Dental incremental lines in Sika deer (Cervus nippon); polarized light and fluorescence microscopy of ground sections

Periodic growth incremental lines are found universally in dental hard tissues. This periodicity theoretically allows for estimation of age, even in days, which would be useful in studies of wild animals. In the present study, enamel and dentin increments of the sika deer (Cervus nippon) were observed in ground sections with a polarized light microscope, and their periodicity was examined by the use of a chronological labeling method with fluorochromes. Enamel increments occurred at a mean interval of 10.6 (SD=1.5) microm, and mean spacing of dentin increments was 17.3 (SD=1.8) microm. Fluorochromic marking revealed that incremental lines form each day in enamel and almost every second day in dentin. The fluorescence-labeled lines suggest that enamel formation of the first molar is complete by the age of 5 months. Due to its longer interval of incremental lines and longer term of formation, we conclude that dentin is more suitable than enamel for day-age estimation in sika deer. Experimental confirmation of incremental growth periodicity in various species can improve the reliability of use of tooth increments for age estimation and life history reconstruction.     

Demonstration of bovine herpesvirus type 1 and Mannheimia haemolytica antigens in specimens stored for up to 22 months in buffered formalin

Bovine herpesvirus type 1 (BHV-1) and Mannheimia haemolytica antigens were demonstrated in lung tissues that were stored in 10% neutral phosphate buffered formalin for 1 to 22 months using the immunoperoxidase method. There were no differences observed in terms of labelling intensity and distribution of M. haemolytica antigens between specimens stored for 1 and 22 months. The labeling intensity in sections from 2-cm thick specimens was comparable to those from 0.2-cm thick specimens. There was no difference observed between pronase-treated and -untreated sections. However, for BHV-1, the labeling intensity in untreated sections was reduced in tissues that had been stored from 12 to 22 months. Sections from thin specimens stored in neutral buffered formalin for 22 months exhibited a stronger staining intensity than those from thick specimens.     

Michel's medium as a preservative for immunofluorescent staining of cutaneous biopsy specimens in dogs and cats

Paired 3-mm skin biopsy specimens from 24 dogs and 11 cats with suspected autoimmune dermatopathies were stained with fluorescein-conjugated antisera. In each case, 1 specimen was immediately embedded in optimum cutting temperature compound, quick frozen to -30 C, processed, stained, and examined. The other sample was placed in Michel's transport medium, held for 7 days, and then processed, stained, and examined. The location, quality, and intensity of immunofluorescent staining (when positive) were then compared in the 2 specimens. There were no differences in the staining patterns between the specimens processed immediately and those held in Michel's medium, thus demonstrating the validity of preserving canine and feline skin biopsy specimens in Michel's medium for immunofluorescent examination.     

Use of destained cytology slides for the application of routine special stains

Background: Special stains to demonstrate microorganisms or intra‐ and extracellular substances have not been evaluated in detail regarding their applicability and usefulness in destained cytologic specimens. Objectives: The aim of this study is to compare the results of routine special stains on destained slides previously stained with Hemacolor and on fresh (unstained) specimens. Methods: Archival Hemacolor‐stained fine needle aspirate specimens of inflammation with infectious agents (bacterial, mycobacterial, and fungal infections), neoplasia (melanoma, myxosarcoma, and mammary adenocarcinoma), and hemorrhage (pericardial effusion) from 14 dogs and 7 cats were selected. Cells in a minimum of 4 fields were photographed and 5 slides from each case were then destained by different methods (alcohol acid or microwave). Seven special stains were applied selectively to the destained slides, depending on the cytologic findings: periodic acid Schiff, Grocott–Gomori methenamine silver, Gram's, Ziehl–Neelsen, Alcian blue, Fontana–Masson, and Prussian blue. The same fields were rephotographed and 2 observers evaluated the slides qualitatively, with comparison to fresh cytologic specimens from similar lesions. Results: Special stains applied to destained slides demonstrated the expected cellular and extracellular material or organisms independent of the destaining method. Staining intensity, nonspecific staining (background), cell morphology, and nuclear counterstaining results were similar to those of special stains applied to fresh unstained slides. Conclusions: Destaining does not appear to affect the results of routine special staining for cytologic specimens. Destaining before special stains may be a valuable diagnostic strategy when few slides are present or only stained slides are available.     

