Morphogenesis of canary poxvirus and its entrance into inclusion bodies

A virus isolated from a natural outbreak of canarypox was replicated on the chorioallantoic membranes of chicken embryos, and its ultrastructure and development were observed. Electron microscopy of thin sections of pocks produced on the chorioallantoic membranes revealed a variety of developmental forms which appear similar to those demonstrated in studies of vaccinia, ie, viroplasm or viral factories; immature, undifferentiated virions partially enclosed by membranes; completely enclosed nondifferentiated spherical or oval virions; immature virions with discrete nucleoids; and the more compact brick-shaped mature virions. Two types of A-type inclusions were noted: those with virions around the periphery, and those filled with virus particles. The appearance of mature viruses within the inclusion bodies and different stages of viruses outside the inclusion indicate that in a course of development, maturing poxvirus may enter the inclusion bodies as they acquire surface tubules on their envelopes. Mature virions also were seen budding out of the cell membrane, apparently enveloped in a portion of the membrane. Studies showing the entrance of poxvirus into inclusion bodies have not been reported. In this report, electron micrographs are shown of viruses entering inclusion bodies.     

Ultrastructure of porcine circovirus in persistently infected PK-15 cells.

The ultrastructure of porcine circovirus was examined in persistently infected porcine kidney (PK)-15 cells. Virus-infected PK-15 cells had large numbers of intracytoplasmic inclusions, and a few cells had intranuclear inclusions. Intracytoplasmic inclusions were dispersed throughout the cytoplasm but were most numerous in the perinuclear cytoplasm. Inclusion were of various sizes, round to oval, and electron dense and were of two general types. Inclusions of the first type were small (0.1-0.5 microm diameter), not surrounded by trilaminar membranes, and granular with indistinct margins that blended with surrounding cytoplasm. Some contained 12+/-2-nm-diameter icosahedral virions in loose aggregates or rarely forming paracrystalline arrays. Small inclusions could be sites of viral assembly or maturation. Intracytoplasmic inclusions of the second type were larger (0.5-5.0 microm diameter) and more numerous and had abrupt margins surrounded by trilaminar membranes. They were more electron dense than small inclusions and were heterogeneous, containing various proportions of aggregated virions, electron-dense crystalline lamellae of 5 nm periodicity, and/or whorls of myelinoid membranes. Virions usually formed paracrystalline arrays and occasionally were loosely aggregated. Larger inclusions were typical of autophagolysosomes. Intranuclear inclusions were not membrane bound and were often associated with reticulated nucleoli or aggregates of heterochromatin. Some inclusions were irregularly shaped aggregates of indistinct, circular 10-12-nm-diameter viruslike particles. Others were 0.1-1.0 microm in diameter, round or ring shaped, dense, and finely granular, with sharply demarcated margins.     

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.     

Spontaneous pustular dermatitis in a newborn piglet associated with a poxvirus

Poxvirus was recovered from pustular skin lesions in a newborn piglet. The lesions were present at birth, suggesting transplacental infection had occurred. The lesions were circular, 1 cm in diameter, and were found over the entire skin surface, bulbs of the heels, coronets, tongue and buccal mucosa. Histologically, cells in the stratum intermedium were undergoing ballooning degeneration and necrosis, coalescing to form vesicles. The lumen of vesicles was filled with fluid and inflammatory cells. Degenerating epithelial cell nuclei were often vacuolated and eosinophilic intracytoplasmic inclusions were in many ballooned cells. The size and morphology of the virions recovered from vesicular fluid, place them in the poxvirus group.     

Structural proteins of two different plaque-size phenotypes of fowlpox virus

Structural polypeptides of two plaque-purified variant isolates of fowlpox virus differing in plaque morphology and size were examined by Coomassie blue-staining and immunoblot analysis of purified virions. A total of 30 structural polypeptides were observed, ranging in molecular weight from 14,100 to 122,600. A late polypeptide of 36,400 molecular weight was quite prominent in the small-plaque clone but absent in the large-plaque clone. Two other polypeptides, of 33,700 and 34,800 molecular weight, were present in virions from large-plaque virus and cell lysates of both clones but were absent in the small-plaque virions. These differences were observed whether the viruses were grown in chorioallantoic membrane or in chicken embryo fibroblast cultures. No difference was observed between the growth curves of the two virus clones. Differences observed in the polypeptides of the two viruses may be due to changes in the less conserved regions of viral DNA and may be used for differentiation of virus isolates.     

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.     

