An ultrastructural study on the Leydig and Sertoli cells in the immature lesser mouse deer (Tragulus javanicus)

Leydig and Sertoli cells of the immature lesser mouse deer testes, obtained in East Malaysia, were observed using light and transmission electron microscopy (TEM). The testes were fixed in 5% glutaraldehyde, post-fixed in 1% OsO4, dehydrated in ethanol, and embedded in Araldite M. Serial semi-thin sections were cut, stained with toluidine blue and observed using light microscopy. Serial ultra-thin sections were cut, stained with uranyl acetate and lead citrate, and examined using TEM. As a result, ultrastructurally, two types of underdeveloped filament bundles were infrequently recognized in Leydig cells, but not in other testicular cells. One type was the underdeveloped bundles of actin filaments (approximately 5 nm in diameter), which were found in the nucleus of Leydig cells. The other type was the underdeveloped bundles of intermediate filaments (approximately 10 nm in diameter), which were found in the cytoplasm of Leydig cells. A multivesicular nuclear body (MNB)--specifically present in the Sertoli cell nucleus of ruminant testes--was infrequently observed. The MNB is situated in the vicinity of nuclear membrane, still in an underdeveloped stage.     

Peculiar bundles of filaments in Leydig cells of the lesser mouse deer (Tragulus javanicus): an ultrastructural study

Leydig cells of lesser mouse deer (Tragulus javanicus) testes were observed using light and transmission electron microscopies. Sexually mature lesser mouse deer were obtained in East Malaysia. The testes were perfused with 5% glutaraldehyde, postfixed with 1% OsO4, dehydrated in ethanol and embedded in Araldite. The semithin sections were cut, stained with toluidine blue and observed under light microscopy. The ultrathin sections were cut, stained with uranyl acetate and lead citrate, and examined using a JEM-1200 transmission electron microscope. As a result, two types of filament bundles were frequently recognized in Leydig cells, but not in other testicular cells. These bundles were clearly seen at even a light microscopic level. One type was bundles of actin filaments (approximately 5 nm in diameter). These structures were found not only in the cytoplasm but also in the nucleus. The other type was bundles of intermediate filaments (approximately 10 nm in diameter). These structures were found only in the cytoplasm. The existence of filament bundles has never been reported in the testicular cells of another mammalian species. Thus, while bundles of actin and intermediate filaments are specifically present in the Leydig cells of the lesser mouse deer, their functions are still unclear.     

Morphological study on the stomach of the lesser mouse deer (Tragulus javanicus) with special reference to the internal surface.

The stomach of the lesser mouse deer (Tragulus javanicus) was observed macroscopically. It consisted of only three compartments, rumen, reticulum and abomasum without omasum. The rumen was S-shaped with large ventral and caudoventral blind sacs and the reticulum was larger than the abomasum. Internally, the rumen was covered with numerous ruminal papillae even on the pillars and the ruminoreticular fold. These papillae were leaf- or tongue-like shaped and varied in size and density. The reticulum had honey-combed crests and the secondary crests were found rarely. The lips of the reticular groove were prominent and more developed in the aboral part than in the oral one. A sac-like transition zone, which had more prominent mucosal folds than had the floor of the reticular groove, was observed between the caudal end of the reticular groove and the abomasum. Mucosal folds of the abomasum were spiral, low but rather thick. These findings were discussed in view of comparison with other ruminants and of possible functional implications.     

Morphology of the intermandibular gland of the Lesser mouse deer, Tragulus javanicus

The morphology of the intermandibular gland of the Lesser mouse deer (Tragulus javanicus), which plays an important function in marking area and territory and in the reproductive behaviour of the animal, was examined using immunohistochemistry, lectin histochemistry and scanning electron microscopy. The gland was composed of sebaceous and apocrine glandular material. Sebaceous glands occupied a greater area of the total gland and consisted of many large lobules with polyhedral cells having a pale cytoplasm. The sebaceous gland, being holocrine, possessed no special secretory ducts. The apocrine gland was lined by cuboid cells and the secretory products were often seen in the apical portions of the cells. Myoepithelial cells contained actin filaments lining the basal membranes of the apocrine gland and were surrounded by nerve fibres which immunostained with protein gene product 9.5. The secretion of the gland appears to be a mixture of larger amounts of lipid material from sebaceous glands, and glycoconjugates secreted by both sebaceous and apocrine glands. Lectin histochemistry detected these as galactose, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, D-mannose and D-glucose. The male gland was larger in size and contained more N-acetyl galactosamine and N-acetyl glucosamine in its secretion than the gland of the female. This implied the presence of sexual differences in secretions in the intermandibular gland of the Lesser mouse deer.     