Glomerular lesions in dogs infected with Leishmania organisms

OBJECTIVE: To histologically identify glomerular lesions in dogs infected with Leishmania organisms. ANIMALS: 41 dogs (17 sexually intact males and 14 sexually intact and 10 ovariohysterectomized females) that had positive results when tested for leishmaniosis as determined by use of serologic evaluation (indirect fluorescent antibody test, titers of 1:80 to 1:640) and direct microscopic identification of the protozoal organisms. PROCEDURE: Urine samples were collected by use of cystocentesis and examined by qualitative SDS-agarose gel electrophoresis (AGE). All dogs had non-selective (glomerular) or mixed (glomerular and tubular) proteinemia. Specimens were obtained from each dog during ultrasound-assisted renal biopsy and used for histologic examination. Each specimen was stained with H&E, periodic acid-Schiff, Goldner's trichrome, methenamine silver, and Congo Red stains. Specimens were adequate for evaluation when they contained at least 5 glomeruli/section, except for specimens stained with Congo Red in which 1 glomerulus/section was adequate. RESULTS: Examination of renal biopsy specimens revealed various glomerular lesions in all dogs and interstitial or tubular (or both) lesions in 23 of 41 (55%) dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Glomerular lesions that develop in dogs during infection with Leishmania organisms can be classified histologically as mesangial glomerulonephritis, membranous glomerulonephritis, membranoproliferative glomerulonephritis, and focal segmental glomerulonephritis. Tubulointerstitial histopathologic conditions were not observed as the primary lesion, despite being evident in 23 of 41 (55%) dogs. Use of SDS-AGE for qualitative evaluation of proteinuria and successive collection of specimens during renal biopsies following diagnosis of nonselective glomerular proteinuria provides the possibility for early identification of renal lesions.     

Pathological studies of cheek teeth apical infections in the horse: 3. Quantitative measurements of dentine in apically infected cheek teeth

Histological measurements of dimensions of primary, regular secondary and irregular secondary dentine, pulp diameter and assessment of the levels of predentine, resting lines and enlarged areas of intertubular dentine were performed in apically infected mandibular and maxillary cheek teeth (CT). These examinations showed significantly reduced regular and irregular secondary dentine thickness in diseased as compared to control CT, with 21/26 infected maxillary CT and 15/18 infected mandibular CT having reduced regular secondary dentine (varying between 27.4% and 89.1% reduced secondary dentine levels compared to age and site matched control CT values). As a result of decreased dentinal deposition, significantly wider pulp horns were present in diseased compared to control CT.No significant differences were found between diseased and control primary dentine thickness in maxillary CT, and minor differences in mandibular CT were not believed to be clinically significant. The significantly reduced presence of predentine and of intertubular dentine and the increased presence of resting lines in diseased compared to control CT confirms that long-term disruption of normal dentine deposition had occurred in many infected CT.     

A study of the thickness of cheek teeth subocclusal secondary dentine in horses of different ages

Reasons for performing study: There is limited knowledge on the thickness of subocclusal secondary dentine in equine cheek teeth (CT). Hypotheses: Subocclusal secondary dentine is of consistent thickness above different pulp horns in individual horses and its thickness increases with age. Methods: 408 permanent CT were extracted post mortem from 17 horses aged 4–30 years, with no history of dental disease. The CT were sectioned longitudinally in the medio‐lateral (bucco‐palatal/lingual) plane through each pulp horn, and the thickness of the secondary dentine overlying each pulp horn was measured directly. Results: The subocclusal thickness of secondary dentine above the pulp horns of CT varied from a mean thickness (above all pulp horns) of 12.8 mm (range 5–33 mm) in a 4‐year‐old to 7.5 mm (range 2–24 mm) in a 16‐year‐old horse. There was wide variation in the depth of subocclusal secondary dentine above different pulp horns, even within the same CT. In contrast to expectations, occlusal secondary dentine thickness did not increase with age. There were no significant differences in occlusal secondary dentine thickness between rostral and caudal, or medial and lateral aspects of the CT, or between contralateral CT. Mandibular CT had significantly thicker subocclusal secondary dentine than maxillary CT. Pink coloured secondary dentine was sometimes found 1–3 mm occlusal to the pulp horn in sectioned CT and this was likely caused by artefactual blood staining from the underlying pulp during sectioning. Conclusions: The thickness of subocclusal secondary dentine varies greatly between individual pulp horns, teeth and individual horses and can be as low as 2 mm over individual pulp horns. Potential relevance: Due to the great variation in the thickness of subocclusal secondary dentine between horses, and even between pulp horns in individual CT, there is a risk of exposure or thermal damage to pulp and thus of apical infection, even with modest therapeutic reductions of CT occlusal overgrowths. In the light of these findings, great care should be taken when reducing equine CT overgrowths and larger dental overgrowths should be reduced in stages.     

