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 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.     

Cutaneous papillomatosis and carcinomatosis in the Western barred bandicoot (Perameles bougainville)

A progressive wart-like syndrome in both captive and wild populations of the Western barred bandicoot (WBB) is hindering conservation efforts to prevent the extinction of this endangered marsupial. In this study, 42 WBBs exhibiting the papillomatosis and carcinomatosis syndrome were examined. The disease was characterized by multicentric proliferative lesions involving cutaneous and mucosal surfaces, which were seen clinically to increase in size with time. Grossly and histologically the smaller skin lesions resembled papillomas, whereas the larger lesions were most commonly observed to be squamous cell carcinomas. Large amphophilic intranuclear inclusion bodies were observed in hyperplastic conjunctival lesions of 8 WBBs under light microscopy. Conjunctival lesions from 2 WBBs examined using transmission electron microscopy contained a crystalline array of spherical electron-dense particles of 45-nm diameter, within the nucleus of conjunctival epithelial cells, consistent with a papillomavirus or polyomavirus. Conjunctival samples from 3 bandicoots that contained intranuclear inclusion bodies also demonstrated a positive immunohistochemical reaction after indirect immunohistochemistry for papillomavirus structural antigens. Ultrastructural and/or immunohistochemical evidence of an etiologic agent was not identified in the nonconjunctival lesions examined. Here we describe the gross, histopathologic, ultrastructural, and immunohistochemical findings of a papillomatosis and carcinomatosis syndrome recently identified in the WBB.     

Ultrastructure of the thyroid gland of the one-humped camel (Camelus dromedarius)

Follicular epithelium of the thyroid gland of the one-humped camel was examined by light and electron microscopy. It consisted of a single type of epithelial cell which varied from flattened to columnar in shape. Follicular epithelial cells were characterized by the presence of markedly dilated cisternae of rough-surfaced endoplasmic reticulum, well developed Golgi apparatus, abundant small vesicles (150 nm to 200 nm in diameter) in the apical cytoplasm, and electron-dense colloid droplets measuring from 250 nm to 1600 nm. Follicular epithelial cells frequently showed apocrine secretion into colloidal lumens. Apocrine protrusions with smooth surface were dome-like or balloon-like structures and contained a fine granular matrix. These findings indicate that the morphological features of the follicular epithelial cells of the thyroid gland of the camel are essentially similar to those of mammals except for the presence of apocrine secretion, which is unique to the camel.     

Respiratory herpesvirus infection in two Indian Ringneck parakeets.

A flock of Indian Ringneck parakeets (Psittacula krameri manillensis) was imported to the United States from Australia. Soon after, 1 parakeet suddenly died, and a second parakeet died after a 2-day course of illness, which consisted of anorexia, lethargy, emaciation, and dyspnea. At necropsy, the affected birds had diffuse consolidation and red discoloration of the lungs, as well as thickened, congested air sacs. The microscopic examination revealed multifocal, necrotizing bronchitis, parabronchitis, and interstitial pneumonia. The lumen of the affected airways contained numerous, large syncytial cells with up to 15 nuclei. The nuclei of these syncytial cells often contained large, eosinophilic inclusion bodies, consistent with herpesvirus. The epithelium of the trachea and air sacs was hypertrophied and contained syncytial cells with intranuclear inclusion bodies similar to the bronchi. In addition, a few intranuclear inclusion bodies were also present in the epithelial cells that line the air capillaries. On ultrastructural examination, the nuclei of degenerating epithelial cells contained clusters of viral nucleocapsid proteins and unenveloped, icosahedral, viral particles that were approximately 90 nm in diameter. In addition, some epithelial cells contained clusters of enveloped viral particles approximately 105 nm in diameter, within the cytocavitary network. These lesions are characteristic of those caused by respiratory herpesvirus of parakeets.     

Association of eosinophilic myositis with an unusual species of Sarcocystis in a beef cow.

The carcass of a mature cow had numerous, disseminated lesions typical of eosinophilic myositis. To elucidate the nature and possible cause of the lesions, histological sections were examined by light microscopy and selected areas were removed and processed for electron microscopy. The lesions were granulomatous in nature. Each granuloma contained at its centre an intact or ruptured sarcocyst associated with degenerate muscle fibers. Surrounding this was a layer of epithelioid cells and an intense accumulation of inflammatory cells, most of which were eosinophils. The primary cyst wall of the sarcocysts in these granulomas consisted of hair-like protrusions that featured many unusual electron-dense bodies. Sarcocysts with ultrastructures characteristic of Sarcocystis cruzi and Sarcocystis hirsuta were also present in muscle from the same animal, but these sarcocysts lacked any associated cellular responses. The eosinophilic myositis in this case appeared to be associated with sarcocystosis of an unknown species. Possibly, the inflammatory reaction was due to the host-parasite interaction in an unusual host.     