Evaluation of semen collected by electroejaculation from captive lesser Malay chevrotain (Tragulus javanicus).

Thirteen sexually mature captive male lesser Malay chevrotains (Tragulus javanicus) were each anesthetized twice with tiletamine-zolazepam for electroejaculation. Viable spermatozoa were collected from all animals. The semen was creamy, milky, pale yellowish, or watery. The mean values for ejaculate volume, sperm concentration, and percentages of sperm motility, normality and viability were 23.7 +/- 2.5 microl, 366.9 +/- 127.8 x 10(6) spermatozoa/ml, 40.0% +/- 3.1%, 71.4% +/- 1.6%, and 59.6% +/- 2.1%, respectively. Semen pH was 7-8. No adverse effects of electroejaculation were noted. These are the first reported values for semen of lesser Malay chevrotain. Electroejaculation should be usable for routine semen collection in this species.     

Cryptosporidiosis and the follicle-associated epithelium over the ileal Peyer's patch in calves

Three calves were studied in stages of spontaneous cryptosporidial infection with particular reference to the relation of the cryptosporidia to the follicle-associated epithelium (FAE) over the ileal Peyer's patch (IPP). In early infection scanning electron microscopy and streptavidin immunoperoxidase staining showed marked predilection of cryptosporidia for the FAE. Cryptosporidial antigen was also found in subepithelial tissue, both in the domes over the IPP and in villi, apparently in macrophages, where the parasites seemed to be progressively degraded. The FAE showed long tightly spaced microvilli, replacing normal low folds and protrusions, particularly in late infection. Endocytosis of indian ink was restricted to the cell periphery in late infection, contrasting the normal, more even distribution of endocytosis in the FAE apical cytoplasm. Few parasites were seen in the intestinal mucosa at this stage. At convalescence the FAE was normal, but all stages of infection were characterised by elongation of microvilli in absorptive cells.     

Experimental superinfection of a Lesser Malayan mousedeer (Tragulus javanicus) persistently infected with bovine viral diarrhea virus.

A Lesser Malayan mousedeer (Tragulus javanicus), persistently infected with noncytopathogenic bovine viral diarrhea virus (BVDV) type 1f, was experimentally superinfected with a cytopathogenic isolate of BVDV type 1c, which antigenically partially matched the endogenous strain. Within the observational period of 125 days after superinfection, the animal did not demonstrate any clinical signs of the disease and/or significant changes in blood values. Neutralizing antibodies were detected at 35 and 42 days postinfection. The isolate causing the superinfection was found in feces, nasal swabs, and saliva starting from day 29 and at various times postchallenge. Macroscopic or histologic examination did not reveal mucosal disease-like lesions, despite the detection of the cytopathogenic isolate in the salivary gland, rumen, abomasum, kidney, and superficial prescapular lymph node. Results indicate that the cytopathogenic BVDV strain, which was used in the superinfection, persisted in the viremic animal without causing disease within the observation period.     

Functional morphology of the mastication muscles in the lesser and greater mouse deer

The mastication muscles were examined in the lesser (Tragulus javanicus) and greater mouse deer (Tragulus napu) to clarify the form of the mastication muscles in these primitive artiodactyls. The M. masseter was well-developed in both species, however the attachment area of its origin was not confirmed in the rostral facial part. The masseter bundles were not observed on the lateral side of the maxilla bone, and their origin was restricted to the zygomatic arch area. This suggests that the M. masseter may not act as a motor raising the mandible rostro-dorsally, but pull the insertion vertically unlike the highly derived grazer of Bovidae. The Crista temporalis was weak and the M. temporalis was thin in the mouse deer, and this indicates that the M. temporalis may not be important in the mastication in the primitive artiodactyls. These findings suggest that the browser such as mouse deer has been adapted for the feeding on soft leaves, and functional-morphologically different in mastication strategy from the grazer such as developed Bovidae species. The architecture of the mastication muscles was not different between the two species. However, in the muscle weight ratios per body weight, the M. temporalis and the M. digastricus were significantly smaller in greater mouse deer than in lesser mouse deer.     