SEM and neurohistological observations of nerve endings in the middle region of the tongue of the collared peccary (Tayassu tajacu): a silver impregnation method

The presence of lingual papillae and the nerve endings in the middle region of the tongue mucosa of collared peccary (Tayassu tajacu) were studied using scanning electron microscopy and light microscopy, based upon the silver impregnation method. The middle region of tongue mucosa revealed numerous filiform and fungiform papillae. The thick epithelial layer showed epithelial cells and a dense connective tissue layer containing nerve fibre bundles and capillaries. The sensory nerve endings, intensely stained by silver impregnation, were usually non-encapsulated and extended into the connective tissue of the filiform and fungiform papillae very close to the epithelial cells. In some regions, the sensory nerves fibres formed a dense and complex network of fine fibrils. The presence of these nerve fibrils may characterize the mechanisms of transmission of sensitive impulses to the tongue mucosa.     

Apoptosis in the cerebellum of dogs with distemper

Canine distemper virus (CDV) may induce multifocal demyelination in the central nervous system of infected dogs. The pathogenesis of this process is not clear. The present work identifies the presence of apoptotic cells in white and grey matter of dogs'cerebellum, naturally infected with CDV. Fifteen dogs with clinical signs of canine distemper that tested positive for CDV nucleoprotein were used. Brain specimens were processed and embedded in paraffin. Sections 5 microm thick were stained with hematoxylin-eosin and Shorr. Other sections were submitted to TUNEL reaction and to immunohistochemistry for CDV nucleoprotein detection. Acute and chronic demyelinated plaques were observed in the white matter, while apoptosis occurred particularly in the granular layer of grey matter. Apoptosis seems to play an important role in the pathogenesis of canine distemper demyelination.     

Donkey dental anatomy. Part 2: Histological and scanning electron microscopic examinations

Ten normal cheek teeth (CT) were extracted at post mortem from donkeys that died or were euthanased for humane reasons. Decalcified histology was performed on three sections (sub-occlusal, mid-tooth and pre-apical) of each tooth, and undecalcified histology undertaken on sub-occlusal sections of the same teeth. The normal histological anatomy of primary, regular and irregular secondary dentine was found to be similar to that of the horse, with no tertiary dentine present. Undecalcified histology demonstrated the normal enamel histology, including the presence of enamel spindles. Scanning electron microscopy was performed on mid-tooth sections of five maxillary CT, five mandibular CT and two incisors. The ultrastructural anatomy of primary and secondary dentine, and equine enamel types-1, -2 and -3 (as described in horses) were identified in donkey teeth. Histological and ultrastructural donkey dental anatomy was found to be very similar to equine dental anatomy with only a few quantitative differences observed.     

The mouse inoculation test in rabies diagnosis: early diagnosis in mice during the incubation period.

Brain tissue from 64 rabies suspect specimens were inoculated intracerebrally into twenty 9-12 gm adult Swiss white mice. Two mice from each specimen were killed on specific days postinoculation and examined for the presence of rabies virus by the fluorescent antibody staining technique. In this way a positive diagnosis was made in the majority of cases between postinoculation days 4 and 12 when the incubation period of these same specimens ranged between eight and 20 days.     

