Histopathological, ultrastructural and molecular phylogenetic analysis of a novel microsporidium in a loggerhead sea turtle Caretta caretta.

Microsporidial spores were identified in the musculature of a loggerhead sea turtle Caretta caretta found dead on the shore in New Brunswick, Canada. Gastroenteritis was diagnosed on gross postmortem examination, with no gross abnormalities detected in the skeletal muscle. Histologically, the microsporidial spores were associated with inflammation and muscular necrosis and measured 1.1-1.7 × 2.2-3.4 µm. Spores were typically identified within sporophorous vesicles and, less often, in sporophorocysts and were weakly Gram positive, had punctate PAS staining, and were occasionally strongly acid-fast. Ultrastructural characteristics included 7-10 polar filament coils and other standard features of microsporidial spores. PCR for the microsporidial small subunit rRNA gene sequence was performed on DNA extracted from the muscle and small intestine, and the resulting amplicon was sequenced and queried against published microsporidial genomes. DNA sequences shared 98.2-99.8% sequence identity to Clade III of the Marinosporidia. This is the first report of a microsporidial infection contributing to the mortality of a sea turtle.

Ultrastructure of trichocysts in Hematodinium spp. infecting Atlantic snow crab, Chionoecetes opilio.

Trichocyst morphology and development were explored using transmission electron microscopy in Hematodinium spp. isolated directly from Atlantic snow crab (Chionoecetes opilio) hemolymph and from in vitro cultures. Appearance of trichocysts defines the initiation of a morphological transition in the parasites life cycle from vegetative stage to the transmission stage. Trichocysts within sporonts were found in distinct clusters near the nucleus in close apposition to the Golgi. As cells transitioned to more mature dinospores however, trichocysts were found randomly distributed throughout the cytoplasm. Clusters contained both primordial and maturing trichocysts at various stages indicating an asynchronous development. The random distribution of mature trichocysts suggests deployment to the cell membrane for future extrusion. Mature trichocysts of Hematodinium spp. appeared structurally similar to trichocysts from photosynthetic dinoflagellates. Hematodinium spp. trichocysts differed by the presence of peripheral tubules associated with novel cuboidal appendages in the apical region rather than a network of central electron dense fibres as found in photosynthetic dinoflagellates. Additionally, the trichocyst membrane of Hematodinium spp. was in close apposition to the square crystalline core. Trichocyst expulsion was not observed during our study which along with features of development and maturation within Hematodinium life stages should provide insight into proposed roles in host attachment or defense that could further our understanding of the mechanisms of pathogenesis and transmission of the parasite.

Histochemical and ultrastructural analysis of pathology and cell responses in gills of channel catfish affected with proliferative gill disease.

Pond-reared channel catfish Ictalurus punctatus with proliferative gill disease (PGD), caused by the myxozoan parasite Henneguya spp., were examined with light and transmission electron microscopy to better characterize the inflammatory response during infection. The early stages of disease are characterized by the destruction of collagen in the matrix of the gill filament cartilage causing weakness and breaks within the gill filaments. These early lesions lacked a notable inflammatory response around the disrupted cartilage, a chondrocyte response was not apparent, and the parasite was not present, suggesting that the cartilage breaks occur prior to inflammation and arrival of the parasite in the gill. In later lesions, a significant inflammatory response was generated in areas of disrupted cartilage, and the inflammatory infiltrate was composed of a mixed population of granulocytes including neutrophils and cells that resembled eosinophils. The majority of eosinophil-like cells demonstrated evidence of degranulation. Trophozoites of Henneguya spp. were surrounded by a uniform population of cells believed to be neutrophils. The granulocytes were infiltrated within the dense collagen layer of the gill filament cartilage and often appeared within chondrocyte lacunae in place of the chondrocyte. The gill lamellae adjacent to the lesions were fused and contained an inflammatory infiltrate containing granulocytes and cells with pericentriolar granules that resembled previous descriptions of Langerhans-like cells. These cells were abundant within damaged lamellar epithelium, but were only rarely found within the gill filament. Lesions that appeared to be recovering lacked the dense collagenous layer around the cartilage and contained hyperplastic and hypertrophic chondrocytes that formed a callus. Other chondrocytes in the lesions had ultrastructural features indicative of cell death.

Ultrastructural and secretory heterogeneity of fa/fa (Zucker) rat islets.

