Characterization of a new densovirus infecting the German cockroach, Blattella germanica.

A new DNA virus (Parvoviridae: Densovirinae, Densovirus) was isolated and purified from descendants of field-collected German cockroaches, Blattella germanica. Viral DNA and cockroach tissues infected with B. germanica densovirus (BgDNV) were examined by electron microscopy. Virus particles, about 20 nm in diameter, were observed both in the nucleus and in the cytoplasm of infected cells. Virus DNA proved to be a linear molecule of about 1.2 microm in length. BgDNV isolated from infected cockroaches infected successfully and could be maintained in BGE-2, a B. germanica cell line. The complete BgDNV genome was sequenced and analysed. Five open reading frames (ORFs) were detected in the 5335 nt sequence: two ORFS that were on one DNA strand encoded structural capsid proteins (69.7 and 24.8 kDa) and three ORFs that were on the other strand encoded non-structural proteins (60.2, 30.3 and 25.9 kDa). Three putative promoters and polyadenylation signals were identified. Structural analysis of the inverted terminal repeats revealed the presence of extended palindromes. The genome structure of BgDNV was compared with that of other members of the family Parvoviridae; the predicted amino acid sequences were aligned and subjected to phylogenetic analyses.

Rafts and synapses in the spatial organization of immune cell signaling receptors.

The multichain immune recognition receptors (MIRRs), including the T cell and B cell antigen receptors and the high affinity receptor for IgE, play an important role in immune cell signaling. The MIRRs have no inherent kinase activity, but rather associate with members of the Src-family kinases to initiate signaling. Although a great deal is understood about the biochemical cascades triggered by MIRRs, the mechanism by which signaling is initiated was not known. The evidence now indicates that the Src-family kinases are concentrated in cholesterol- and sphingolipid-rich membrane microdomains, termed lipid rafts, that exclude the MIRRs. Upon ligand-induced crosslinking the MIRRs translocate into rafts where they are phosphorylated. The MIRRs subsequently form highly ordered, polarized structures termed immunological synapses that provide for prolonged signaling. An understanding of the biochemical composition of rafts and synapses and the mechanisms by which these form should lend insight into the regulation of immune cell activation.

Floating the raft hypothesis: the roles of lipid rafts in B cell antigen receptor function.

The initiation of antibody responses to foreign antigens requires that B cells receive and integrate a variety of signals through an array of cell surface receptors including the B cell antigen receptor (BCR) as well as a number of essential coreceptors. Recent evidence indicates that cholesterol-rich plasma membrane microdomains, referred to here as lipid rafts, serve as platforms for BCR signaling and trafficking in B cells. The existence of rafts suggests a previously unappreciated level of organization at the B cell surface that may explain, at least in part, how BCR signaling is coordinated. Here the current evidence that lipid rafts play a key role in B cell responses is reviewed.

Ultrastructural morphometry of bovine blastocysts produced in vivo or in vitro.

The objective of this study was to compare the ultrastructure of bovine blastocysts produced in vivo or in vitro by using morphometric analysis. Blastocysts produced in vivo (multiple ovulations, MO) were obtained from superovulated Holstein cows. For blastocysts produced in vitro, cumulus-oocyte complexes aspirated from ovaries of Holstein cows were matured and fertilized in vitro. At 20 h postinsemination (hpi), zygotes were distributed into one of three culture media: 1) IVPS (in vitro produced with serum): TCM-199 + 10% estrous cow serum (ECS); 2) IVPSR (in vitro produced with serum restriction): TCM-199 + 1% BSA until 72 hpi, followed by TCM-199 + 10% ECS from 72 to 168 hpi; and 3) mSOF (modified synthetic oviductal fluid): mSOF + 0.6% BSA. At 168 hpi, six or seven grade 1 blastocysts from each of the four treatments (MO, IVPS, IVPSR, and mSOF) were fixed and prepared for transmission electron microscopy. Random micrographs of each blastocyst were used to determine the volume density of cellular components. Overall, as blastocysts progressed in development, the volume densities of cytoplasm and intercellular space decreased (P < 0.05) and the volume densities of mature mitochondria, nuclei, blastocoele, and apoptotic bodies increased (P < 0.05). Across treatments, the proportional volumes of nuclei and inclusion bodies were increased in inner cell mass cells compared with trophectoderm cells for mid- and expanded blastocysts. For blastocysts produced in vitro, the volume density of mitochondria was decreased (P < 0.05) as compared with that of blastocycts produced in vivo. The proportional volume of vacuoles was increased (P < 0.05) in blastocysts from the mSOF treatment as compared with blastocysts produced in vivo. For mid- and expanded blastocysts from all three in vitro treatments, the volume density of lipid increased (P < 0.05) and the volume density of nuclei decreased (P < 0.05) compared with those of blastocysts produced in vivo. In conclusion, blastocysts produced in vitro possessed deviations in volume densities of organelles associated with cellular metabolism as well as deviations associated with altered embryonic differentiation. However, the specific nature of these deviations varied with the type of culture conditions used for in vitro embryo production.