Occurrence of Piscirickettsiosis-like syndrome in tilapia in the continental United States.

From 2001 to 2003, tilapia (Oreochromis sp.) farms in Florida, California, and South Carolina experienced epizootics of a systemic disease causing mortality. The fish exhibited lethargy, occasional exophthalmia, and skin petechia. The gills were often necrotic, with a patchy white and red appearance. Grossly, the spleen and kidneys were granular with whitish irregular nodules throughout. Granulomatous infiltrates were observed in kidney, spleen, testes, and ovary tissues, but not in the liver. The granulomas contained pleomorphic coccoid bacteria, measuring 0.57 +/- 0.1 x 0.8 +/- 0.2 microm, that were Giemsa-positive, acid-fast-negative, and Gram-negative. The bacteria had a double cell wall, variable electron-dense and -lucent areas, and were present in the cytoplasm and within phagolysosomes. The syndrome was associated with cold stress and poor water conditions. These findings are consistent with an infectious process caused by a Piscirickettsia-like bacterium described previously in tilapia in Taiwan and Hawaii. This report involves the first identified cases of a piscirickettsiosis-like syndrome affecting tilapia in the continental United States.     

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.     

Demonstration of colonies of Anaplasma marginale in the midgut of Rhipicephalus simus

Rhipicephalus simus nymphs were allowed to feed on a cow experimentally infected with the BW-strain of Anaplasma marginale from Republic of South Africa, and they were studied as adults. Colonies were demonstrated by light microscopy in midgut epithelial cells of adult ticks that were unfed (as adults), incubated, or prefed for 72 hours on a cow. The colonies occurred in 5 different morphologic types (1 to 5) that were similar to those described previously for a Virginia isolate of A marginale in Dermacentor andersoni. The colony density (number of colonies/0.001 mm2 midgut tissue examined) ranged from 0 to 2.0 and was highest in unfed ticks that were not incubated (mean 0.566). Colonies observed by light microscopy were sectioned for study with the electron microscope. The colonies contained both electron-dense forms and reticulated forms. The organisms in type 2 and 3 colonies appeared to be attached to one another, and those in type 4 and 5 colonies occurred separately. Small particles were seen within the limiting membrane of some organisms. A few colonies contained a dense matrix and were surrounded by many small electron-dense particles.     

Feline cystic thymoma: a clinicopathologic,immunohistologic, and electron microscopic study of 14 cases

Cystic thymoma was diagnosed in 14 cats in a period of 6 years. The most common clinical sign was laboured breathing. The tumours were characterized by various-sized cystic spaces with central vessels. The epithelial cells varied from oval to spindle to polygonal cells enclosing cystic spaces or in pure epithelial cell components. The nuclei of the neoplastic cells had scattered chromatin and small nucleoli. The cytoplasm was pale eosinophilic. The concentration of mature lymphocytes varied from area to area with rare germinal centres. Immunohistochemically, the epithelial cells stained only with AE(1)/AE(3). The central vessels were positive for vimentin, smooth muscle actin, and factor VIII antigen. Electron microscopy revealed that the cyst walls were lined by epithelial cells that were joined by desmosomes, and the walls were well separated from the cystic spaces by a well-defined basement membrane. The neoplastic epithelial cells contained scattered tonofilaments. Three of the cats had metastasis to the lymph nodes and lungs. Two novel cases of ectopic cystic thymoma have also been described. Results of this study reveal that cystic thymoma is uncommon in cats, and that the histomorphologic, immunohistochemical, and electron microscopic features are similar to those of cystic thymoma in humans.     

Light and scanning electron microscopic studies of the extrahepatic bile duct of sheep with experimentally induced Fasciola hepatica infection.

Changes in the common bile duct that contained adult Fasciola hepatica of sheep were evaluated by light (LM) and scanning electron microscopy (SEM). Nine ewes were inoculated with F hepatica metacercariae and necropsied 18 weeks after inoculation. The proximal portion of the common bile duct (CBD) that contained adult flukes was recovered and examined by LM and SEM. The CBD from 2 noninoculated ewes were used for control. On gross examination, CBD were markedly large because of the adult flukes, which were free in the lumen of the ducts. Extensive hemorrhage was not found either in intrahepatic or in extrahepatic bile ducts of any sheep. Histologic examination revealed changes, such as villous hyperplasia and hypertrophy of the epithelium; cell infiltration, predominantly with eosinophils or macrophages; and arterial intimal proliferation. By SEM, the epithelial surface of the CBD appeared intact. Villous hyperplasia and hypertrophy of the epithelium observed by LM was clearly seen by SEM. Epithelial damage, seen as small areas of denuded surface by LM and SEM, was confined to a few areas of the mucosa. Lack of extensive hemorrhage and confined epithelial damage were evaluated relative to the mode of feeding of adult flukes.     