Many previous studies of obese rodents documented biochemical changes in pancreatic islets that contribute to hyperinsulinemia in vivo. Those studies used heterogeneous populations of islets, although the size of islets from obese rats ranges from < 100 to > 500 microm. Here, functional and morphological changes in size-sorted (< 125 and > 250 microm diameter) islets from obese Zucker (fa/fa) rats were correlated. Ultrastructural examination revealed that > 250 microm cultured islets had an increased number of immature secretory granules in the beta cells. The number of degranulated beta cells in > 250 and < 125 microm cultured islets from fa/fa rats was higher than in lean rat islets (33 vs 25%). The glucose EC50 values for cultured islets were 4.64 +/- 0.43, 7.9 +/- 0.70 and 7.29 +/- 1.64 mmol.l(-1) for > 250 microm, < 125 microm, and lean groups, respectively. Inhibition of insulin secretion by 10 mmol.l(-1) mannoheptulose was reduced by 50% in > 250 microm islets compared with small islets. Studies of individual beta cells by reverse hemolytic plaque assay revealed 3-fold more cells from > 250 microm islets were stimulated by 1.4 mmol.l(-1) glucose than cells from < 125 microm islets. We conclude that functional defects in mixed size populations of islets from fa/fa rats are mainly due to alterations in the large islets, whereas smaller islets have relatively normal function. Exposure to high glucose exacerbates morphological and functional differences of large islets, which could have important implications in the transition to noninsulin-dependent diabetes when beta cell insulin production is unable to compensate for hyperglycemia.

Ultrastructural changes in the lungs of neonatal rats intratracheally inoculated with meconium.

Meconium aspiration syndrome has been for many years an important cause of neonatal respiratory distress in newborn babies and sporadically reported in animals. This investigation was designed to study the ultrastructural and morphometric changes in the lungs of neonatal rats following the intratracheal inoculation of meconium. Seven-day-old Fischer-344 rats (n = 24) were randomly allocated in two groups. One group was intratracheally inoculated with saline solution and the second group received homologous meconium. Neonates were euthanatized at 1, 3 and 7 postinoculation days (PID) and lungs were examined by light and electron microscopy. Saline solution did not induce any ultrastructural changes in the lung. In contrast, meconium induced deciliation, recruitment of neutrophils and pulmonary alveolar macrophages to the bronchoalveolar space, intravascular sequestration of neutrophils and aggregation of platelets at PID 1 and 3. Other ultrastructural changes at PID 1 and 3 included interstitial edema and escape of red cells and fibrin into the alveolar space and interstitium. Interstitial edema and sequestration of neutrophils were responsible for the significant increase in thickness of alveolar septa. At PID 7 there was hyperplasia and enlargement of type II pneumocytes as well as interstitial proliferation of mesenchymal cells with intra-alveolar fibrosis. It was concluded that intratracheal inoculation of meconium in neonatal rats induces acute ultrastructural changes followed by a reparative response.

Microscopic changes induced by the intratracheal inoculation of amniotic fluid and meconium in the lung of neonatal rats.

Meconium aspiration syndrome is a major contributor to neonatal respiratory distress in infants and it has been sporadically recognized in neonatal animals. This investigation was designed to study the short and long term effects of meconium and amniotic fluid in the lungs of neonatal rats. Seven-day-old rats (n = 123) divided in three groups were intratracheally inoculated with saline solution, amniotic fluid or meconium. Rats were euthanatized on 1, 3, 7, 14, 28, 56 and 112 postinoculation days (PID) and the lungs were examined by light microscopy. Saline solution did not induce any change while amniotic fluid elicited only a mild foreign body response which disappeared by PID 14. In contrast, meconium induced an exudative alveolitis characterized by recruitment of neutrophilsn in the bronchoalveolar spaces. Meconium also induced atelectasis, hyperinflation and thickening of alveolar septa all of which had disappeared by PID 14. Starting at PID 7, neutrophils were progressively replaced by macrophages, giant cells, and some fibroblasts. There were sporadic foci of mineralization starting at PID 14 and lasting up to PID 112. Some mineralized foci became lined with cuboidal epithelial cells at PID 28. Meconium was slowly degraded but still evident by PID 112. It was concluded that inoculation of meconium in neonatal rats induces acute microscopic changes typical of meconium aspiration syndrome. The long term lesions induced by meconium consisted of persistent multifocal histiocytic alveolitis and bronchiolitis reaction with occasional foci of calcification.