Immunoperoxidase labeling of canine distemper virus replication cycle in Vero cells

An indirect immunocytochemical labeling technique, using horseradish peroxidase-conjugated antibody was used to detect the intracellular and surface membrane localization of canine distemper virus (R252-CDV) antigens during productive virus replication in infected Vero cells. Specific labeling of intracellular viral antigens was restricted to rough nucleocapsid aggregates. Surface membrane labeling correlated directly with the appearances both of virus-specific membrane spikes and buds and of mature virions. Syncytial cell formation was associated with labeled cytoplasmic nucleocapsid, but there was no evidence of productive CDV formation on surface membranes. The immunoperoxidase technique provided precise ultrastructural antigenic localization with concomitant preservation of excellent ultrastructural detail within single virus-infected cells during CDV replication cycle in vitro.     

A selection of naturally occurring pathological phenomena in the brush-tail possum,Trichosurus vulpecula

During a study of the brush-tail possum, Trichosurus vulpecula, from three different sites in New Zealand, two of which are endemic for bovine tuberculosis, several pathological entities were encountered. Some of these are differential diagnoses for tuberculosis. The list included: bite abscess, foreign body granulomas, calcification of the caudal aorta, lymphoid nodules in the lung, pulmonary lipid plaques, lipid pneumonia, pneumoconiosis, focal fibrosing alveolitis, adiaspiromy-cosis, collagenous hamartoma, focal dystrophic calcification in the lung, mineralised casts in renal tubules, hyaline nephrosis with proteinuria, focal interstitial nephritis, pyelonephritis, renal and hepatic amyloidosis, lymphoid nodules around sublobular veins in the liver, multi-focal non-suppurative hepatitis, multi-focal bacterial hepatitis, bile ductular hyperplasia and peri-portal fibrosis, hepatocellular fatty change, intranuclear inclusions and cytoplasmic invaginations in hepato-cytes, hepatocellular lipopigmentation and gastrointestinal parasitism.     

Experimental infection of lambs with ovine adenovirus isolate RTS-151: lesions

Young lambs, inoculated with ovine adenovirus isolate RTS-151, were killed sequentially (to postinoculation day 21), and their morphologic lesions were studied. Lesions were found only in the respiratory tract and were most severe in the terminal airways. Many cells of the respiratory epithelium had severe cytomegaly and karyomegaly with intranuclear inclusions. Desquamation of affected cells was accompanied by hyperplasia of airway and alveolar epithelium and by accumulation of necrotic cellular debris and neutrophils in terminal airways and alveoli. Virus replicated in the nucleus of enlarged epithelial cells in the airways, but not in alveolar epithelium. Intranuclear inclusions representing viral replication progressed from clumps of finely granular material without virions to a granular material with virions and finally to a dense inclusion with numerous virions.     

An encephalopathy with argyrophilic inclusions in a Holstein-Friesian cow

The brain of a 6-year-old Holstein cow, which showed progressive neurologic symptoms during several months, was examined by histopathologic methods. Many round or oval-shaped cytoplasmic inclusions were observed, mainly in neurons of the temporal lobe and the hippocampus. Those inclusions were faintly eosinophilic with hematoxylin and eosin and positive with Bielschowsky's silver stain. Immunohistochemically, the inclusions were recognized by antiubiquitin and antiphosphorylated tau antibodies. Ultrastructurally, the inclusions were globular and well demarcated from the rest of the cytoplasm, lacked limiting membranes, and were mainly composed of straight fibrils about 15 nm in width. The structure of the inclusions was similar to that of Pick bodies in Pick's disease of humans. The pathogenesis of this bovine condition is not known.     

传染性法氏囊病病毒（IBDV）人工感染SPF鸡的病理学观察

取３２０只３０～４０Ｈ龄ＳＰＦ鸡，经滴鼻和点眼接种传染性法氏囊病病毒（ＩＢＤＶ），接种后２４～７２ｈ出现死亡，病死率５１．２５％，死亡鸡剖检变化主要为法氏囊肿胀、出血，甚至呈紫葡萄样，脾脏肿大、出血，骨骼肌出血，腺胃肌胃交界处出血。病理组织学变化，呈现以法氏囊淋巴滤泡内髓质淋巴细胞坏死为主的特征性病变，并可在巨噬细胞浆内发现病毒包涵体。电镜观察，在法氏囊内淋巴细胞、异染性细胞、巨噬细胞浆内，见大量晶格状排列的病毒粒子和包涵体，表明ＩＢＤＶ首先损害法氏囊淋巴滤泡髓质内未成熟的Ｂ淋巴细胞，病毒在淋巴细胞内以包涵体方式增殖。     

Etiology of the stomatitis pneumoenteritis complex in Nigerian dwarf goats.

