Demonstration of colonies of Cowdria ruminantium in midgut epithelial cells of Amblyomma variegatum

The development of colonies of Cowdria ruminantium was studied in midgut epithelial cells of adult Amblyomma variegatum that had become infected by feeding as nymphs on cattle with experimentally induced heart-water disease. Colonies were not observed in gut tissues obtained from nymphs during the feeding period, but were present in midgut epithelial cells of ticks obtained at 15 days after they were replete through molting to the adult stage. Colonies were small (1 to 10 micron) initially, but as tick development progressed, their diameter increased to as much as 60 micron. With electron microscopy, colonies were observed to be membrane bound and contained pleomorphic organisms that were reticulated. The organisms seemed to be dividing by binary fission. Many colonies contained a large, electron-dense inclusion that was morphologically similar to hemoglobin deposits found in the cytoplasm of midgut epithelial cells of recently fed ticks. Cowdria ruminantium was often observed adhered to these inclusions.     

Ultrastructural morphology of Cowdria ruminantium in midgut epithelial cells of adult Amblyomma hebraeum female ticks

Amblyomma hebraeum male and female ticks, experimentally infected as larvae with the Ball 3 stock of Cowdria ruminantium, were fed on a heartwater susceptible sheep. The initial attachment of the males was required as a pre-requisite for female attachment. Reticulate bodies were the predominant morphologic form of Cowdria observed in gut epithelial cells after 1-3 days of feeding. Single intermediate bodies and no elementary bodies were observed. Organisms were found within a membrane-bound vacuole and each organism had a double-unit membrane. Infrequently colonies contained homogeneous electron-dense inclusions. Groups of Cowdria organisms within a haemocyte suggested a possible dissemination of organisms from the gut to various other tissues by haemocytes.     

The tick-borne rickettsia Cowdria ruminantium has a Chlamydia-like developmental cycle.

The development of the tick-borne rickettsial pathogen Cowdria ruminantium (S stock) was studied in bovine umbilical endothelial (BUE) cell cultures and in goat choroid plexus, by light- and electron microscopy. Cowdria divided by binary fission within intracytoplasmic vacuoles resulting in large colonies of reticulate bodies. After three to four days in culture, reticulate bodies developed into smaller intermediate bodies characterized by an electron-dense core. Shortly before disruption of the host cells, intermediate bodies condensed further into electron-dense elementary bodies, which were released into the culture medium. Elementary bodies invade other endothelial cells thus initiating a new infectious cycle which lasts between 5 and 6 days. In the infected goat choroid plexus similar reticulate and intermediate bodies were identified within vacuoles of capillary endothelial cells. However, extracellular elementary bodies were not detected. Another stock of Cowdria (W) showed an identical developmental cycle as that of the S stock. The W isolate was also pathogenic for mice, making it possible to test the infectivity of reticulate and elementary bodies in these animals. Reticulate bodies appeared to be less infective than elementary bodies. The developmental cycle of Cowdria resembles the cycle known to occur in Chlamydia. Moreover, Cowdria has other similarities with Chlamydia. It has a Gram-negative envelope, it does not store iodine-stainable carbohydrates and may lack peptidoglycan as does Chlamydia. It is concluded, that Cowdria and Chlamydia are to a certain extent related, confirming a recent report that both organisms have certain antigenic determinants in common. Since Cowdria is also related to Ehrlichia it may well be that Cowdria takes an intermediate position between Chlamydia and Ehrlichia. The phylogenetic relationship between Cowdria and Chlamydia and also with Ehrlichia should be further elucidated by molecular analysis using 16S ribosomal DNA sequences.     

Fluorescent antibody technique to detect Cowdria ruminantium in in vitro-cultured macrophages and buffy coats from cattle, sheep, and goats

Fluorescent antibody tests, Giemsa stain, and electron microscopy were used to detect colonies of Cowdria ruminantium in in vitro-cultured macrophages and buffy coats from heartwater-infected cattle, sheep, and goats. Antibodies were obtained from C ruminantium-infected cattle, sheep, and goats treated with a small dose of oxytetracycline HCl. Cowdria ruminantium elementary bodies were small-coccus forms (0.14 micron) and large-coccus forms (0.22 micron to 0.6 micron). The size of inclusion bodies varied from 1.5 micron to 2 micron. Inclusion bodies and elementary bodies were observed in the cytoplasm of macrophages and neutrophils.     