Pathology associated with an aquareovirus in captive juvenile Atlantic halibut Hippoglossus hippoglossus and an experimental treatment strategy for a concurrent bacterial infection.

A large-scale mortality of larval and juvenile halibut Hippoglossus hippoglossus occurred at a semi-commercial halibut farm in Atlantic Canada. Investigation of the cause revealed aquareovirus particles in necrotic liver tissue of affected fish. Cytopathic effect on CHSE-214 cell lines occurred from all fish cultured for viruses, and the viral morphology of the particles in culture was consistent with that observed in necrotic host tissue. The virus was placed in the family of Reoviridae, genus Aquareovirus based on morphology and RT-PCR results. Multifocal hepatocellular necrosis was a consistent finding in all fish as well as acute necrosis of proximal renal tubules. Concurrent bacterial infections were present in some specimens. Fish experimentally treated with oxytetracycline or a combination of oxytetracycline and chloramine-T had a significantly lower mortality rate than untreated fish. Fish treated with chloramine-T alone had a significantly elevated mortality rate compared to controls. Despite supportive medical therapy, mortality levels in treated and untreated groups remained elevated, supporting the hypothesis that the primary pathogen was of viral origin. This is the first report of elevated mortalities in Atlantic halibut associated with an aquareovirus.

Isolation and identification of infectious salmon anaemia virus (ISAV) from Coho salmon in Chile.

The isolation of infectious salmon anaemia virus (ISAV) from asymptomatic wild fish species including wild salmon, sea trout and eel established that wild fish can be a reservoir of ISAV for farmed Atlantic salmon. This report characterizes the biological properties of ISAV isolated from a disease outbreak in farmed Coho salmon in Chile and compares it with ISAV isolated from farmed Atlantic salmon in Canada and Europe. The virus that was isolated from Coho salmon tissues was initially detected with ISAV-specific RT-PCR (reverse transcription-polymerase chain reaction). The ability of the virus to grow in cell culture was poor, as cytopathology was not always conspicuous and isolation required passage in the presence of trypsin. Virus replication in cell culture was detected by RT-PCR and IFAT (indirect fluorescent antibody test), and the virus morphology was confirmed by positive staining electron microscopy. Further analysis of the Chilean virus revealed similarities to Canadian ISAV isolates in their ability to grow in the CHSE-214 cell line and in viral protein profile. Sequence analysis of genome segment 2, which encodes the viral RNA polymerase PB1, and segment 8, which encodes the nonstructural proteins NS1 and NS2, showed the Chilean virus to be very similar to Canadian strains of ISAV. This high sequence similarity of ISAV strains of geographically distinct origins illustrates the highly conserved nature of ISAV proteins PB1, NS1 and NS2 of ISAV. It is noteworthy that ISAV was associated with disease outbreaks in farmed Coho salmon in Chile without corresponding clinical disease in farmed Atlantic salmon. This outbreak, which produced high mortality in Coho salmon due to ISAV, is unique and may represent the introduction of the virus to a native wild fish population or a new strain of ISAV.

Ultrastructural study of the late stages of Loma salmonae development in the gills of experimentally infected rainbow trout.

The main objective of this investigation was to examine the ultrastructural features of gills from rainbow trout experimentally infected with Loma salmonae to determine the morphological events that occur during the late stages of development of this parasite. Peripheral distribution of the mature parasites inside round xenomas was observed at weeks 5 and 6 postexposure (PE), but eventually the parasite occupied the entire xenoma. Degenerative changes were observed only in immature parasites at week 7 PE, and eventually an inflammatory reaction with a cellular infiltration was directed against mature spores. Round, flattened, and irregular shaped xenomas were observed at week 8 PE. The round xenomas showed a severe inflammatory response with disintegration of the xenoma membrane. This event was accompanied by eversion of polar tubes within the attacked xenoma and by the simultaneous presence of 2 tubular appendages, the type I and II tubules. Flattened xenomas were observed below the endothelium of gill lamella arteries. The irregular xenomas were located in the connective tissue of the gill filament and showed multiple projections occupied by spores. Both flattened and irregular xenomas showed no evidence of inflammatory reaction. An earlier proposed hypothesis is expanded to explain how L. salmonae is implanted beneath lamellar endothelium and within filament connective tissue.

