PAS-positive inclusions in the alveolar type II cells of the caprine lung.

Histochemical analysis of inclusions in the alveolar type II cells was done with the alcian blue-PAS technique. PAS-positive inclusions, which were absent in the sheep and buffalo, were detected in the alveolar type II cells of the goat lung. Mixed diamine-sodium chloride did not stain these inclusions which also showed orthochromatic blue reaction with toluidine blue. The high percentage of alveolar type II cells showing these inclusions suggested that this difference could be explained by the environmental habitat of the goat.     

Hepatic lesions in cetaceans stranded in the Canary Islands

This article describes the gross, histopathologic, and ultrastructural findings of the livers of cetaceans stranded on the coast of the Canary Islands between 1992 and 2000. A total of 135 cetaceans were included in the study, among which 25 were common dolphins (Delphinus delphis), 23 Atlantic spotted dolphins (Stenella frontalis), 19 striped dolphins (Stenella coeruleoalba), and 15 other species of dolphins and whales. The most common lesion observed in these animals was a nonspecific chronic reactive hepatitis (47/135), followed by hyaline intracytoplasmic inclusions in hepatocytes (33/135). Parasitic cholangitis was detected in 8/135 animals, whereas hepatic lipidosis was presented in 7/135 animals. The ultrastructure of hyaline hepatocytic cytoplasmic inclusions is described, and possible causes of these inclusions are discussed.     

Fine structure of secondary granule inclusions in fowl heterophils after ruthenium tetroxide fixation

Bone marrow from domestic fowls was initially fixed for electron microscopy in glutaraldehyde and post-fixed with either ruthenium tetroxide or osmium tetroxide. Inclusions with distinct outlines were revealed in the secondary granules of heterophil leucocytes after ruthenium tetroxide but not with osmium tetroxide fixation. In longitudinal orientation, these inclusions were rod-shaped and composed of microfilaments measuring 3.7 nm in diameter. In transverse section, the outline of some of these inclusions was hexagonal and therefore the inclusions may be crystalline in nature.     

Complex polysaccharide inclusions in the skeletal muscle of stranded cetaceans

Skeletal muscle samples were examined post-mortem in 148 cetaceans over a 12-year period. Histological analysis included haematoxylin and eosin (HE) and periodic acid-Schiff (PAS) staining with and without diastase digestion. In addition, histological muscle sections were immunostained for ubiquitin and fast and slow heavy-chain myosin isoforms. PAS-positive, diastase-resistant inclusions were detected in 26 animals from 11 different species. Older cetaceans were preferentially affected. These intrafibre inclusions varied from large aggregates to multiple coarse granules and were typically associated with type II fibres. All diastase-resistant inclusions were positive for ubiquitin. These features resembled those inclusions described as complex polysaccharide in horses. Based on these histological findings and the ubiquitin staining pattern, a morphological diagnosis of complex polysaccharide storage myopathy is proposed.     

Immunohistochemical and ultrastructural analyses of cytoplasmic blood plasma inclusions of rat hepatocytes

In the present study, we investigated the histological, immunohistochemical and ultrastructural characteristics of cytoplasmic blood plasma inclusions that spontaneously occurred in a rat liver. Histologically, a number of cytoplasmic inclusions were observed in the liver of an 8-week-old female SD rat. These inclusions were strongly positive for PAS staining and resistant to diastase digestion. Immunohistochemical analyses revealed that these inclusions were positive for albumin and IgG; however, most of them were negative for LAMP-1 and LAMP-2. Ultrastructurally, the inclusions were surrounded by limiting membranes and composed of moderately electron dense, homogenous materials. These characteristics described here represent valuable information for pathological examination in toxicity studies.     