饮水硼对非洲雏鸵鸟肝脏组织的影响

本研究旨在探究饮水硼对非洲雏鸵鸟肝脏组织的影响。本试验选择36羽10日龄非洲雏鸵鸟,随机分成6组,每组6羽,饲喂相同的基础日粮及不同添加量的饮水硼,各组饮水中硼含量分别为0、40、80、160、320、640 mg/L。试验期80 d。试验结束后,称体重,颈动脉放血致死。处死后立即取出肝脏称重,计算肝脏指数。取肝脏标本于4%多聚甲醛常温固定,制作石蜡切片,HE染色,显微观察并照相。结果证实,低剂量的饮水硼（40、80 mg/L）有利于肝脏发育,改善肝脏组织结构,使肝血窦里的枯否氏细胞增多,肝血窦增大,最佳添加量为80 mg/L。高剂量的饮水硼（160、320和640 mg/L）则会对非洲雏鸵鸟肝脏发育产生不良影响,使肝脏组织出现明显的病理学变化。     

检测细胞凋亡的常用方法

细胞凋亡的主要形态学特征是凋亡小体的形成，过去常用普通光学显微镜和透射电子显微镜进行观察。实际上，细胞凋亡的发生远远早于典型的形态变化。怎样将病理形态和代谢结合在一起来观察细胞的凋亡，是多年来人们研究的重点。本文将近年来常用于细胞凋亡研究的十种新方法作以简介。这些方法包括碘化丙啶荧光染色法、吖啶橙荧光染色法。吖啶橙活细胞悬液染色法、Hoechst33258荧光染色法，简易末端标记法、5’末端^32P标记法、荧光原位末端标记技术、酶原位末端标记技术、PI染色的流式细胞分析术和Hoechst-P1染色的流式细胞分析术等。     

SKULL RADIOGRAPHS: SILVER IMPREGNATION AS AN INTERPRETATION AID

Individual skull bones of newly killed young cats were stained with an aqueous solution of 0.5% silver nitrate. These bones became blackened grossly and more radiodense than adjacent unstained skull bones. It was possible to correlate the gross and radiographic appearance of selected bones stained with silver nitrate. This was a useful teaching aid.     

Ultrastructural observations on different developmental stages of Goussia sinensis (Chen, 1955), a parasite of the silver carp (Hypophthalmichthys molitrix Valenciennes, 1844)

The development of Goussia sinensis, a coccidium parasitizing the intestine of the silver carp (Hypophthalmichthys molitrix) was studied by electron microscopy. All stages developed in the epithelial cells, less frequently in the goblet cells, and were located within a parasitophorous vacuole. In some cases one cell was invaded by several merozoites. Eight to sixteen merozoites were formed within the meront by ectomerogony. The ultrastructural processes characteristic of gamogony were the same as those found for Goussia spp. parasitizing other species of fish. A hitherto unknown mechanism of oocyst wall formation was observed. The oocyst membrane developing within the zygote surrounded only part of the zygote material. Thus, a small part of the zygote material left the oocyst proper. It is suggested that this zygote residue and the necrotic host cell constitute the so-called "yellow bodies" which include the excreted oocyst. The oocyst wall was 40 to 60 nm thick. Oocyst sporulation took place within the fish. The sporocysts consisted of two hemispheres connected by sutures and had a 100 to 120 nm thick double wall. They were surrounded by sporocyst veils fixed to the oocyst wall by membranes.     

Dental wear in horses in relation to the microhardness of enamel and dentine.

The microhardness of enamel, primary dentine and secondary dentine was determined in the incisor teeth of 39 horses of three different breeds, trotter horses, Belgian draft horses and Arab horses. Using a microhardness tester fitted with a Knoop diamond indenter, the overall Knoop Hardness Number was determined for each tissue, and the influence of breed and age on the hardness were evaluated. Enamel and secondary dentine were significantly harder in Arab horses than in trotters and Belgian draft horses, but there were no significant differences between draft horses and trotter horses in the hardness of their enamel and dentine.     