Ultrastructural study of the early development and localization of Loma salmonae in the gills of experimentally infected rainbow trout.

The early ultrastructural stages of Loma salmonae were studied in the gills of experimentally infected rainbow trout. No parasitic stages were identified during the first 2 wk of the infection. By week 3 postexposure (PE), uninucleate and binucleate meronts were recognized within host cells (no xenomas) associated with the capillary channels of secondary lamellae and lamellar arteries. An inflammatory reaction was absent. In secondary lamellae, infected cells were isolated from the capillary lumen, and some were recognized as pillar cells. In lamellar arteries, infected cells were localized beneath the endothelium and not in the lumen. Inflammatory reaction and destruction of parasites inside blood cells in the lumen of secondary lamellae were observed by week 4 PE. Three hypotheses, i.e., isolation, internalization, and evasion, are proposed to explain the localization of the infected cells in the gills. It is concluded that meronts are the earliest parasitic stage observed by week 3 PE, pillar cells are secondarily infected by phagocytosis of infected cells in the blood, endothelial cells of gills are not infected, and inflammatory response to the parasite starts by week 4 PE.

Multicentric undifferentiated spindle-cell sarcoma with virus-like particles in an aged dairy cow.

A 10-year-old Holstein dairy cow was slaughtered because of weight loss and ataxia. In addition to a neoplastic mass in the animal's forehead, there was extensive neoplastic infiltration of the skeleton and liver. Other viscera were spared. Tumours were composed of sheets and interlacing fascicles of poorly differentiated, vimentin-positive cells in a fibrillar matrix. Intracytoplasmic virus-like particles, 80 nm in diameter, with a central electron-dense core were found in many neoplastic cells. This neoplasm had an unusual predilection for bone. The significance of the virus-like particles requires further investigation.

Establishment of conditions for the detection of bovine herpesvirus-1 by polymerase chain reaction using primers in the thymidine kinase region.

Polymerase chain reaction (PCR) for detection of bovine herpesvirus-1 (BHV-1) was developed and optimized using 22 bp sense and 20 bp antisense primers in the thymidine kinase (TK) coding region. The amplification product is 183 bp long. The PCR optimization was done using BHV-1 tissue culture supernate (BHV-1TCS), concentrated BHV-1 tissue culture supernate (cBHV-1TCS) and sucrose gradient purified BHV-1 (pBHV-1). The sensitivity of four methods of sample preparation which are standard DNA extraction, modified proteinase K (PK) digestion, GeneReleaserTM + 34 cycles or + 44 cycles, and boiling were compared with virus isolation (VI) using BHV-1TCS. The incorporation of 10% glycerol in the reaction mixture, the incubation in PK for 18 hours and predenaturation of samples and cooling in ice prior to PCR were essential for the amplification of BHV-1 DNA for samples prepared by standard DNA extraction and modified PK digestion. The preparation of samples by Gene-ReleaserTM, a proprietary nucleic acid releasing cocktail, showed 10 to 1,000-fold increase in sensitivity compared to standard DNA extraction and modified PK digestion. No amplification was observed in samples prepared by boiling. The sample preparation of BHV-1 LA strain by GeneReleaserTM showed sensitivity equivalent to virus isolation. The BHV-1 TK PCR using GeneReleaserTM has a detection limit of 1 picogram and 10 fentograms of purified BHV-1 DNA using ethidium bromide stained gel and Southern blot hybridization, respectively. It could detect viral DNA in 1,000 infected cells in a total suspension of 10,000 cells using either ethidium bromide stained gel or Southern blot hybridization.

Formation of virus-like particles when the polyprotein gene (segment A) of infectious bursal disease virus is expressed in insect cells.

The baculovirus expression vector system was used to examine the expression of the full-length infectious bursal disease virus (IBDV) segment A cDNA, which encodes the structural proteins in a polyprotein precursor that is autocatalytically cleaved to VPX, VP3, and VP4. No VP2 was observed in lysates of recombinant baculovirus infected cells indicating the lack of processing of VPX to VP2 in this system. Virus-like particles (VLP) were purified from the infected insect cells, and on negative staining electron microscopy, looked very similar to authentic IBDV particles in shape and size, suggesting that processing of VPX to VP2 is not necessary for capsid assembly.