Comparative studies on the secondary lymphoid tissue areas in the chicken bursa of Fabricius and calf ileal Peyer's patch

The chicken bursa of Fabricius and calf ileal Peyer's patch are thought to be the primary lymphoid organs of B cell development. In the bursa, the existence of secondary lymphoid tissue, called the diffusely infiltrated area, has been recognized. Recently, we have found the presence of a region of secondary lymphoid tissue in the ileal Peyer's patch at the period of the most rapid growth of this organ. In this study, we compared the development of these secondary lymphoid tissue regions in the bursa and ileal Peyer's patch histologically. Before hatching, lymphatic follicle formation occurred in the bursa, but not in the diffusely infiltrated area, where only a small number of lymphoid cells were found. However, during fetal calf development, lymphatic follicle formation occurred not only in the primary lymphoid organ but also in the secondary lymphoid tissue regions. Therefore, the prenatal development of the secondary lymphoid tissue regions of the bursa and ileal Peyer's patch were distinct. After hatching, formation of the germinal center, which contained many CD4⁺ cells, was observed in the diffusely infiltrated area of the bursa. After birth, many CD4⁺ cells and IgG mRNA expression were observed in the lymphatic follicle of the secondary lymphoid tissue regions in the ileal Peyer's patch, but rarely in the ileal Peyer's patch lymphatic follicles. The change of character of these secondary lymphoid tissue regions at the postnatal stage might be dependent on external antigens.     

Reduced apoptosis in sheep ileal Peyer's patch is associated with low levels of follicle centre carbonic anhydrase reactivity

Apoptosis in lymphoid follicles of the ileal Peyer's patch (IPP) in 21 sheep of two different age groups was visualized by the TdT-mediated dUTP nick end-labelling (TUNEL) method, and quantified using computer-assisted image analysis. The IPP follicle carbonic anhydrase (CA) reactivity was evaluated in the same samples. No significant differences with respect to apoptosis and CA reactivity were found between sheep aged 5 and 11 months. Individual variation in apoptotic activity correlated with the follicle centre CA reactivity. The group of animals found to have predominantly atypical ileal lymphoid follicles (more than 80% of total number of follicles) with features resembling jejunal Peyer's patch follicles, had lower number of apoptotic cells and reduced CA reactivity compared to the rest of the animals. The differences in CA reactivity in the follicle centres probably represent a variation in the presence of CA rich approximately 50 nm membrane-bounded particles known to be a feature of the sheep IPP. The present results suggest that the particles are involved in the modulation of the lymphocyte proliferation of the IPP follicles.     

Salmonella enterica serovar Choleraesuis infection of the porcine jejunal Peyer's patch rapidly induces IL-1beta and IL-8 expression

Salmonella enterica serovar Choleraesuis is an enteric pathogen of swine, producing septicemia, enterocolitis, pneumonia, and hepatitis. The initial molecular events at the site of Salmonella infection are hypothesized to be critical in the initiation of innate and adaptive immune responses; however, the acute immune response elicited by porcine intestinal tissues is not well understood. To address this need, we employed explants of jejunal Peyer's patch (JPP) mucosa from pigs to examine Salmonella-induced immune responses under controlled conditions as well as to overcome limitations of whole animal approaches. JPP explants mounted in Ussing chambers maintained normal histological structure for 2 h and stable short-circuit current and electrical conductance for 2.5 h. After ex vivo luminal exposure to Salmonella serovar Choleraesuis, JPP responded with an increase in mRNA expression of IL-1beta and IL-8, but not TNFalpha. Increased IL-1beta and IL-8 expression were dependent on efficient Salmonella adhesion and internalization, whereas mutant Salmonella did not induce inflammatory cytokine expression. Commensal enteric bacteria, present in some experiments, also did not induce inflammatory cytokine expression. These findings indicate that Salmonella uptake by Peyer's patch is important in the induction of an innate response involving expression of IL-1beta and IL-8, and that ex vivo intestinal immune tissue explants provide an intact tissue model that will facilitate investigation of mucosal immunity in swine.     