Polysaccharide storage myopathy in Morgan, Arabian, and Standardbred related horses and Welsh-cross ponies

Polysaccharide storage myopathy is an equine neuromuscular disorder characterized by accumulation of glycogen-related polysaccharide inclusions within skeletal muscle fibers. The pathologic criteria for diagnosis of this disorder are somewhat controversial; however, periodic acid-Schiff-positive, amylase-resistant inclusions are considered pathognomonic. Although these inclusions are most often found in affected horses related to the Quarter Horse, draft horse, and Warmblood breeds, this report describes these characteristic inclusions in muscle of five horses from nonrelated breeds (two Morgans, one Arabian, one Arabian x Thoroughbred, and one Standardbred) and two Welsh cross ponies. Affected horses had histories of recurrent exertional rhabdomyolysis, and one developed progressive weakness leading to increased recumbency. The affected ponies were part of an unrelated research project and had no apparent clinical signs.     

Recovery of viral agents from the central nervous system of cats

Isolation of viruses from the central nervous system (CNS) of cats was attempted using an explant culture technique and subsequent co-cultivation with Crandell feline kidney (CRFK) or Vero cells. Feline syncytia-forming virus was isolated from the CNS of 11 of 16 cats where the initial co-cultivation was with CRFK cells. Feline panleucopaenia virus was isolated from the CNS of 2 adult cats. Co-cultured cells from the CNS of 3 cats contained eosinophilic cytoplasmic and intranuclear inclusions. The cytoplasmic inclusions consisted of tubular structures, 16-18 nm in diameter and up to 500 nm in length, which were similar in morphology to paramyxovirus nucleocapsids. The 3 co-cultured cells with cytoplasmic and intranuclear inclusions showed haemadsorption of guinea pig erythrocytes. The possible identity of these structures, and their association with a previously described primary focal demyelinating lesion in the CNS of cats, is discussed.     

Atrophy of the primary lymphoid organs of meat pigeons in Italy associated with circoviruslike particles in the bursa of Fabricius

During a survey effected in a meat pigeon slaughterhouse of central Italy, atrophy of primary lymphoid organs (bursa of Fabricius and thymus) and hypoplasia of bone marrow were observed. Histologic, ultrastructural, and hematologic examinations were performed on a total of 80 randomly selected 30-day-old meat pigeons. By histologic studies, lymphocytic depletion and necrosis with cyst formations in the bursa of Fabricius were detected in all subjects that showed thymus and bursa atrophy at necropsy. Basophilic intranuclear inclusions were also observed in bursal cells. After ultrastructural studies, these inclusions were proved to be viral particles resembling circoviruslike particles in morphology and size. Severe lymphocytic depletion of the bursa was plausibly associated with the presence of the viral particles.     

Viral matrix inclusion bodies in myocardium of lymphoid leukosis virus-infected chickens

Chicken embryos and healthy adult chickens naturally infected with lymphoid leukosis virus were used to investigate viral inclusion bodies in myocardial cells by light and electron microscopies and by immunocytochemical technique. Intracytoplasmic viral matrix inclusion bodies frequently appeared in the myocardium of adult chickens, but not in that of embryos. In light microscopic preparations, inclusions were irregularly distributed, were basophilic, and contained ribonucleic acid. Ultrastructurally, inclusions in myocardial cells were in areas containing numerous interstitial C-type particles. Early inclusions were composed of clusters of ribosomes associated with sarcoplasmic tubules; spherical bodies developed among these ribosomes. Mature inclusions were composed of numerous spherical bodies (50 to 75 nm) with interspersed ribosomes and of ribosomes clustered at the periphery. Inclusions were not membrane-enclosed. Occasionally, spherical bodies were in paracrystalline arrays. Multiple budding occurred on cell membranes adjacent to matrix inclusions. The viral group-specific protein, p27, was demonstrated by the peroxidase-antiperoxidase method and by the protein A-gold method in the spherical bodies, in nucleoids of mature virus particles, and among ribosomes of inclusions. The results indicate that the matrix inclusions were the result of lymphoid leukosis virus infection and were the product of viral protein synthesis on ribosomes.     

