Morphological characterization of the immune organs in...

H. SONG, K. PENG, S. LI, Y. WANG, L. WEI, L. TANG

89

Turk. J. Vet. Anim. Sci.

2012; 36(2): 89-100

© TÜBİTAK

doi:10.3906/vet-0910-128

Morphological characterization of the immune organs in

ostrich chicks*

Hui SONG, Kemei PENG**, Shenghe LI, Yan WANG, Lan WEI, Li TANG

Department of Animal Science and Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, P.R. CHINA

Received: 14.10.2009

Abstract: Th is study describes the anatomical, light microscopic, and ultrastructural characterization of the immune

organs in ostrich chicks. In this study 6 healthy 8-week-old ostrich chicks were processed for light and transmission

electron microscopy. Hematoxylin and eosin as well as improved Weigert staining were used for light microscopy. Th e

small thymus, which clustered caudally along both sides of the neck, contained Hassall’s corpuscles in the medulla. Th e

cloacal bursa, which formed the hanging and lateral walls of the cloaca, enveloped the cloaca but did not form a truly

independent bursa. Inside the bursa convex papillae were densely distributed on the surface of the folds, each with a

single bursal nodule in the lamina propria. Th ere were several round or elliptical structures, similar to ellipsoids, in the

parenchyma of the spleen. Homogeneous acidophilic material was found to exist between the ellipsoid and the lymph

tissue, and intermittent transverse striations were detected by transmission electron microscopy. Th ese data indicate that

the immune organs of ostrich chicks, especially the spleen and cloacal bursa, have numerous distinct, conformational,

and structural features. Th e acidophilic material between the ellipsoid analog and circum-lymph tissue of the spleen,

which was identifi ed as collagen fi ber, has not been reported in other animals.

Key words: Ostrich chicks, immune organs, anatomy, microstructure, ultrastructure

* Th is research was supported by the National Natural Science Foundation of China (Project Number: 30471249).

** E-mail: [email protected]

Research Article

Introduction

Th e immune or lymphatic organs, including the central and peripheral lymphoid organs, can produce and cultivate lymphocytes. Th e central lymphoid organs of birds, namely the thymus and cloacal bursa, exist to generate, diff erentiate, and mature T and B lymphocytes, respectively. Th e central lymphoid organs maintain normal immunological function and enhance resistance against disease. Th e spleen is an important peripheral lymphoid organ, and is the location of immune response production. Among

the immune organs of animals diff erences exist in terms of size, morphology, and structure. With the development of immunology the histo-conformation and physiological functions of the immune organs in mammals (1-4), birds (5-7), and fi sh (8) have been detailed.

Ostriches are an economically valuable cultivated animal, and researches on ostriches which focused on pharmacology (9), nutrition (10), epidemiology (11,12), breeding (13), disease control (14-16) and the morphological features of their digestive (17),

Morphological characterization of the immune organs in ostrich chicks

90

genitourinary (18,19), and gustatory organs (20) have been reported. However, little information is available regarding their immune organs. Ostrich chicks 90 days of age or less (post-hatching) are defi ned as immature ostriches. Th e condition of ostrich chicks’ growth and developmental is directly related to the quality of the mothball breeding bird and infl uences the value of the breeder ostrich.

Here, we report the anatomical and histological characteristics of the immune organs of ostrich chicks, based on transmission electron microscopy and histochemical analysis. We sought to elucidate the morphological, positional, and structural features of the immune organs in ostrich chicks and to provide a morphological reference for their physiological function.

Materials and methods

Samples and treatments

Randomly selected for use in this study were 6 healthy ostrich chicks (8 weeks old, male and female) weighing 3.2-4.3 kg. Th e animals were obtained from an ostrich farm and euthanized with an overdose of sedatives administered by a veterinarian. Th e thymus, spleen, and cloacal bursa were removed aft er being stripped of all surrounding tissue and fi xed for light and transmission electron microscopy.