Ultrastructural characterization of gills in juveniles of the Argentinian Silverside, Odontesthes bonariensis (Valenciennes, 1835) (Teleostei: Atheriniformes)

An ultrastructural study was performed on the gills of juvenile Argentinian silverside, Odontesthes bonariensis. The gills are composed of two sets of four holobranchs and, in turn, each holobranch consists of a gill arch and two rows of caudolaterally projecting branchial filaments. From the dorsal and ventral surfaces of each filament, branchial lamellae radiate out as foldings of the epithelial layer. Gill rakers are present on each of the gill arches, on the anteromedial side of the arch opposite to the filaments. Gill rakers, gill arches and branchial filaments are covered by a stratified epithelium, whereas branchial lamellae essentially consist of a thin epithelial envelope containing capillaries. In the stratified epithelium, mucous cells, rodlet cells, granular cells, pavement epithelial cells and mitochondria-rich cells are identified. The thin epithelium that lines the lamellae comprises two cell types, outer and inner epithelial cells, and the capillary walls on the inside of the epithelial envelope are defined by pillar cells. The ultrastructure of all these cell types is described and our findings are discussed in light of the existing data on fish gill morphology. In the gills of juvenile Argentinian silverside is of particular interest the characteristics showed by mitochondria-rich cells, such as their arrangement in clusters of 2-3 cells and their small and depressed surface in contact with the aquatic milieu, features which strongly resemble those of euryhaline species.     

Cultivation of bovine adenovirus type 3 in tracheal organ cultures

A growth curve study of the WBR-1 strain of bovine adenovirus type 3 (BAV-3) in bovine foetal tracheal organ cultures showed that the virus replicated at high titres, was mainly cell-associated and persisted in the cultures. The histopathological changes were characterized by the progressive swelling, rounding and shedding of the epithelial cells which often showed intranuclear inclusions. The indirect immunoperoxidase reaction demonstrated that the virus replicated only in the epithelial cells of the organ cultures. Ultrastructural studies demonstrated that BAV-3 induced three different types of nuclear inclusions in the epithelial cells. A network of highly electron-dense, finely granular material was interspersed with patches of granular material of low electron density in the centre of the infected nuclei. In some nuclei, there were also some small circular very dense bodies near the central inclusion. The virions were either dispersed or packed into crystals, and they were released into the cytoplasm through breaks in the nuclear membrane. These ultrastructural changes were comparable with those induced by other oncogenic adnoviruses.     

Epizootic outbreaks of gizzard erosion associated with adenovirus infection in chickens

Two outbreaks of gizzard erosion in slaughtered broiler chickens in Japan were examined pathologically and microbiologically. The prevalences of such lesions were 9%-11% and 4%-50% in the affected flocks. Affected chickens had no clinical signs. Group I fowl adenovirus (FAV) serotype 1 was isolated from gizzard lesions. Histologically, gizzard mucosa were necrotic. Intranuclear inclusion bodies were seen in the enlarged nuclei of degenerating epithelial cells of the gizzard. The keratinoid layer in the erosion was edematous and desquamated and contained degenerative cells. Moderate to marked inflammatory cell infiltration was observed in the lamina propria and perivascular connective tissue in the submucosa and muscle layer. Immunohistochemical staining showed evidence of FAV antigens in the intranuclear inclusion bodies within degenerating epithelial cells. Ultrastructurally, numerous viral particles were demonstrated in the inclusions.     

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.     

Inclusion body hepatitis caused by fowl adenovirus in broiler chickens in Japan, 2009-2010

From January 2009 to June 2010, many broiler chicks suddenly died without clinical signs. The mortality rates were from 1.2% to 17.0% in affected flocks. Inclusion body hepatitis (IBH) was detected in 13 prefectures (northern, eastern, western, and southern areas) in Japan. The livers were enlarged and pale. The bursa of Fabricius and thymus had not atrophied. Multifocal necroses of hepatocytes with basophilic intranuclear inclusions were seen in the liver. Eosinophilic intranuclear inclusion bodies in hepatocytes were rare. Focal necrosis of acinar cells with basophilic intranuclear inclusions was found in the pancreas. Basophilic intranuclear inclusion bodies were detected in intact surface epithelial cells of gizzard and epithelial cells of the small intestine. The intranuclear inclusions of liver, pancreas, gizzard, and small intestine were stained positively for immunohistochemistry of fowl adenovirus (FAV) antigen. Ultrastructurally, basophilic intranuclear inclusions consisted of viral particles approximately 70 nm in diameter and arranged in a crystalline array. FAV was isolated from the liver of chickens affected with IBH. The serotype of most isolates was 2. This study suggests that IBH produced by FAV is epidemic in broiler chicks in Japan and that the present cases occurred as the primary disease without the association of infectious bursal disease virus or chicken anemia virus.     