Floating the raft hypothesis for immune receptors: access to rafts controls receptor signaling and trafficking.

The B cell antigen receptor (BCR) is a member of an important family of multichain immune recognition receptors, which are complexes composed of ligand-binding domains associated with signal-transduction complexes. The signaling components of these receptors have no inherent kinase activity but become tyrosine phosphorylated in their cytoplasmic domains by Src-family kinases upon oligomerization, thus initiating signaling cascades. The BCR is unique in this family in that, in addition to its signaling function, it also serves to deliver antigen to intracellular compartments where the antigen is processed and presented bound to major histocompatibility complex (MHC) class II molecules. Recent evidence indicates that both the signaling and antigen-trafficking functions of the BCR are regulated by cholesterol- and sphingolipid-rich plasma membrane microdomains termed rafts. Indeed, upon oligomerization, the BCR translocates into rafts that concentrate the Src-family kinase Lyn and is subsequently internalized directly from the rafts. Thus, translocation into rafts allows the association of the oligomerized BCR with Lyn and the initiation of both signaling and trafficking. Significantly, the access of the BCR to rafts appears to be controlled by a variety of B lymphocyte co-receptors, as well as factors including the developmental state of the B cell and viral infection. Thus, the translocation of the immune receptors into signaling-competent microdomains may represent a novel mechanism to initiate and regulate immune-cell activation.

Epstein-Barr virus coopts lipid rafts to block the signaling and antigen transport functions of the BCR.

The B cell antigen receptor (BCR) functions to initiate signaling and to internalize antigen for processing from within Lyn kinase-enriched membrane lipid rafts. The signaling function of the BCR is blocked by Epstein-Barr Virus (EBV) latent membrane protein 2A (LMP2A), which is constitutively phosphorylated by Lyn. Here, we show that LMP2A resides in lipid rafts and excludes the BCR from entering rafts by Lyndependent mechanisms, thus blocking both BCR signaling and antigen transport. Mutant LMP2A that permits BCR signaling and raft translocation still blocks antigen trafficking, indicating independent control of these BCR functions. Thus, EBV coopts the lipid rafts to disarm both the signaling and antigen-processing functions of the BCR by independent mechanisms.

High mortality in a large-scale zebrafish colony (Brachydanio rerio Hamilton & Buchanan, 1822) associated with Lecythophora mutabilis (van Beyma) W. Gams & McGinnis.