Development of ileal Peyer's patches and follicle associated epithelium in bovine foetuses

The development of the ileal Peyer's patches (ilPP) and follicle associated epithelium (FAE) was examined in 30 bovine foetuses ranging from 73 to 271 days of gestation by light and transmission electron microscopic methods. The first primordial ilPP was encountered in the foetus at 164 days of gestation The ilPP were found to have been formed from the aggregation of lymph follicles in the foetus at 227 days of gestation whereas in the foetus at 271 days of gestation the follicular development was observed to have been completed. While the cells in the FAE in the foetus at 164 days of gestation and those older were cuboidal, those of the foetus at 271 days of gestation were columnar. As from the foetus at 227 days of gestation, however, the FAE was found to be composed of uniform lymphoepithelial cells with an increase in the number of intraepithelial leukocytes. In the early stages, whereas the apical surfaces of the FAE cells appeared shorter with microfolds, with advancing age the apical surfaces of the FAE cells were observed to be heterogeneous. Our results suggest that bovine ilPP and FAE cells are histologically and functionally mature before birth.     

Distribution of T and B lymphocytes in jejunal and ileocaecal Peyer's patches of lambs

Jejunal (JPPs) and ileocaecal (IPP) Peyer's patches in lambs were studied by employing the indirect and the avidin-biotin-peroxidase methods, using antibodies to sheep IgM and thymocytes. Thymocyte sera absorbed with IPP germinal centre cells showed specificity for T cells in the thymus, lymph nodes and Peyer's patches. Whereas JPPs had large interfollicular T cell areas, IPP mainly contained B cells, although small triangular T cell areas were found at the apex of the follicles. Some T cells were also found in the dome and corona region of JPPs and IPP. While germinal centres of JPPs had about 40 per cent of IgM-positive cells, IPP germinal centres contained about 80 per cent of such cells. Negative or weak IgM-positive cells were seen in the peripheral zone of IPP germinal centres, contrasting with surface IgM-positive cells in the central zone, and indicating a centripetal migration of maturing lymphocytes.     

Immunolocalization of gastrin-releasing peptide (GRP) in the uteroplacenta of the mouse deer

The considerable phylogenetical differences between mouse deer and other ruminants have been established by means of DNA sequence analysis and anatomical observations. To clarify the physiological role of the uteroplacenta of the mouse deer, immunohistochemical observation was attempted by using GRP, which has been suggested as a novel regulatory peptide in the female reproductive tract, as an indicator to compare with other ruminants. Strong positive reactions for the GRP were detected in the uterine glands of the pregnant animals, but not in the non-pregnant ones. Although the placenta of the mouse deer is categorized as a diffuse placenta that is different from other ruminants' polycotyledonary placenta, in terms of GRP immunoreactivity, the mouse deer placenta can be classified as a synepithecholial placenta like the other ruminants'. The secretion of GRP from the uterine glands is of some importance to the fetus in the mouse deer.     

Flow cytometric characterization of Peyer's patch and cecal tonsil T lymphocytes in laying hens following challenge with Salmonella enterica serovar Enteritidis

Two trials were conducted to determine T cell changes in Peyer's patches (PP) and cecal tonsils (CT) of specific-pathogen-free Single-Comb White Leghorn hens challenged with Salmonella enterica serovar Enteritidis (SE). Each week, crop lavage samples were obtained from 4 or 3 hens in Trials 1 and 2, respectively. These birds were then sacrificed and their intestinal tracts excised. The crop sample and contents of one cecum from each hen were cultured for the presence of SE. Cells were purified from proximal and distal PP along with both CT and then aliquots of cells were incubated with antibodies to CD4, CD8, and the three T cell receptors (TCR). The T subsets were identified via flow cytometric analysis. Crop and cecal samples were 100% culture positive for SE at week 1 post challenge and a percentage of samples remained positive throughout the study. Some differences in TCR subsets between or within tissues were observed at various times relative to SE challenge but over-all the subsets remained similar during the study. The predominant TCR was TCR2 (vβ1) followed by TCR3 (vβ2). Low numbers of TCR1 (γδ) cells were observed. CD4/CD8 ratios increased in the PP and CT tissues by week 1 post challenge and the ratio elevation persisted throughout the experiment. These results indicate that T cell populations are comparable between PP and CT and enteric SE infection can affect the cellular dynamics of these lymphoid tissues.     