Amplification of strains of bovine herpesvirus 1 by use of polymerase chain reaction with primers in the thymidine kinase region.

A primer pair was designed from the published nucleotide sequence of the coding region of the bovine herpesvirus 1 (BHV-1) thymidine kinase (tk) gene for use in detection of the virus by use of polymerase chain reaction (PCR) amplification of 12 BHV-1 strains (3 ATCC and 9 local isolates). A tk deletion mutant BHV-1, and 2 BHV-4 strains from ATCC were used as negative controls. One strain each of feline herpes-virus, equine herpesvirus, and bovine adenovirus, and 2 noninoculated bovine cultured cells--bovine fetal testis and Madin-Darby bovine kidney--also were examined to verify specificity of the primers. A PCR product, 183 bp long, was detected by ethidium bromide staining after agarose gel electrophoresis, when purified DNA from cell cultures infected with BHV-1 strain LA was used as template. Specificity of the PCR product was confirmed by restriction digestion with Sac II enzyme and Southern blot hybridization. Amplification was detected by ethidium bromide staining of agarose gels and/or Southern blot hybridization with the radiolabeled PCR product of the LA strain in similarly prepared DNA templates of 5 other BHV-1 strains, 2 obtained from ATCC and 3 of the 9 local isolates. In a modified PCR protocol, using virus suspensions treated with a nucleic acid-releasing cocktail, substantial amplification was obtained for the 3 BHV-1 strains from ATCC and for all 9 local bovine herpesvirus field isolates.(ABSTRACT TRUNCATED AT 250 WORDS)

Proliferative pododermatitis associated with virus-like particles in a northern gannet.

Small multifocal lesions of proliferative pododermatitis were observed in an emaciated adult male northern gannet (Morus bassanus). Ultrastructurally, these lesions were associated with numerous virus-like particles with a size and morphology suggestive of Papovaviridae. DNA in situ hybridization with probes for avian polyomaviral and papillomaviral nucleic acid and an immunohistochemical test for the presence of papillomaviral antigen failed to identify this virus further. To our knowledge, papovavirus-like particles have not been recognized previously in this avian species.

Ultrastructural examination of the host cellular response in the gills of Atlantic salmon, Salmo salar, with amoebic gill disease.

Gills from Atlantic salmon with experimentally induced amoebic gill disease (Neoparamoeba spp.) were examined with transmission electron microscopy to assess pathology and host-cell responses. Amoebae were found either on the surface epithelium or with pseudopodia extending deeply into invaginations of epithelial cells. The amoebae had various densities along the plasma membrane and contained electron-dense deposits within their cytoplasm. Surface epithelial cells sloughed from the gills and had features consistent with apoptosis, including rounded shape, loss of surface microridges, and hypercondensation of nuclear chromatin. Affected areas of gills had fusion of secondary lamellae with interlamellar spaces occupied by mitotic epithelial cells and eosinophils. Eosinophils contained abundant fusiform-shaped granules that measured approximately 1 microm long and 360 nm wide. The granule consisted of an electron-dense matrix with a central inclusion that was less electron-dense, consisting of particulate and fibrillar material. In many instances, the central inclusion appeared empty and 90% of the eosinophils had morphology suggestive of piecemeal degranulation. Also observed within affected areas were a few neutrophils, mucous cells releasing mucus, and a small number of dendritic-like cells.

Morphological characterization and notes on the life cycle of a newly discovered variant of Loma salmonae (Putz, Hoffman & Dunbar) from a natural infection of chinook salmon, Oncorhynchus tshawytscha (Walbaum).

Two variants of Loma salmonae occur in net-pen reared chinook salmon, Oncorhynchus tshawytscha. The typical variant (OA) has a host specificity for salmonids of the genus Oncorhynchus whereas the atypical variant (SV) has a host specificity for brook trout, Salvelinus fontinalis, and in this study, the ultrastructure of the two are compared. In fish at 8 weeks post-exposure xenomas of the SV variant have a very high proportion of mature spores compared with other developmental stages, while in xenomas of the OA variant there are fewer spores and many other developmental stages. Spores of the SV variant had up to 20 turns of their polar tube whereas those of the OA variant only had 17. Furthermore, the spores of the SV variant were significantly larger than those of the OA variant. The sporophorous vesicle for both variants appears to form around a sporogonial plasmodia, which results in many spores developing within the vesicle.