Identification and morphological characteristics of dental neonatal line in sika deer (Cervus nippon)

The dental neonatal line of the sika deer (Cervus nippon) was identified experimentally using chronological labeling methods. In the enamel, prominent dark lines were observed under transmitted light, and the number of increments between the dark line and labeling line was almost consistent with the day-age at the time of labeling injection. Therefore, we identified the dark line as the enamel neonatal line. In the dentin, the bright line was observed under polarized light. Since the bright line corresponded to the enamel neonatal line, we recognized the bright line as the dentin neonatal line. Neonatal lines intersected with the enamel-dentin junction at approximately one-third cervical in the first molar. Using these features, it would make possible to distinguish the neonatal line in wild sika deer.     

Histopathological study of feline eosinophilic dermatoses

A retrospective study was conducted on skin specimens from 24 cats with eosinophilic granuloma complex. The specimens were stained with haematoxylin and eosin and Gallego's trichrome stain. In all specimens, flame figures and/or large foci of so-called "collagen degeneration" were detected and histopathological features were not predictive of the clinical picture. Use of the term eosinophilic dermatosis was advocated in diagnostic dermatopathology. On trichrome-stained sections, normally stained collagen fibres were identified in the middle of both flame figures and large foci of "collagen degeneration" and the debris surrounding collagen bundles showed the same tinctorial properties as eosinophil granules. Eosinophil degranulation around collagen bundles seemed to represent the major pathogenetic event in these lesions, analogous with human flame figures. The term flame figures might therefore be more accurately used to designate those foci of eosinophilic to partly basophilic debris commonly referred to as "collagen degeneration".     

Natural occurrence of amyloid-Abeta deposits in the brain of young common marmosets (Callithrix jacchus): a morphological and immunohistochemical evaluation

The spontaneous incidence and distribution of beta-amyloid (Abeta) peptide and argyrophilic deposits were extensively investigated in the common marmoset (Callithrix jacchus). In 12 young marmosets, up to 67 coronal step sections were evaluated in the brain. One brain hemisphere was stained by a silver method and also immunohistochemically with a primary antibody directed against Abeta. Argyrophilic deposits were observed in 4 (33%) of 12 animals with silver stain, and Abeta was seen in 4 (33%) of 12 animals with anti-4G8 antibody. The overall incidence was 6 (50%) of 12 affected animals. These results show that the young marmoset brain displays Abeta deposits at a low incidence and that immunohistochemistry is more suitable than silver staining to reveal rare amyloid plaques. These findings also provide evidence for the early onset of amyloid deposits in marmoset brain.     

Establishment of two vaccine-associated feline sarcoma cell lines and determination of in vitro chemosensitivity to doxorubicin and mitoxantrone

OBJECTIVE: To establish 2 vaccine-associated feline sarcoma (VAFS) cell lines and to determine their in vitro sensitivity to the chemotherapeutic agents doxorubicin and mitoxantrone. SAMPLE POPULATION: Tumor specimens collected from 2 cats undergoing surgery for removal of vaccine-associated sarcomas. PROCEDURES: Tumor specimens were minced and treated with trypsin under aseptic conditions to obtain single-cell suspensions, which were then cultured in vitro in medium supplemented with 5% heat-inactivated fetal bovine serum. Growth rates and sensitivity after 24 hours of exposure to various concentrations (0.1 to 100 microg/ml) of doxorubicin and mitoxantrone were assessed for each cell line. Survival of cells was estimated 3 days after exposure to the 2 agents, and the concentration of each drug that resulted in a 50% reduction in the number of viable cells (IC50) was calculated. RESULTS: Two tumor-derived cell lines (FSA and FSB) were successfully established and determined to be sensitive to doxorubicin and mitoxantrone. Under the conditions tested, the IC50 of doxorubicin were 0.6 and 1.5 microg/ml for cell lines FSB and FSA, respectively. The IC50 of mitoxantrone was 0.4 microg/ml for both cell lines. CONCLUSIONS AND CLINICAL RELEVANCE: The establishment of VAFS cell lines provides a tool for the in vitro screening of antitumor drugs. Doxorubicin and mitoxantrone were effective in decreasing the number of viable cells in the 2 cell lines tested. Both of these anthracycline antibiotics have been used to treat various neoplasias in cats, and their efficacy for adjuvant treatment of vaccine-associated sarcomas should be further evaluated.