The causative agent of stomatitis pneumoenteritis complex was isolated in domesticated goats and Vero cell culture. It was identified immunologically and morphologically as identical with the "Peste des Petits Ruminants" virus. There were cross reactions between stomatitis pneumoenteritis complex virus isolate and rinderpest virus by immunodiffusion and complement fixation tests but no cross neutralization. Goats recovered from stomatitis pneumoenteritis complex were protected against a challenge with rinderpest virus that was lethal to control goats. Ultrastructural morphology revealed intracytoplasmic and intranuclear inclusions made up of random arrays of fibrillar strands. Pleomorphic particles budded from the plasma membrane of infected cells and enveloped virions were seen extracellularly. Specific ferritin tagging was demonstrated in the stomatitis pneumoenteritis complex virus infected cells treated with homologous and peste des petits ruminants viral antibody systems but little, if any, tagging in the heterologous rinderpest system.     

山羊痘病毒粒子的电子显微镜观察

本研究室2003年以来对山羊痘病毒感染多种细胞进行了透射电镜观察。在感染的皮肤和黏膜上皮细胞浆中观察到大量不同成熟阶段的典型山羊痘病毒粒子，病毒粒子也见于巨噬细胞、成纤维细胞、血管内皮细胞胞浆内，甚至少量散在血管腔中。用山羊痘病毒分离物感染鸡胚绒毛尿囊膜形成痘斑，其上皮细胞和成纤维细胞中也可见成熟和不成熟的病毒粒子，同时感染BHK-21细胞也观察到典型的山羊痘病毒粒子。     

Ultrastructural study of the liver and kidney in ochratoxicosis A in young broiler chicks

Ultrastructural changes are reported in the kidney and liver of 20-day-old broiler chicks fed ochratoxin A (OA), incorporated in the diet at levels of 2 and 4 ppm. Changes in the kidney included the presence of abnormally shaped mitochondria in the proximal convoluted tubules. There was an increase in the size and number of mitochondrial dense granules and cytoplasmic peroxisomes. Intranuclear and cytoplasmic lipid droplets and electron dense round bodies in the dilated smooth endoplasmic reticulum were also noted. Regional thickening and degeneration of the glomerular basement membrane was observed in some cases. In the liver from OA fed birds there was an increased accumulation of cytoplasmic glycogen in the hepatocytes. Abnormal mitochondrial ring forms in the kidney and the accumulation of glycogen in the liver are considered to be of diagnostic significance in ochratoxicosis of young broiler chicks. The severity of the changes was found to be dose related. These results suggest that the mitochondria in the proximal convoluted tubules of kidney were most sensitive to OA toxicity.     

Outbreak of cutaneous form of poxvirus on a commercial turkey farm caused by the species fowlpox

The present report documents the occurrence of a poxvirus infection in commercial meat turkeys. The affected farm had six flocks, with a total of 11,680 birds at different ages; birds from two of these flocks were affected. The clinical picture was characterized by severe epithelial lesions and proliferations on the head and neck regions as reported for the cutaneous form of poxvirus infection. Except for these lesions, no adverse clinical signs or gross pathologic lesions were observed. Only a low number of birds was affected (n = 20) and no increase of mortality could be seen. Bacteriologic investigations from the lesions revealed multiresistant Staphylococcus aureus. Eosinophilic inclusions (Bollinger bodies) in histologic examinations in the cytoplasm of keratinocytes were noticeable. Typical pox virions were demonstrated by electron microscopy, and poxvirus was isolated on the chorioallantoic membrane of specific-pathogen-free chicken eggs. Further identification of the poxvirus species was carried out by PCR and sequencing, revealing an infection with the species fowlpox. Layers in vicinity of the turkey farm that also were affected by fowlpox were considered as potential source of infection. Although it is assumed that avian poxviruses are strongly species specific, the present case report reinforces the changing picture of poxvirus infections in turkeys. Furthermore, it supports the assumption of previous data that fowlpox virus has to be seen as recently emerging pathogen in turkeys.     

Neuronal Ceroid-lipofuscin Storage in Siamese Cats

The clinico-pathological findings of a nervous disease in two mature Siamese cats are described. In one animal intermittent convulsions and mania over a period of three weeks terminated in death, while in the other irritability and hind leg weakness were the main clinical features. Neuronal cytoplasmic inclusions composed of a complex lipid material were detected microscopically. The ultrastructure and staining reactions of these inclusions in one cat were similar to the curvilinear bodies described in neurovisceral storage disease in humans and in a neuronal lipodystrophy of cattle. The deposit was considered likely to be related to ceroid-lipofuscin. No differences in brain lipid quantity or quality could be detected when compared to two control cats.     

Spontaneous Accumulation of Globotriaosylceramide (Gb3) in Proximal Renal Tubules in an ICR Mouse

This report describes spontaneous cytoplasmic vacuolation in the proximal renal tubules of a 7-week-old male ICR [Crlj:CD1(ICR)] mouse. The contents of vacuoles were positively stained with periodic acid-Schiff (PAS) and Sudan black, and the membranes were positive on immunohistochemical staining for lysosomal-associated membrane protein-2 (LAMP-2), a marker of lysosomal membrane. Electron microscopy revealed electron-dense lamellar bodies in the proximal tubular epithelial cells. These histopathological features are similar to those in α-galactosidase A-deficient mice, in which globotriaosylceramide (Gb3), a glycosphingolipid, accumulates in lysosomes. When we performed immunohistochemical staining for Gb3, the contents of vacuoles were positively stained. From these results, spontaneous cytoplasmic vacuolation in the proximal renal tubules in the mouse was identified as lysosomal accumulation of Gb3.     