Anatomical,histological and biochemical studies of desert rodent Gerbillus tarabuli (Thomas, 1902) kidney

The present work aimed to study the anatomy, histology, cytology and some biochemical parameters (urea, osmolality, haematocrit, serum natrium, serum kalium) of the kidney of Gerbillus tarabuli. The investigated animals (n = 16) were collected from the desert, weighed and transferred alive to the laboratory in separate cages. A blood sample was taken by puncture at the retro-orbital sinus of each animal using a Pasteur-type capillary pipette capillary. They were anaesthetized with urethane injection (25%), after which they were carefully dissected; their organs were taken out and prepared for the histological and cytological studies. Pasteur pipette capillary type the kidney of the Gerbillus tarabuli is subdivided into three regions: Cortex (1193.625±60μm), Outer Medulla (1316.72±73μm), Inner Medulla (2525.08±85 μm). Pasteur pipette capillary type the kidney of the Gerbillus tarabuli is subdivided into three regions: Cortex (1193.625±60μm), Outer Medulla (1316.72±73μm), Inner Medulla (2525.08±85 μm). The concentration of the biochemical parameters of urea (0.41 ± 0.02 g/L), osmolality (300.75 ± 3.33 mOs/kg), haematocrit (34.18 ± 1.3%), serum natrium (141.37 ± 2.31 mmol/L) and serum kalium (7.69 ± 0.39 mmol/L) is in the interval of the norm compared with several studies on desert and semi-desert rodents and also on the Wistar rat. These findings revealed the adaptive morphology and physiological function in the kidney of G. tarabuli to the desert environment.     

Development of Peyer's patch and cecal tonsil in gut-associated lymphoid tissues in the chicken embryo

It is well known that chicken B cells develop in the bursa of Fabricius (BF), which is categorized as gut-associated lymphoid tissue (GALT). Chicken GALT also includes Peyer's patch (PP) and cecal tonsil (CT). The relationship between these tissues in GALT during B cell development is currently unknown. In this study, we conducted comparative examination of PP, CT and BF development during embryogenesis using immunohistochemical staining. On day 13 of embryogenesis (E13), accumulation of MHC class II(+) cells was observed in the intestine. Thereafter, Bu-1(+) cells and IgM(+) cells appeared, and their number continuously increased at the same sites where MHC class II(+) cells were present. Similar results were obtained in the CT. The locations of embryonic PP were limited to two sites; near the Meckel's diverticulum and the ileocecal junction. Anlage of bursal follicles first appeared at E13 and developed thereafter. Immigration of Bu-1(+) cells to bursal follicles began at E13, and the number of Bu-1(+) cell subsequently increased. When the follicle of BF was eliminated from the embryo by treatment with testosterone, development of PP and CT were observed. We concluded therefore that the development of PP and CT start during late embryogenesis at the same time as the follicle of BF, and that appearance of surface IgM(+) cells in PP and CT is independent form the development of the follicle of BF.     

Prevalence and molecular characterization of Anaplasmataceae agents in free-ranging Brazilian marsh deer (Blastocerus dichotomus)

Anaplasmataceae organisms comprise a group of obligate intracellular gram-negative, tick-borne bacteria that can infect both animals and humans. In the present work we investigate the presence of Ehrlichia, Anaplasma, and Neorickettsia species in blood samples from Brazilian marsh deer (Blastocerus dichotomus), using both molecular and serologic techniques. Blood was collected from 143 deer captured along floodplains of the Paraná River, near the Porto Primavera hydroelectric power plant. Before and after flooding, marsh deer were captured for a wide range research program under the financial support of S?o Paulo State Energy Company (CESP), between 1998 and 2001. Samples were divided into four groups according to time and location of capture and named MS01 (n=99), MS02 (n=18) (Mato Grosso do Sul, before and after flooding, respectively), PX (n=9; Peixe River, after flooding), and AGUA (n=17; Aguapeí River, after flooding). The seroprevalences for Ehrlichia chaffeensis and Anaplasma phagocytophilum were 76.76% and 20.2% in MS01, 88.88% and 5.55% in MS02, 88.88% and 22.22% in PX, and 94.12% and 5.88% in AGUA, respectively. Sixty-one animals (42.65% of the total population) were PCR-positive for E. chaffeensis PCR (100.0% identity based on 16S rRNA, dsb, and groESL genes). Seventy deer (48.95% of the total population) were PCR-positive for Anaplasma spp. (99.0% of identity with A. platys, and in the same clade as A. phagocytophilum, A. bovis, and A. platys based on 16S rRNA phylogenetic analysis). Our results demonstrate that Brazilian marsh deer are exposed to E. chaffeensis and Anaplasma spp. and may act as reservoirs for these rickettsial agents, playing a role in disease transmission to humans and other animals.