Ultrastructural pathology of Bordetella avium infection in turkeys

One-day-old turkeys were infected intranasally with Bordetella avium, and tracheas were examined by scanning and transmission electron microscopy at 1 to 5 weeks post-inoculation (PI). The predominant ultrastructural lesions were progressive loss of ciliated epithelium with replacement by nonciliated cells, bacterial colonization of ciliated cells, membrane-bound crystalline inclusions in cytoplasma of epithelial cells, depletion of mucous granules, and distortion of tracheal rings and the mucosal surface. Tracheal surface exudates consisted of mucus, necrotic cells, heterophils, and fibrin. Ciliated cells were replaced by immature cuboidal cells characterized by abundant rough endoplasmic reticulum with small numbers of electron-dense mucous granules in the apical cytoplasm. Bacterial surfaces were rough and contained numerous pleomorphic, knob-like structures, 20-50 nm in diameter. Other changes included enlarged mucosal gland openings, cell extrusion marks, pleomorphic microvilli, and cells with small numbers of short cilia.     

Ultrastructural Studies of Paratuberculosis (Johne’s Disease) in Goats

Biopsy material of the ileum and corresponding mesenteric lymph nodes from 10 naturally infected goats was studied. In ileum a loss of epithelial cells and infiltration of epitheloid cells, macrophages and a low number of lymphocytes, plasma cells and occasional eosinophils were seen. Ultrastructurally, the epithelial cells showed degenerative changes. Epitheloid cells were characterized by a large nucleus and a wide cytoplasm rich in free ribosomes.Macrophages had been fixed in the process of engulfing bacteria or contained bacteria in phagosomes and phagolysosomes. Large phagolysosomes were common. In macrophages with many or large phagolysosomes, few or no lysosomes were observed. Degenerative changes were seen in macrophages containing many bacteria. Degenerative changes of capillary endothelium were observed. The intercellular spaces were distended by oedema and contained cell debris.The mesenteric lymph nodes were infiltrated with epitheloid cells and macrophages. The ultrastructural picture of these cells was almost identical to that of the ileum.The differences between the changes found in naturally infected and experimentally infected animals are discussed. It is concluded that the mode of infection, the number of bacteria to which the animal is exposed and the intervals between events of exposure may play a role.     

Development of Anaplasma marginale in male Dermacentor andersoni transferred from parasitemic to susceptible cattle.

The development and transmission of Anaplasma marginale was studied in Dermacentor andersoni males. Laboratory-reared male D andersoni were allowed to feed for 7 days on a calf with ascending A marginale parasitemia. The ticks were then held in a humidity chamber for 7 days before being placed on 2 susceptible calves. Anaplasmosis developed in the calves after incubation periods of 24 and 26 days. Gut and salivary glands were collected from ticks on each day of the 23-day experiment and examined with light and electron microscopy. Colonies of A marginale were first observed in midgut epithelial cells on the sixth day of feeding on infected calves, with the highest density of colonies found in gut cells while ticks were between feeding periods. The first colonies contained 1 large dense organism that subsequently gave rise to many reticulated organisms. Initially, these smaller organisms were electron-lucent and then became electron-dense. On the fifth day after ticks were transferred to susceptible calves for feeding, A marginale colonies were found in muscle cells on the hemocoel side of the gut basement membrane. A final site for development of A marginale was the salivary glands. Colonies were first seen in acinar cells on the first day that ticks fed on susceptible calves, with the highest percentage of infected host cells observed on days 7 to 9 of that feeding. Organisms within these colonies were initially electron-lucent, but became electron-dense.     

The Structure of the Harderian Gland of the Guinea Fowl at Embryonic and Post Embryonic Stages

The Harderian gland of the guinea fowl is a bright, pink and relatively large orbital organ situated at the ventromedial aspect of the orbit. It possesses a single duct that has its exit on the convex medial surface. The outline is irregular with its caudal half narrower than the rostral half, and possessing a slight constriction about the mid point. Histologically, the gland had been outlined with the existence of a large contorted lumen by day 18 of incubation. The surface epithelial lining showed two layers of cells all through and lymphocytes were also present. By day 19 of incubation, certain zones of the surface epithelium had become pseudostratified and some of these cells contained vacuoles indicative of secretory materials within their cytoplasm. By day 21, few definitive acini with lumina had been observed and at day 23, the epithelium had assumed only a single layer of cells that were tall columnar cells, except at the junctions of the folds. By day 24, the tunica propria seemed to have completely disappeared with the acini occupying every available space. Plasma cells were seen three days after hatching.