Spermatid giant cells, tubular hypospermatogenesis, spermatogonial swelling, cytoplasmic vacuoles, and tubular dilatation in the testes of normal rabbits

Testes of 36 normal New Zealand white rabbits (8, 15, 18, 26, and greater than 52 weeks of age) were examined by light and electron microscopy. The incidence and number of affected tubules were determined for spermatid giant cells, focal tubular hypospermatogenesis, cytoplasmic swelling of spermatogonia, intracytoplasmic vacuoles in seminiferous epithelium, and tubular dilatation. Spermatogenesis commenced at 15-18 weeks of age and was present in all rabbits by 18 weeks. Spermatid giant cells occurred in 96% of rabbits 15 weeks of age and older. Focal hypospermatogenesis was present in 14-57% of testes once active spermatogenesis began. Ninety-seven percent of testes in all age groups combined had spermatogonial swelling. Infrequent dilated seminiferous tubules were present in five rabbits. Ultrastructurally, spermatid giant cells were round cells with multiple nuclei that appeared to arise by widening of narrow intercellular bridges that normally connect spermatogenic epithelial cells. Pale-staining spermatogonia consisted of cytoplasmic and nuclear swelling with disruption of plasma and nuclear membranes. Tubules with spermatogonial swelling were more numerous in 15- and 18-week-old rabbits. There were no significant differences in incidence or extent of spermatid giant cells, focal hypospermatogenesis, cytoplasmic vacuoles, or tubular dilatation between age groups after spermatogenesis commenced. Although the cause of these changes is not known, they were frequent findings in normal rabbits 15 weeks of age and older.     

Electron Microscopy of Intestinal Epithelial Cells of Piglets Infected with a Transmissible Gastroenteritis Virus

An electron microscopic study of intestinal epithelial cells of neonatal piglets infected with transmissible gastroenteritis (TGE) virus revealed a unique parasite-host cell interaction. Entry of the TGE virus into intestinal epithelial cells of newborn piglets is mediated through a network of cytoplasmic tubules of plasmalemma origin. the tubules, called microcanaliculi, are morphologically distinct from endoplasmic reticulum and Golgi. In uninfected animals similar tubules appear to be responsible for the indiscriminate uptake of large quantities of macromolecules from colostrum during the first few days of life.

Thin-section profiles of plasmalemma invaginations resembled tubules or canals and frequently contained viral particles. TGE viral particles developed and accumulated within cytoplasmic vacuoles. Initially the vacuoles were continuous with the microcanaliculi formed by deep plasmalemma invagination. Mature viral particles were 60 to 85 mu in diameter with an electron dense doughnut-shaped nucleoid surrounded by a trilaminar membrane which resembled the vacuolar wall. Abundant evidence of viral effects was observed in absorbtive epithelial cells of the jejunum and ileum but not of the duodenum.

The ability of absorbtive intestinal epithelial cells to form deep cytoplasmic tubular invaginations is temporally related to the pathogenesis of TGE and may explain in part why pigs usually are fatally affected by TGE only during the neonatal period.

     

Characterization of the extracellular matrix in the chorioallantoic membrane of water buffalo (Bubalus bubalis) in early gestation

Placenta is formed by a parenchyma rich in extracellular matrix (ECM), and this structure guarantees the proper development of the embryo and placental functioning. Recently, studies have focused on the characterization of ECM in the placenta and foetal membranes of different species. This work aimed to analyse the composition of the ECM and to quantify the types of collagens in its composition. For this, 33 chorioallantoic membranes were used at different gestational ages, which were grouped into five groups. Subsequently, haematoxylin–eosin staining, Masson trichrome and picrosirius were performed for histological analysis. Through the technique of polarized light, it was possible to quantify the total collagen present in the membranes and finally the immunohistochemical technique was performed to verify the presence of collagens and glycoproteins. It was possible to verify that the chorioallantoic membranes have, in all the gestational periods of the initial third of gestation, the same histological structures, being the most significant difference the membrane thickening that occurs gradually during the gestation. However, we notice the appearance of binucleate cells only from group II. In addition, it was verified that a gradual increase of collagen occurs until the group IV, yet from the group V begins to occur a decrease of this protein. In addition, collagen I, collagen III, fibronectin and laminin were present in all membranes. With this, we concluded that the buffalo chorioallantoic membrane presents ECM in constant remodelling at the beginning of gestation and can be used as biomaterial in works on regenerative biology.