Amylopectinosis in fetal and neonatal Quarter Horses

Three Quarter Horses, a stillborn filly (horse No. 1), a female fetus aborted at approximately 6 months of gestation (horse No. 2), and a 1-month-old colt that had been weak at birth (horse No. 3), had myopathy characterized histologically by large spherical or ovoid inclusions in skeletal and cardiac myofibers. Smaller inclusions were also found in brain and spinal cord and in some cells of all other tissues examined. These inclusions were basophilic, red-purple after staining with periodic acid-Schiff (both before and after digestion with diastase), and moderately dark blue after staining with toluidine blue. The inclusions did not react when stained with Congo red. Staining with iodine ranged from pale blue to black. Their ultrastructural appearance varied from amorphous to somewhat filamentous. On the basis of staining characteristics and diastase resistance, we concluded that these inclusions contained amylopectin. A distinctly different kind of inclusion material was also present in skeletal muscle and tongue of horse Nos. 1 and 3. These inclusions were crystalline with a sharply defined ultrastructural periodicity. The crystals were eosinophilic and very dark blue when stained with toluidine blue but did not stain with iodine. Crystals sometimes occurred freely within the myofibers but more often were encased by deposits of amylopectin. This combination of histologic and ultrastructural features characterizes a previously unreported storage disease in fetal and neonatal Quarter Horses, with findings similar to those of glycogen storage disease type IV. We speculate that a severe inherited loss of glycogen brancher enzyme activity may be responsible for these findings. The relation of amylopectinosis to the death of the foals is unknown.     

Studies on the replication of a bovine parvovirus

Optimal replication of a bovine parvovirus type 1 was found to occur when parasynchronous bovine embryonic lung cells were infected during the S phase of the cell cycle, just prior to maximum DNA synthesis. Viral antigen was first detected in the cytoplasm by immunofluorescence at 8 h post-infection, reaching a maximum at this location by 16 h and then disappearing. In the nucleus, antigen was first detected at 12 h, concurrent with early inclusion body formation and first detection of intracellular virus production. Intranuclear antigen then increased rapidly to a maximum at 20 h, as the inclusions progressively matured, large amounts of virus were produced within the cell, with some release to the environment. From 24 h, the nuclear inclusions became increasingly shrunken and basophilic as virus migrated to the cytoplasm and was progressively released to the exterior concurrent with cell degeneration and fragmentation. The majority of virus remained cell associated, even at 28 h post-inoculation. Two morphological types of early and late stage intranuclear inclusions were produced by the virus, these appearing to be a distinct feature of bovine strains. In other aspects, the replication of bovine parvovirus appeared similar to that of other members of the genus.     

Age-related histological changes in the canine substantia nigra

Age-related changes in the canine substantia nigra, were examined using immunostaining for tyrosine hydroxylase (TH), glial fibrillary acidic protein (GFAP), neurofilament (NF), ubiquitin, single stranded DNA (ssDNA), and alpha-synuclein (alphaSN). Brain sections from 34 necropsied dogs, ranging from 2 months to 18 years old, were used for this study. On general histological examinations, several age-related changes, including lipofuscin deposition, polyglucosan bodies, amorphous basophilic inclusions and eosinophilic crystal inclusions, were found in the aged dogs. Immunohistochemically, TH-positive neurons were located only in the substantia nigra. The number of TH-positive neurons was well preserved in all dogs examined, however, the ratio of TH-positive neurons to GFAP-positive glial cells tended to show slight decrease in aged dogs. By ssDNA immunostaining for apoptotic cells, there were no significant results in the number of ssDNA-positive neurons. The number of ubiquitin- and NF-positive swollen neurites was increased markedly in aged dogs. Ubiquitin immunostaining revealed a small number of basophilic and eosinophilic inclusions, although both types of inclusions were negative for NF. By alphaSN immunostaining, no neurons were immunoreactive and no basophilic or eosinophilic intracytoplasmic inclusions were revealed. These results indicate that in the substantia nigra of aged dogs the dopaminergic neurons are well preserved, but intracytoplasmic inclusions and ubiquitin-positive degenerative neurites are commonly found.     