Psittacine beak and feather disease in three captive sulphur-crested cockatoos (Cacatua galerita) in Thailand

Three sulphur-crested cockatoos (Cacatua galerita) were diagnosed as psittacine beak and feather disease (PBFD). Histopathology of the feather pulp and follicles showed intracytoplasmic botryoid clusters or granular inclusion bodies in epithelial cells and macrophages. Electron microscopy revealed multiple cytoplasmic clusters of electron dense viral particles corresponding to the inclusions. PBFD virus (circovirus) DNA-specific product was detected from formalin-fixed paraffin-embedded feathers by nested polymerase chain reaction (PCR) method.     

Heartwater. The morphology of Cowdria ruminantium and its staining characteristics in the vertebrate host and in vitro

The morphology of Cowdria ruminantium is described and its staining characteristics in the vertebrate host and in vitro are summarized. Morphologically, the organisms are characterized in the cytoplasm of endothelial cells, macrophages and reticulo-endothelial cells. Based on the morphology of the internal structure of the organisms, elementary (electron-dense), intermediate and reticulate bodies are identified. Each organism is surrounded by a double membrane and a "capsule" is evident around a few organisms in vitro. Usually, only organisms of the same form are found within a particular vacuole, although mixed colonies are described in the in vitro studies.     

Ultrastructural features of Cowdria ruminantium in midgut epithelial cells and salivary glands of nymphal Amblyomma hebraeum

Colonies of Cowdria ruminantium were studied in midgut epithelial cells and salivary gland acini of nymphal Amblyomma hebraeum that were infected experimentally as larvae. Colonies were found in both tissues and studied with light and electron microscopy. Colonies observed within gut cells frequently contained 2 types of the organism: electron-dense and reticulated forms. The morphology of colonies from salivary glands, as seen with light microscopy, varied from compact, densely-staining, small colonies to larger ones in which individual organisms were apparent. With electron microscopy, most organisms in salivary glands were reticulated and appeared to be dividing by binary fission. In both types of host cells, colonies often contained a dense inclusion to which reticulated organisms were adhered.     

Heartwater. The development and life cycle of Cowdria ruminantium in the vertebrate host, ticks and cultured endothelial cells

Various aspects of the development and life cycle of Cowdria ruminantium are discussed. C. ruminantium is transmitted transstadially by certain Amblyomma species. Apparently organisms initially develop in the gut epithelial cells of ticks and subsequent stages of C. ruminantium invade and develop in the salivary gland acini cells of the vector. Stages at which transmission to the final host are attained appear to be coordinated with the feeding cycle of the ticks and the vertebrate host is infected via salivary glands of the tick. In the vertebrate host, ticks and cultured endothelial cells, different morphological forms of C. ruminantium (electron-dense and reticulated forms) are found. Organisms enter cells through a process resembling phagocytosis and reticulated forms of the organisms appear to be the main vegetative stage. In the vertebrate host, organisms proliferate in vascular endothelial cells, neutrophils, macrophages and reticulo-endothelial cells.     

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.     

Ehrlichia platys (Anaplasma platys) in dogs from Maracaibo,Venezuela: an ultrastructural study of experimental and natural infections

Since 1982 Ehrlichia platys, now emended as Anaplasma platys, has been diagnosed in dogs from Maracaibo, Venezuela, using buffy coat smears stained with Dip Quick. Three dogs were inoculated with an A. platys strain. When parasitemia reached 60-97%, blood samples obtained from the inoculated dogs and from two naturally infected dogs were centrifuged to obtain platelet-rich plasma, which was mixed with 0.1% glutaraldehyde at 37 C for 10 minutes. Platelet pellets were fixed in 3% glutaraldehyde for 72 hours and processed for conventional transmission electron microscopy. Platelets contained pleomorphic organisms with a distinct double membrane that was not observed when the bodies were in a determinate developmental stage. There were 1-15 individual bodies included in a host cell vacuole. The organisms had an electron-lucent inner area, whereas the internal surface of their inner plasma membranes exhibited an electron-dense rough substance. In naturally infected dogs, organisms with different ultrastructural features were found inside the same platelet. Some organisms contained central dense material surrounded by a pale zone, which was in turn surrounded by a moderately dense peripheral area. Other organisms contained an eccentrically electron-dense material. The intravacuolar space appeared fully electron-lucent. Each organism usually exhibited inner fine strands. Empty structures displaying junctions with the vacuolar membrane were observed. Our results indicate that distinct ultrastructural characteristics are associated with different stages of A. platys development and may differ among A. platys strains.     