Zebrafish (Brachydanio rerio) have become an important model system for studying vertebrate embryonic development and gene function through manipulation of genotype and characterization of resultant phenotypes. An established research zebrafish colony without substantial disease problems for more than 7 years of operation began experiencing appreciable mortalities in November of 1997. Young fish (fry), from five to 24 days after hatching, spontaneously developed elongate strands of organic material protruding from the mouth, operculum, and anal pore, leading workers in the laboratory to describe the infected fish as "bearded." Unlike typical freshwater fish fungal infections, the skin surface did not have evidence of fungal colonization. The disease was associated with progressive lethargy, reduced feeding, and subsequent mortality. From 10 to 100% of the fry in a given tank were affected. Initial examination indicated that the biofilm around the head of affected fry consisted of bundles of septate fungal hyphae, large numbers of mixed bacterial populations, and protozoans. Environmental samples of air and water in the laboratory were obtained to ascertain the source of the infective agent and to isolate and identify the fungus. A fungus identified as Lecythophora mutabilis was isolated repeatedly from infected fish and water samples from infected fish tanks, and from the main laboratory water supply tanks, but not from laboratory air. Some biofilm beards on fish were found to consist of relatively pure bacterial populations, and beards on occasional fish examined in the later part of the study consisted of hyphae and spores of the oomycete genus Aphanomyces. Lecythophora mutabilis did not invade tissues; however, elimination of the epizootic correlated with reduction in the number of L. mutabilis conidia in the water following modification of the laboratory water system by use of new filtration and sterilization systems. We conclude that the dense hyphal strands of L. mutabilis composing the predominant biofilm type, along with mixed bacteria and protozoa, contributed to the die-off in young fry by occluding the oral cavity and/or gills, leading to starvation and/or asphyxiation.

The effects of the Epstein-Barr virus latent membrane protein 2A on B cell function.

Epstein-Barr Virus (EBV) infects B-lymphocytes circulating through the oral epithelium and establishes a lifelong latent infection in a subset of mature-memory B cells. In these latently infected B cells, EBV exhibits limited gene expression with the latent membrane protein 2A (LMP2A) being the most consistently detected transcript. This persistent expression, coupled with many studies ofthe function of LMP2A in vitro and invivo, indicates that LMP2A is functioning to control some aspect of viral latency. Establishment and maintenance of viral latency requires exquisite manipulation of normal B cell signaling and function. LMP2A is capable of blocking normal B cell signal transduction in vitro, suggesting that LMP2A may act to regulate lytic activation from latency in vivo. Furthermore, LMP2A is capable of providing B cells with survival signals in the absence of normal BCR signaling. These data show that LMP2A may help EBV-infected cells to persist in vivo. This review discusses the advances that have been made in our understanding of LMP2A and the effects it has on B cell development, activation, and viral latency.

Ultrastructural morphometry of bovine compact morulae produced in vivo or in vitro.

The objective of this study was to compare the ultrastructure of bovine compact morulae produced in vivo or in vitro using morphometric analysis. Compact morulae produced in vivo were obtained from superovulated Holstein cows. Compact morulae produced in vitro were obtained from cumulus-oocyte complexes aspirated from ovaries of Holstein cows. The complexes were matured and fertilized in vitro. At 20 h postinsemination (hpi), zygotes were distributed into 1 of 3 culture media: 1) IVPS (in vitro produced with serum): TCM-199 + 10% estrous cow serum (ECS); 2) IVPSR (in vitro produced with serum restriction): TCM-199 + 1% BSA until 72 hpi followed by TCM-199 + 10% ECS from 72 to 144 hpi; 3) mSOF (modified synthetic oviductal fluid): SOF + 0.6% BSA. At 144 hpi, five grade 1 compact morulae from each of the four treatments were prepared for transmission electron microscopy. The volume density occupied by cellular components was determined by the point-count method using a sampling of seven to nine random micrographs from each compact morula. The volume density of lipid was greater (P < 0.05) in compact morulae from IVPS, IVPSR, and mSOF treatments compared with those produced in vivo. There was a reduced proportional volume of total mitochondria in compact morulae from the IVPS treatment compared with those produced in vivo (P < 0.05). For compact morulae from the IVPS culture treatment, the volume density of vacuoles was greater than that for compact morulae produced in vivo (P < 0.05). The cytoplasmic-to-nuclear ratio for compact morulae from the IVPS treatment was increased (P < 0.05) compared with the ratio for those produced in vivo. In conclusion, compact morulae produced in vitro differed ultrastructurally from those produced in vivo. Compact morulae produced in IVPS culture medium possessed the greatest deviations in cellular ultrastructure.