Studies on the cytopathology of bovine adenoviruses in calf testis and calf kidney cell cultures

The sequential development of intranuclear inclusions in calf kidney and calf testis cells infected with nine bovine adenovirus (BAV) serotypes is described. Haematoxylin and Eosin (H & E), immunofluorescent and electron microscope (EM) studies indicated two distinct subgroups of viruses. Serotypes 1, 2, 3, 7 and 9 comprised subgroup-1, while types 4, 5, 6 and 8 comprised subgroup-2. Differences were noted in the early stages of infection. With subgroup-1 viruses, irregular patches of eosinophilic material were first to appear, followed by small refractile inclusions and basophilic inclusions. In EM studies, the eosinophilic material was thought to correspond to the irregular type II inclusions, and the refractile bodies to type I inclusions. Eventually a basophilic inclusion, consisting of aggregated virus-associated inclusions and virus particles, was formed in the centre of the nucleus.With subgroup-2, the refractile inclusions were more prominent, larger, and were the first to appear. These were thought to correspond to type I inclusions, which were larger and denser than with subgroup-1. Circular basophilic bodies developing later were similar in size and distribution to type II inclusions, which with subgroup-2 viruses were seen in prominent circular or lobulated aggregates.Several other types of inclusion including tubular structures and paracrystals, which have thus far not been reported in BAV infected cells, are described.     

Biological properties and genetic analysis of the ompA locus in chlamydiae isolated from swine.

Eight strains of Chlamydia psittaci isolated from swine with pneumonia, pleuritis, pericarditis, and enteritis were characterized through analysis of the major outer membrane protein gene ompA by a two-step polymerase chain reaction, by their interactions with cells in culture, and by the morphologic features and ultrastructure of intracellular inclusions. Amplified chlamydial ompA DNA fragments were differentiated by restriction endonuclease digestion. Chlamydial isolates were separated into 2 types on the basis of ompA restriction fragment length polymorphism. Strains of type L71 had finely granular inclusions, whereas those of type 1710S contained pleomorphic reticulate bodies (RB) in the inclusions, which are characteristic of aberrant chlamydial developmental forms. Chlamydial types L71 and 1710S required centrifuge-assisted inoculation for efficient infection of cell cultures. Cultivation in cell culture medium containing cycloheximide increased the numbers of chlamydial inclusions about 1.5-fold. These strains formed few elementary bodies in yolk sac cells of chicken embryos. Ultrastructurally, unique doublet RB were observed, particularly in strains of the ompA type L71. These doublets consisted of 2 RB, bounded by a cytoplasmic membrane, contained within a common cell wall and an extended periplasmic space. Ultrastructural examination of strains of the ompA type 1710S confirmed the aberrant chlamydial developmental forms, but evidence of viral infection of the RB as a cause of these aberrant forms was not found. The strain S45 isolated from intestinal sites of swine was a trachoma restriction fragment length polymorphism type. With the mouse biotype, it represented the second isolate from animals of Chlamydia trachomatis.     

Light and electron microscope studies of the growth of ovine adenoviruses in primary lamb kidney cell cultures

The growth of five ovine adenovirus serotypes in lamb kidney cell cultures is described. All five serotypes exhibited similar changes although nuclear enlargement and deformity was most marked with types 3 and 5. The first inclusions seen by H & E staining were irregular eosinophilic bodies. These increased and coalesced, and later refractile ‘pearl-like’ inclusions and granular basophilic inclusions were also seen. A single large basophilic inclusion was eventually formed. The first fluorescent inclusions were small and granular. Later, fluorescent rings and fluorescent reticular-like networks were observed. Electron-microscope studies with types 1 and 5 showed virus particles and associated inclusions of several types accumulating in the centre of infected nuclei. The sequence of changes observed was similar to those described for the human adenovirus 3, 4, 7 subgroup, and the Bartha group A bovine adenoviruses.     