Intraerythrocytic schizogony of Theileria sergenti in cattle

The characteristics of developing intraerythrocytic stages of T. sergenti were studied by light and transmission electron microscopy. The parasites with many ribosomes, acristate mitochondria, cytostome, and food vacuoles were morphologically regarded as the trophozoite stage. Although this type of parasites was frequently detected, intraerythrocytic merozoite stage with electron dense cisternae, rhoptries and small electron dense bodies was rarely observed in high parasitaemia. The intraerythrocytic stages of T. sergenti were divided mainly into four daughters by schizogony, and alternatively into two by binary fission. The daughter parasites in each division had the same ultrastructural features as of merozoites. As a result, it was suggested that T. sergenti trophozoites multiplied by schizogony to four organisms or by binary fission in the peripheral erythrocyte, and differentiated to the merozoites which acquired penetrating ability into the erythrocytes.     

Morphology and development of Cowdria ruminantium in Amblyomma ticks

The morphology and development of Cowdria ruminantium have been studied in Amblyomma hebraeum and A. variegatum. Colonies of C. ruminantium have so far been demonstrated microscopically in gut, salivary gland cells, haemocytes and malphighian tubules of infected Amblyomma ticks. Colonies in gut cells were seen in both unfed and feeding ticks but colonies in salivary gland acini were observed only in nymphs that had fed for 4 days. Although the predominant type seen in both tick stages was the reticulated form that appeared to divide by binary fission, electron dense forms were also present. The latter are similar to those forms documented in endothelial cells of the vertebrate host as well as in cell culture. The presence of colonies of C. ruminantium in salivary glands of feeding ticks, along with the demonstration of different morphologic forms of the organism, suggests that a developmental cycle of the organism occurs in its invertebrate host. It is thought that organisms first infect and develop within gut cells. From there subsequent stages continue their development in haemolymph and salivary glands and are then transferred to the vertebrate host during tick feeding. Further studies are needed to completely understand the development of C. ruminantium in ticks and its subsequent transmission by these parasites.     

Morphological studies of esophageal papilloma naturally occurring in cattle

Esophageal papillomas naturally occurring in 35 cattle were examined by light and electron microscopy. The characteristic histological lesions noted were acanthosis and parakeratosis of the mucous membrane with formation of basophilic intranuclear inclusion bodies in cells of the stratum corneum. Electron microscopically, viral particles were observed in the strata spinosum and corneum, and larger aggregations of viral particles appeared to correspond to the histological inclusions. The virus particles had a hexagonal profile, measured 35-40 nm in diameter and were arranged in crystalline arrays in some nuclei. The viral particles had morphologic characteristics indistinguishable from those of other papilloma viruses. The inclusions were stained positively by immunohistochemical methods using anti-bovine papilloma virus serum.     

Heartwater: an in vitro study of the ultrastructure of Cowdria ruminatium

Notwithstanding morphological differences, the ultrastructure of Cowdria ruminatium cultured in vitro concurred to a large extent with that in previous in vivo studies. Two distinct forms of the organism, elementary and reticulate bodies, and a 3rd group of intermediate organisms were identified. Organisms within a particular vacuole were generally a specific form, but in cells containing many colonies different forms were present in the same colony. Most organisms were enveloped by 2 membranes and a few were surrounded by a 'capsule'. C. ruminantium multiplies mainly by binary fission, but it appears that multiplication can also take place by means of budding. The taxonomy of C. ruminantium is briefly discussed.     

The Sertoli cell of the water buffalo--an electron microscopic study.