Light microscope samples

Th e thymus, spleen, and cloacal bursa were cut into 1.5 × 1.0 × 0.3 cm pieces, fi xed in 4% paraformaldehyde-0.1% phosphate buff er, dehydrated in a graded ethanol series, clarifi ed in xylene, and embedded in paraffi n wax. Th e tissues were then sectioned by a microtome (LEICA-RM2245) at a thickness of 4-6 μm.

Transmission electron microscope samples

Th e thymus, spleen, and cloacal bursa were fi xed in 2.5% glutaraldehyde (prepared with 0.1 M phosphate buff er) for 3 h at room temperature. Aft er fi xation, the specimens were rinsed with 0.1 M phosphate buff er, post-fi xed in 1% osmium tetroxide and rinsed in 0.1 M phosphate buff er again, dehydrated in a graded ethanol and acetone series, and embedded in Araldite. Semi-thin (1 μm) and ultra-thin (70 nm) sections were then cut using an ultramicrotome. Each

semi-thin section was stained with toluidine blue,

while the ultra-thin sections were double-stained

with uranyl acetate and lead citrate for transmission

electron microscopy (Tecnai G212, FEI, Eindhoven,

the Netherlands) (21).

Histology and histochemistry

Th e paraffi n sections were prepared with hematoxylin

and eosin (HE), as well as improved Weigert’ staining

for light microscopic analysis (Olympus, Tokyo,

Japan).

Improved Weigert’ staining: Paraffi n-embedded

sections were treated fi rst with 95% ethanol and

then a resorcine-roseine solution for 1-2 h. Th e

sections were rinsed with 95% ethanol until no color

defl uviumed and diff erentiated in 1% hydrochloric

acid-ethanol. Finally, the sections were rinsed with

water for 5 min, post stained with Van Gieson’s

Solution for 30 s, diff erentiated in 95% ethanol,

dehydrated in absolute alcohol, clarifi ed in xylene,

and mounted with balsam neutral. Th rough this

procedure the collagen fi bers were stained red and

the elastic fi bers appeared dark blue (Figure 6A).

Results

Anatomy of the immune organs

Th e thymus ran along each side of the 5-6 cervical

vertebrae and ventrally across the sides of the neck,

reaching the costal margin of the costa prima. Th ere

was individual variation, and sublobes diff ered in

size and number across the 2 sides of the organ. On

the left side 2-4 sublobes weighing 2.3783 ± 0.0620 g

were identifi ed, and 1 to 5 sublobes weighing 2.4133

± 0.0460 g were identifi ed on the right. Th e volume

of each sublobe decreased from the cranial to caudal.

Th e pars cranialis of the thymus was connected to the

thyroid in 2 of the 6 ostrich chicks (Figure 1A).

Th e cloacal bursa connected directly with the

cloaca and was situated at the upper portion of the

cloaca. Th e cloacal bursa and cloaca combined to

form the scrotiform. Th e forepart of the cloacal

bursa formed hermetization, and the pars dorsalis

constituted the hanging wall of the urodaeum and

proctodaeum. Th e cloacal bursa was covered with

musculus rectococcygeus. Th ere were 15-19 plica

bursalis on the internal surface of the capsule wall


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in the cloacal bursa. Th e folds were valviform and

distributed intensively by the papillas (Figures 2A,

2B and 1C).

Th e spleen of each chick was dark red and

elliptical and was wedged against the right kidney, the

posterior of the vena caudalis, and the proventriculus.

Th e dorsal aspect was curved, while the segmented

venter was cavate, forming a port. Each spleen was

about 4.8 cm in length and 1.5 cm in diameter. Th ere

were accessory spleens in 2 of 6 experimental animals

(Figure 1B).

Microstructural characteristics of the immune

organs

A connective tissue tunica covered the surface of the

thymus. Th e tunica extended into the parenchyma

formed by the interlobular septum, dividing the

parenchyma into several leafl ets. Th e lobule of the

thymus included a cortex and a medulla comprised

of epithelial reticular cells that mainly served as

structural support for lymphocytes, macrophages,

etc. Th e cortex, situated along the periphery of

the parenchyma, was enriched with lymphocytes

TG

AS

S

A B

T

C

PB

Figure 1. A-C. Anatomical structure of thymus (T) and thyroid gland (TG) (A). Anatomical structure of spleen

(S) and auxiliary spleen (AS) (B) Anatomical structure of median section through the cloacal bursa (C),

showing the plica bursalis (PB).