Dynamics of the development of Chlamydophila psittaci inclusions in epithelial and fibroblast host cells

The development of Chlamydophila psittaci (formerly Chlamydia psittaci, avian strains) inclusions in fibroblast L-929 and epithelial BGM cell lines was studied along the bacterial growth cycle using a BGM cell-adapted strain in the presence or absence of cycloheximide and cycloheximide + polyethylene glycol. Evolution of the inclusions was determined in terms of their number and size at 24, 30, 36, 48 and 54 h after infection. Significant differences in the chlamydial growth were found between both host cells, throughout the study. Higher numbers of inclusions (P < 0.05) were observed in L cells while larger inclusions (P < 0.01) were found in BGM cells. In both fibroblast and epithelial cells, inclusions showed a significant (P < 0.001) increase in size at the later times studied. Free extracellular chlamydial particles were noticed at 48 and 54 h post-infection (p.i.) in infected L cells, and at 54 h p.i. in BGM cells. Addition of cycloheximide or cycloheximide + polyethylene glycol had no significant effect on the number of inclusions or their size. The results suggest that host cell characteristics and innate compatibility between Chlamydophila strain and host cell are more important than host cell adaptation for the development of the microorganism.     

应用免疫胶体金标记技术对狂犬病病毒形态发生学的抗原定位研究

利用包埋后免疫胶体金标记技术，通过透射电子显微镜，对感染ＢＨＫ２１细胞的鹿狂犬病病毒进行了形态发生的抗原定位。结果：感染细胞的细胞质内有５种包涵体，其中４种被免疫胶体金特异性标记，这些病毒抗原是多成分的，另一种未被免疫胶体金标记的包涵体可能是病毒核酸；感染细胞的细胞质内大量堆积的病毒前体物质包涵体，其中可能有病毒装配后多余的成分，表明病毒各组分并不是按比例需要而合成的。     

Electron microscopy of Cowdria-infected macrophages suggests that in the absence of binary fission a mosaic of organisms develops from an amorphous electron dense matrix.

Electron microscopy of mouse peritoneal macrophages infected with the Kümm stock of Cowdria ruminantium suggests that in the final stage of intracellular growth, a mosaic of organisms develops from an amorphous matrix of varying electron density by a process in which double unit membranes portion off the Cowdria particles. This stage is preceded by inclusions consisting of a network of aggregated electron dense granules and these in turn by homogeneous dense bodies. The study failed to show how these dense bodies develop from internalized Cowdria particles introduced in the infective inoculum. The replication of the heartwater agent in macrophages differs from that in vascular endothelial cells in two important respects. First, at no stage during the course of development in macrophages is binary fission in evidence and second, in the absence of a limiting membrane the inclusions and colonies of organisms throughout the cycle of development in macrophages are in intimate contact with the host cell cytoplasm.     

Acidophilic macrophage pneumonia in laboratory mice

Acidophilic macrophage pneumonia, characterized by an accumulation of characteristic crystalloid-laden alveolar macrophages, was seen in 30/7,500 NMRI, 7/600 T x HT, 2/100 C57BL and in no cases of 1,500 CBA and 1,100 BALB/c mice. Histologically, there was a focal accumulation of large numbers of eosinophilic macrophages, generally associated with granulocytes. Macrophages could be mononucleate or multinucleate and had a crystalline cytoplasm. Free-lying crystals were sometimes observed. Ultrastructurally, macrophages had a cytoplasmic accumulation of needle-shaped and rhomboidal crystals, often showing a clear lattice structure with a repeat of 3-5 nm. The crystalloid inclusions may be derived from the breakdown products of granulocytes and appear similar to inclusions in macrophages in other parts of the hematopoietic system. That these inclusions are probably derived from eosinophils is based on the appearance within macrophages of structures resembling eosinophil granules at various stages of degradation and the similarity between the lattice repeat of the crystalloids and that of the crystalline core of the eosinophil granule. The crystalloid inclusions may be related to the Charcot-Leyden crystals found in human beings.