A role for MHC class II antigen processing in B cell development.

For mature B cells, the encounter with foreign antigen results in the selective expansion of the cells and their differentiation into antibody secreting cells or memory B cells. The response of mature B cells to antigen requires not only antigen binding to and signaling through the B cell antigen receptor (BCR) but also the processing and presentation of the BCR bound antigen to helper T cells. Thus, in mature B cells, the ability to process and present antigen to helper T cells plays a critical role in determining the outcome of antigen encounter. In immature B cells, the binding of antigen results in negative selection of the B cell, inducing apoptosis, anergy or receptor editing. Negative selection of immature B cells requires antigen induced signaling through the BCR, analogous to the signaling function of the BCR in mature B cells. However, the role of class II antigen processing and presentation in immature B cells is less well understood. Current evidence indicates that the ability to process and present antigen bound to the BCR is a late acquisition of developing B cells, suggesting that during negative selection B cells may not present BCR bound antigen and interact with helper T cells. However, the expression of class II molecules is an early acquisition of B cells and recent evidence indicates that the expression of class II molecules early in development is required for the generation of long lived mature B cells. Here we review our current understanding of the processing and presentation of antigen by mature B cells and the role for antigen processing and class II expression during B cell development.

Histologic and immunohistochemical characterization of lens capsular plaques in dogs with cataracts.

OBJECTIVE: To determine histologic and immunohistochemical characteristics of the multifocal adherent plaques that commonly develop on the internal surfaces of the anterior and posterior lens capsules in dogs with cataracts. SAMPLE POPULATION: 31 anterior and 4 posterior capsular specimens collected during lens extraction surgery in dogs with cataracts. PROCEDURE: Specimens were evaluated, using light and transmission electron microscopy. Immunohistochemical techniques were used to localize cytokeratin, vimentin, alpha-smooth muscle-specific actin, fibronectin, tenascin, and transforming growth factor-beta (TGF-beta) within plaques. RESULTS: Histologically, plaques comprised elongated spindle-shaped cells that formed a placoid mass. Cells were embedded in an extracellular matrix containing collagen fibrils, often with duplicated or split basement membranes. Immunohistochemically, normal lens epithelial cells and cells within plaques stained for vimentin. Most cells and some areas of the extracellular matrix within plaques stained for TGF-beta and alpha-smooth muscle-specific actin. Fibronectin and tenascin were also detected in the extracellular matrix. CONCLUSIONS AND CLINICAL RELEVANCE: Canine lens capsular plaques are histologically and immunohistochemically similar to posterior capsule opacification and subcapsular cataracts in humans, which suggests that the canine condition, like the human conditions, is associated with fibrous metaplasia of lens epithelial cells. Transforming growth factor-beta may play a role in the genesis of capsular plaques. Because severity of plaques was correlated with stage of cataract development, earlier surgical removal of cataracts may be useful to avoid complications associated with plaque formation.

Etiologies, observations and reporting of estuarine finfish lesions.

Lesions in estuarine finfish are associated with a variety of organisms including parasites and bacterial, viral, and fungal infectious agents. In addition, trauma, suboptimal water quality, and other abiotic stress factors may result in the loss of homeostasis. We have observed solitary ulcerative lesions on menhaden sampled from the Chesapeake Bay, Maryland, the Pimlico River, North Carolina, and the St. Johns River, Florida. Histologically, the lesions demonstrated a marked chronic inflammatory infiltrate and granulomas in response to fungal hyphae throughout large areas of exposed necrotic muscle. Gram-negative rod-shaped bacteria were also observed in the lesions, a common finding in ulcers of aquatic organisms. Similar observations in menhaden and other species have been described previously in the literature as ulcerative mycosis, mycotic granulomatosis, red spot disease, and epizootic ulcerative syndrome. Despite the many different known causes of fish lesions, the popular press and the scientific literature have recently emphasized Pfiesteria piscicida and other Pfiesteria-like dinoflagellates (and their bioactive compounds) as the primary causative agent for finfish lesions, particularly mycotic granulomatous ulcers in Atlantic menhaden. While some laboratory data suggest that Pfiesteria may play a role in field-observed lesions, much more cause-and-effect evidence is needed to determine the importance of other risk factors, both alone or and in combination with Pfiesteria. In order to better understand the etiology of lesion initiation and progression in estuarine finfish, accurate assessments of environmental conditions collected on appropriate temporal and spatial scales, and fish morphological indicators consistent with gross and histological pathologic terminology, should be used for reporting fish lesion observations and kills. Further, this outlook will help to avoid bias and may foster a broader perspective for examining the health of estuarine systems in general.