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(Figure 3A). Th ere were a small number of epithelial reticular cells in the region, identifi able by their pale-stained cytoplasm and round or ovate nuclei. Epithelial reticular cells were relatively large, and the interlobular septum was obvious. Th e medulla, which was located in the middle of the thymic parenchyma, had more epithelial reticular cells than lymphocytes. Th e medullary region was lightly stained and confl uent. Several Hassall’s corpuscles were observed in the medulla (Figure 3B).

Th e capsule wall of the cloacal bursa was composed of a mucosa, a submucosa, a muscular layer, and an adventitia. Th e mucosa included a pseudostratifi ed columnar epithelial layer (Figure 4B). A single bursal nodule was observed in the lamina propria of every mamelon on the surface of the plicae. Th e bursal nodule included 3 zones: the external zone, zona intema, and zona intermedia. Th e zona externa, namely the cortex, was thin, deeply stained, and contained a large number of lymphocytes and a small number of epithelial reticular cells and macrophages. Th ere was a well-developed zona intema, which consisted of the medulla. Th e medulla was thick and deeply stained, and it contained a large number of lymphocytes, with only a few scattered reticulocytes and macrophages. Th e zona intermedia (Figure 4A), which was located between the cortex and the medulla, was thick and mainly composed of loosely arranged, lightly stained epithelial cells (Figure 4B). Th e thin submucosa was composed of loose

Cloacal

bursa

Ecarteur

Ecarteur

Ecarteur

Ecarteur

Oviduct

Cloacal

bursa

Anal

gland

Ureter

Ureter

Papillas

B

A

Papillas

Figure 2. A-B. Schematic diagram of cloacal chamber and

cloacal bursa (dorsal). Anatomical structure of the

cloacal chamber (A), showing rectum, coprodaeum,

urodaeum, proctodeum, anal gland, ureter, oviduct,

and cloacal bursa. Anatomical structure of the cloacal

bursa (B), showing cloacal bursa, plica bursalis, and

papillas.

Figure 3. A-B. Microstructure of the thymus. Transverse section of a thymus (A) (HE ×100), showing the cortex (Co),

medulla (M), and capsule (Ca). Medulla of the thymus (B) (HE ×400), with Hassall’s corpuscles (HC).


93

connective tissue and contained many blood vessels. Th e muscular layer was well developed, while the adventitia was simply a fi brous membrane.

Th e tegument of spleen in the ostrich chicks was thin, ranging from 25.0 to 41.5 μm. It extended into the splenic parenchyma, where an undeveloped trabecula lienis was formed. Th e parenchyma included both white and red pulp, with no obvious demarcation between them (Figure 5A). In the white pulp the periarterial lymphoid sheath was thin (12.5 μm). Th e splenic corpuscle, which lay across the side of the periarterial lymphatic sheath, was deeply stained, revealing that lymphocytes were intensively distributed. Th e germinal center was not obvious. Th ere were numerous round or elliptical structures in the region that resembled ellipsoids (Figure 5B). Th e volume of each ellipsoid was small, and the diameter ranged from 25.0 to 44.5 μm. Th e ellipsoids were observed to be distributed diff usely or gathered together at the junction between the red pulp and the periarterial lymphatic sheath. Each ellipsoid was composed of 2 to 4 layers of cells with large round or ovate, lightly stained nuclei (Figure 5C). Th e area between the ellipsoid and lymph tissue was strongly acidophilic and formed an obvious border. Th e border was stained red by the Weigert method, whereas the elastic fi bers in the blood vessels were dark blue (Figures 6A and 6B). Each of the sheathed capillaries held a tiny lumen, and the endothelial cells

were closely arrayed. Erythrocytes were observed within the cavity and ellipsoid. Th e red pulp was composed of alternating bands of splenic cord and sinus lienis. Th e segments of the splenic cord were interlaced, forming a meshwork fi lled with lymphocytes, macrophages, and plasma cells. Th e sinus lienis was developed and distributed under the tegument near the trabeculum and splenic cord. A large cavity containing several scattered erythrocytes lay under the tegument or near the trabeculum.