The ultrastructure of Sertoli cells in the seminiferous tubules of water buffaloes before and during sexual maturity was studied by transmission electron microscopy, with emphasis on the intranucleolar vesicular elements. Sertoli cells of animals under 12 months of age were distinguished from the germ cells by the presence of electron dense membrane bound bodies within their cytoplasm. These cells, referred to as basal indifferent supporting cells, were probably involved in the phagocytosis and elimination of degenerating spermatocytes, which failed to differentiate into spermatids and spermatozoa in animals under one year of age. In 12 month old animals, a few Sertoli cells exhibiting the vesicular elements appeared in the nucleolar region while in animals over 15 months of age Sertoli cells could be positively identified by the characteristic cytoplasm containing microtubules, elongated and electron dense mitochondria, extensive granular endoplasmic reticulum and the presence of spermatids in various stages of spermiogenesis. The vesicular elements in the nucleolar region of the Sertoli cells were most prominent at this stage. Ultrastructural features of the Sertoli cells revealed an abundance of ribosome-like particles surrounding the vesicles of varying size. Some of these vesicular elements contained amorphous material suggesting that they represent the products sequestered in the nuclear region for transport to the cytoplasm and that the process of spermiogenesis may be dependent on the ability of Sertoli cells to generate these products at sexual maturity.     

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.     

Immunocytochemical labeling of Anaplasma marginale Theiler in Dermacentor andersoni Stiles with peroxidase- antiperoxidase technique

Colonies of Anaplasma marginale in midgut epithelial cells of adult ticks that had been infected as nymphs were specifically labeled, using the unlabeled antibody peroxidase-antiperoxidase method of immunocytochemistry. Visual comparison of infected and control tissue sections with the electron microscope demonstrated deposition of ring-like peroxidase-antiperoxidase complexes over organisms within the colonies. The intensity of labeling differed among organisms within a single colony, possibly as a result of varying antigenicity. The labeling observed on organisms in the colonies was similar to that seen on anaplasmal initial bodies in inclusions of infected bovine erythrocytes examined concurrently.     

Development of a hamster model of Chlamydophila pneumoniae infection

The aim of this study was to develop a new experimental model of Chlamydophila pneumoniae infection in the hamster. Intraperitoneal injection of C. pneumoniae purified elementary bodies (EBs) in the hamsters caused a systemic infection, since it was possible to isolate viable chlamydiae from several organs up to 14 days after infection. In particular, spleen infection was detectable up to 7 days post infection in 100% of animals. In contrast, cultures of the organs obtained from intranasally infected animals were far less frequently positive. Systemic infection probably occurred via macrophages, as demonstrated by the presence of intracellular chlamydial inclusions in peritoneal macrophages of peritoneally inoculated animals four days after infection. Furthermore, by infecting LLC-MK2 cells with supernatant preparations obtained from these macrophages, it was possible to observe the development of chlamydial intra-cytoplasmic inclusions after 96 h. Immunization of 18 hamsters with heat-inactivated purified EBs completely protected 16 animals and substantially reduced infection levels in the remaining two. Sera obtained from immunized hamsters prior to challenge reacted mainly against two C. pneumoniae proteins of about 60 kDa, when tested by immunoblot.     

Coxiella-like infection in psittacines and a toucan

Seven psittacine birds and a toucan (Ramphastos toco) were diagnosed as infected with Coxiella-like bacteria, based on polymerase chain reaction and bacterial 16S rRNA gene sequence obtained from each bird's liver tissue. Most of the birds exhibited lethargy and weakness for several days prior to death. Gross lesions included mild to moderate emaciation and severely enlarged and mottled pale livers and spleens. Microscopically, there was multifocal necrosis of hepatocytes with infiltration of a mixed population of inflammatory cells, including lymphocytes, heterophils, plasma cells, and macrophages randomly scattered throughout in most birds. In several birds within the macrophages there were vacuoles containing basophilic small cocco-bacilli organisms measuring about 0.5-1 microm. The spleens had increased numbers of mononuclear phagocytic system cells, some of which had vacuoles that contained similar organisms, as observed in the liver. There was inflammation in the epicardium and endocardium, interstitium of the lungs, kidney, adrenal and thyroid glands, lamina propria of the intestine, and in occasional birds in the brain, bursa of Fabricius, and bone marrow associated with similar organisms in the macrophages. Transmission electron microscopy of the liver and lungs in most birds and in the thyroid glands of one bird revealed pleomorphic round to elongated bacteria measuring about 0.45 microm in diameter and more than 1.0 microm in length. Most of these organisms contained a peripheral zone of loosely arranged electron dense material that was located immediately beneath a trilaminar membrane. Occasional organisms contained nucleoids. This is the first documentation of disease presumptively associated with Coxiella-like bacteria in birds.     

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.