet lingering on the threshold in a most comfortable frame of
mind, when the door was thrown open. Imagine my horror when the
first figure I saw was my aunt herself, not in the drapery of the grave,
but bedizzened with ribbons from head to heel, and leaning her
withered hand on the arm of the Reverend Mr Sickerscreed. I gasped
for breath—my tongue swelled and clung to the roof of my mouth—
my eyes literally started from their sockets as if they would leave
their bony casements altogether. Had I not caught hold of the porch,
down I should have dropped.
“Am I in my senses, aunt? Do I see you really alive? Is this no
unreal mockery—no cruel hallucination? Resolve me, for Heaven’s
sake, else I go mad.”
“Dear me, nephew,” said the old lady, “what agitates you so? I feel
so glad that you have paid me this visit ere I set off on my marriage
jaunt with the elect of my heart, your worthy connection, Mr
Sickerscreed.”
“Marriage!” thundered I, “marriage!—I came to mourn over your
bier, not to laugh at your bridal. O, the infernal cruelty, Mr What’s-