Ultrastructural characteristics of the immune

organs

Transmission electron microscopy revealed that

the lymphocytes in the thymus contained several

round or oval mitochondria, a few free ribosomes,

and rough endoplasmic reticulum (RER). Th e

lymphocyte nuclei were rich in euchromatin, and

nucleoli were clearly visible, as was the nuclear

bilayer, with several nucleopores. Heterochromatin

was distributed along the nuclear membrane (Figure

7). Th e lymphocytes in the cloacal bursa were less well

developed, and abundant euchromatin was observed

in their nuclei (Figure 8A). Th e epithelial reticular

cells with the processes in the bursal nodules were

stellate, with desmosomes between adjacent cells.

Th e mucosal epithelium was comprised of columnar

cells with microvilli on the surface and lysosomes

and mitochondria in the cytoplasm (Figure 8B).

Figure 4. A-B. Microstructure of the cloacal bursa. Bursal nodule (A) (HE ×100), showing the cortex (Co), medulla (M),

and mid-zone (MZ). Bursal nodule (B) (HE ×400), showing the mucosal epithelium (ME).


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Th e lymphocytes of the spleen were similar to those in the thymus, with prominent nucleoli, free ribosomes, round or ovate mitochondria, and RER in the cytoplasm. In addition, a nuclear bilayer was clearly observed (Figure 9A). Th e lymphocytes in the periarterial lymphatic sheath of the spleen were immature, with abundant heterochromatin, RER, free ribosomes, and mitochondria in the copious cytoplasm. Euchromatin was abundant in the nuclei of the endotheliocytes in the sheathed capillaries, and nucleoli were clearly observed. Th e plasma

membrane was enate with several invaginations, and several sites of contact were observed between adjacent cells. Mitochondria, RER, and free ribosomes were observed in the cytoplasm of the cells. Th e cells surrounding the sheathed capillaries were rich in euchromatin, while the cytoplasm of the cells contained scattered RER, free ribosomes, and mitochondria. Th e fi bers between the ellipsoid and circum-lymph tissue were 0.033 μm in diameter, and they were interlaced with one another, forming a transverse band every 0.025-0.066 μm (Figure

Figure 5. A-C. Microstructure of the spleen. Microstructure of the spleen (A) (HE ×100), showing the capsule (Ca), red

pulp (RP), white pulp (WP), and ellipsoids (E). Parenchyma of the spleen (B) (HE ×100), showing periarterial

lymphatic sheath (PALS), and splenic nodule (SN). Parenchyma of the spleen (C) (HE ×400), showing ellipsoids

(E), trabeculae (T), and the homogeneous structure (HS) around the ellipsoids.


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9C). Large and irregularly shaped macrophages, whose nuclei contained branches or processes, were also observed. Th e cytoplasm of each macrophage held several lysosomes, free ribosomes, and RER in addition to a small number of mitochondria, a few vacuoles, and the engulfed foreign particles (Figure 9B).

Discussion

Th e thymus

In this study, it was found that the thymus of the ostrich was distributed posteriorly along each side of the neck, its sublobes were not obvious, and the number of lobes per thymus was small. In contrast, chicken thymus contains 14 lobes, and duck thymus has 10 lobes arranged like beads on a string, which are distributed along the jugular vein from the throat to the front of the chest. Th us, there are diff erences in the number of thymic lobes and in the location of the thymus in ostrich chicks and other domestic fowls. Th e thymus produces and cultivates T lymphocytes and functions in cell-based immunity. Th e diff erentiation and development of T lymphocytes in the thymus is completed during the process of T cell migration from the cortex to the medulla. Lymphoblasts, the precursors of T lymphocytes, enter the thymus and undergo selection in the thymus during diff erentiation. Th ose thymocytes that recognize

Figure 6. Parenchyma of the spleen (A) (HE ×200), showing collagenous fi bers (CF) and elastic fi bers (EF). CF in the

splenic parenchyma (B) (HE ×400).