A role for lipid rafts in B cell antigen receptor signaling and antigen targeting.

The B cell antigen receptor (BCR) serves both to initiate signal transduction cascades and to target antigen for processing and presentation by MHC class II molecules. How these two BCR functions are coordinated is not known. Recently, sphingolipid- and cholesterol-rich plasma membrane lipid microdomains, termed lipid rafts, have been identified and proposed to function as platforms for both receptor signaling and membrane trafficking. Here we show that upon cross-linking, the BCR rapidly translocates into ganglioside G(M1)-enriched lipid rafts that contain the Src family kinase Lyn and exclude the phosphatase CD45R. Both Igalpha and Lyn in the lipid rafts become phosphorylated, and subsequently the BCR and a portion of G(M1) are targeted to the class II peptide loading compartment. Entry into lipid rafts, however, is not sufficient for targeting to the antigen processing compartments, as a mutant surface Ig containing a deletion of the cytoplasmic domain is constitutively present in rafts but when cross-linked does not internalize to the antigen processing compartment. Taken together, these results provide evidence for a role for lipid rafts in the initial steps of BCR signaling and antigen targeting.

A three-year study of viable airborne fungi in the North Carolina Zoological Park R.J.R. Nabisco Rocky Coast Alcid Exhibit.

Aspergillosis is a common cause of mortality in captive birds, particularly in recently imported birds or captive chicks and their parents. Use of the Andersen N-6 single-stage viable air sampler in the North Carolina Zoological Park (NCZP) R.J.R. Nabisco Rocky Coast Alcid Exhibit before and after the introduction of birds allowed a unique study of the mycological content of the air in a developing self-contained ecosystem. The Alcid Exhibit had a median count of 17 colony-forming-units (CFU)/m3 of air in comparison to 200-500 CFU/m3 and 1,000-3,500 CFU/m3 reported in human dwellings and the NCZP R.J. Reynolds Forest Aviary, respectively. Cladosporium and Penicillium represented 21.3% and Aspergillus 1.08% of the fungi collected. During the study, no respiratory mycoses were reported in any of the alcids. Continuous high-efficiency particulate air filtration, maintenance of low exhibit air temperatures, and an environment with little residual organic material capable of supporting fungal growth were important factors contributing to low colony counts. All colony counts >100 CFU/m3 in the exhibit were related to the apparent introduction of fungi from outside the facility. A reduction in the number of fungi transported from an external source into enclosed cool-temperature aviaries may be sufficient to avoid outbreaks of aspergillosis.

A description of cutaneous-subcutaneous pythiosis in fifteen dogs.

Information regarding signalment, duration of clinical signs, history of swimming, results of CBC and serum biochemical analyses, biopsy findings and mycological results, together with treatments and outcome, was retrieved from the medical records of 15 dogs with a diagnosis of pythiosis made between 1985 and 1995 at the Colleges of Veterinary Medicine, North Carolina State University and the University of Florida. Most of the dogs were young (median age 22 months) and represented larger breeds (> 20 kg). Lesions were characteristically chronic, ulcerated, and nodular with multiple draining tracts on the limbs, thoracic wall or perineal regions. The median duration of these lesions was 3 months with a range of 2 weeks-6 months. Seven dogs had a history of swimming. Peripheral eosinophilia was observed in 14 of the dogs. Cytological evaluation of discharge, aspirates, or impression smears made from biopsy specimens revealed hyphae in five of 11 dogs (45%). Histopathological evaluation using the Gomori Methenamine-Silver (GMS) stain was the most useful test for providing presumptive evidence of cutaneous pythiosis. Immunotherapy or antifungal therapy using either amphotericin B, liposomal nystatin, itraconazole, or ketoconazole were all unsuccessful. The only dog to survive underwent amputation of the affected limb; thus, the prognosis for cutaneous pythiosis in the dog is poor.