Figure 7. Ultrastructure of the lymphocytes in the thymus,

showing the nucleus (N), mitochondria (Mi) and RER

(×10,000).


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autoantigens or are incompatible with the major histocompatibility complex are inactivated; only a few lymphocytes actually mature into T lymphocytes (22). Histological analysis showed that the cortex is larger than the medulla at this stage, meaning that a greater number of T lymphocytes are available for selection by the thymus. Based on some data, Hassall’s corpuscles in the thymic medulla of many animals are thought to play an important role in generating the antigenic properties of the surrounding cells and in disposing of the dead lymphocytes and cellular debris generated during the process of T lymphocyte cultivation and selection. As a result, any thymus that lacks Hassall’s corpuscles cannot function normally (22,23). Bodey et al. (2000) indicated that the Hassall’s corpuscles, constituting multicellular components of the nonlymphocytic, cellular micro-environment of the thymic medulla, and participating in the physiological activities of the prenatal and adult thymus, are unique and functionally active (24). Th ymic capsules exist in avifauna. Th e thymic capsule does not participate in the diff erentiation of the cells in the thymus (25). Observing the submicrostructure of the Chinese soft shell turtle Pelodiscus sinensis,

Guo (1999) inferred that the thymic capsule may be involved in thymocyte selection and rejection (23). Concentric circles were observed in the Hassall’s corpuscles of the ostrich chicks, which were mainly cystic Hassall’s corpuscles. Th e occurrence of typical Hassall’s corpuscles presumed that thymocytes began to become mature to scavenging apoptotic cells and cell debris, and the existence of Hassall’s corpuscles may be concerned with the evolution of the species.

Th e cloacal bursa

Th e cloacal bursa is the site of B lymphocyte diff erentiation and development. If the cloacal bursa is cut during embryo development, the B lymphocyte defi ciency results in immature poults. B cell-defi cient immature poults are unable to mount a proper humoral immune response to disease, resulting in poults death. In this study diff erences in the shape and structure of the cloacal bursa in ostrich chicks versus that in other animals were observed. For example, the cloacal bursa of the ostrich chicks was cyst-like, with 15-19 convex papillae-covered strips of plicae on the intima; however, it did not form a truly independent vesicle. In comparison, the cloacal

Figure 8. Ultrastructure of the lymphocytes in the cloacal bursa (arrow) (A) (×2500). Mucosal epithelium of a bursal

nodule (B), showing pseudostratifi ed ciliated cells (PCC) and microvilli (M) (×1000).


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Figure 9. Ultrastructure of the lymphocytes in the spleen (A) (×5000), showing the N, Mi, RER, and free ribosomes (FR).

Macrophages in the spleen (B) (×2500), showing the N, lysosomes (Ly), vacuoles (V), and foreign particles (FP).

Acidophilic matter between the cells surrounding the sheathed capillary (C) (×10,000), showing transverse

striations (F).


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bursa of chickens is oval with 12-14 strips of plicae,

and cloacal bursa in ducks and geese are cylindrical,

with only 2 strips of plicae (26). Furthermore, the

cloacal bursa in pigeons is spherical, and in the

oriental white stork it is oblong and cecal. Finally,

the plicae in the cloacal bursa of chickens, ducks,

geese, pigeons, and oriental white storks are smooth

(i.e. no convex papillae). Light microscopy revealed

that the bursal nodules of the ostrich chicks were

singly distributed in the lamina propria of the convex

papillae, while the bursal nodules of chickens,

ducks, geese, and pigeons are located in the lamina

propria of the plicae. Based on our observations

of the structural features of the cloacal bursa in

ostrich chicks we infer that the convex papillae on

the surface of the plicae increase the surface area of

the folds, thereby multiplying the number of bursal

nodules in the plicae. Davenport and Allen (1995)

inferred that lymphoid follicles (bursal nodules)

could represent a morphological “pore complex” that

matured early in posthatching development and may

be related to the immunological function of the bursa

(27). Th e very abundant bursal nodules may be one

reason that ostrich chicks show stronger immune

responses than most other domestic fowls. Th e B

lymphocytes produced by the cloacal bursa in birds

mature as they move from the medulla to the cortex

(22). During this period the medullary regions of the

ostrich chicks showed signifi cant staining, indicating

the proportion of undeveloped lymphocytes in the

medulla was greater than that in the cortex, which

may be related to the selection of B lymphocytes.

Th e spleen

Th e spleen is the largest peripheral lymphoid organ in

most organisms, and it functions in hematogenesis,

blood fi ltration and storage, and immunity.

Immunocompetent cells proliferate and diff erentiate

in the spleen aft er antigenic stimulation, and there

are interspecifi c diff erences in the morphology of the

spleen. For example, the ostrich chick spleens were

elliptical, while those of chickens are globular, and

those of ducks are triangular (26). In ostriches, as

in other fowls, the tegument of the spleen was thin,

the trabeculum was undeveloped, and the smooth

muscle content of the tegument and trabeculum was

small. Interspecies diff erences exist in the size and

number of ellipsoids among domestic animals. Pig

spleens contain numerous large ellipsoids distributed

throughout the marginal zone. Th e ellipsoids of cats,

dogs, and horses are similar to those of pigs in terms

of location, but their volumes are smaller (28). Other

experts considered the existence of a relationship

between the contents of tegument, trabeculum, and

smooth muscle in spleen and the volumes of ellipsoids.

Th e smooth muscles play a role in the ellipsoids,

which could provide a favorable mechanism for

blood entering circulation (28). Accordingly, in

mammals (such as pigs) that have large ellipsoids the

number of smooth muscles in the spleen was larger.

While the volume of ellipsoid in the spleen of ostrich

chicks was small, the smooth muscle content of the

tegument and trabeculum was lower, which confi rms

the relationship between the smooth muscle content

and the ellipsoid volume in the spleen (28). Th ese

data also indicate that the blood storage capacity

of the spleen in ostrich chicks is weak, but the link

between ellipsoid number, volume, and age requires

further study. Light and transmission electron

microscopy revealed several diff erences in splenic

structure between ostrich chicks and other animals.

Th e cells outside the sheathed capillaries were lightly

stained and contained abundant euchromatin,

and little heterochromatin was observed in the

nuclei. In addition, the nucleus of each cell was

situated off to the side, while the cytoplasm was

rich with RER, suggesting a high level of cellular

activity. Th ese observations are diff erent from those

obtained by electron microscopy, which showed

that the perivascular cells surrounding the sheathed

capillaries were mainly composed of macrophages

(22). In addition, improved Weigert staining and

transmission electron microscopy revealed that the

strongly acidophilic material between the ellipsoid

and circum-lymph tissue contained collagen fi bers.

Th e collagen fi bers in the ellipsoid were compact

and situated correctly to serve as a fi ltration barrier,

indicating that an ostrich chick spleen is capable of

eff ectively fi ltering out bacteria, extraneous materials,

antigens, and senescent blood cells, and this may

explain why the immune response of ostrich chicks

is stronger than that of other fowls.


99

In conclusion, our results indicate several clear diff erences in immune organ shape and structure between ostrich chicks and other animals. Th e number of lobes in the ostrich chick thymus is small, and the thymus runs posteriorly along both sides of the neck. Th e cloacal bursa of the ostrich chicks is not truly an independent capsular space, and convex papillae are distributed along the surface of the plicae. Th e acidophilic material between the ellipsoid and circum-lymph tissue is composed of collagen fi bers, which has not been reported in other animals.

Acknowledgements

Th is work was supported by the National Natural Science Foundation of China (30471249). Th anks to the professors of the ultramicro-pathology lab, the TongJi Medical College of Huazhong University of Science and Technology, for the observation of ultra-thin sections. And thanks to Professor Jin-kai Lin, the Animal Science and Veterinary Medicine College of Huazhong Agricultural University, for invaluable help in drawing the schematic diagram of the cloacal chamber and cloacal bursa.

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Morphological characterization of the immune organs in...

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