Effect of alpha-chymotrypsin on breaking strength and ultrastructural morphology of canine ciliary zonules.

OBJECTIVE: To determine the effect of alpha-chymotrypsin treatment on breaking strength and ultrastructural morphology of canine ciliary zonules. SAMPLE POPULATION: Eyes from young random-source dogs from an animal shelter. PROCEDURE: Eyes were obtained immediately after euthanasia of dogs. The enzyme alpha-chymotrypsin was applied to the ciliary zonules of 1 eye of each dog; the other eye was treated with saline solution as a control. The breaking strength of ciliary zonules was measured, using a linear actuator and force transducer. The lenses and ciliary bodies were then analyzed by scanning and transmission electron microscopy. RESULTS: alpha-Chymotrypsin reduced the breaking strength of ciliary zonules by a mean +/- SD 44 (+/- 20)%, compared with that for saline-treated control eyes. Increasing the volume of enzyme further decreased the breaking strength of the zonules. Differences in the appearance of the ciliary body by electron microscopy were not apparent between enzyme- and saline-treated specimens. CONCLUSIONS AND CLINICAL RELEVANCE: Application of alpha-chymotrypsin to enucleated canine eyes at a concentration used in people significantly reduces the breaking strength of canine ciliary zonules without any apparent damage to the ciliary body. alpha-Chymotrypsin may be useful in the removal of subluxated canine lenses and in removal of cataractous lenses in young dogs, in which phacoemulsification often results in appreciable post operative capsular opacification.

A comparison of sampling methods for airborne fungal spores during an outbreak of aspergillosis in the forest aviary of the North Carolina Zoological Park.

An outbreak of aspergillosis with the death of six birds in the North Carolina Zoological Park R. J. Reynolds Forest Aviary in the spring of 1993 led to an investigation of the concentration of Aspergillus fumigatus spores in the air. No Aspergillus sp. was found in the facility through use of the drop plate method (gravitometric sampling) along with swab-sampling of selected surfaces within the exhibit and plating of food samples and nesting material onto petri dishes of nutrient media. A number factors that could stress the avian population were identified. These included excessive heat in the upper portion of the aviary due to the failure of an air handling system, a malfunctioning cooling tower, and large numbers of visitors to the facility (an average of 3,500/day). In addition, the outbreak occurred during a period of increased nesting behavior. Sampling of the fungal population of the air was conducted 1 year later, when no disease was noted, to compare the sensitivity of the commonly used drop plate method (open plates of nutrient media) with a volumetric impaction method (Andersen N-6 Air Sampler). The volumetric method delivered quantitative as well as qualitative data and exhibited more sensitivity for fungal spores of size similar to those of Aspergillus sp.

The experimental antipsychotic agent 1192U90 targets tapetum lucidum in canine eyes.

To assess the potential adverse effects in people of the antipsychotic agent 1192U90, we dosed mice, rats, beagles, and cynomolgus monkeys for up to 3 mo. In dogs, but not the other species, 1192U90 caused ocular changes detectable ophthalmoscopically as loss of tapetal reflectivity, altered tapetal color, and the appearance of black pigmentation on the tapetal fundus. Eyes from affected dogs had atrophic tapeta lucidum due to cell loss. Rodlets in remaining tapetal cells were separated by electron-lucent spaces or finely granular material, varied in size and shape, and often contained irregularly shaped electron-dense inclusions. Nontapetal ocular structures were unaffected. Because 1192U90 caused no ocular changes in nontapetal species, we hypothesized that it targeted only tapetum lucidum and spared other ocular structures. We tried to test this hypothesis by dosing congenitally atapetal dogs; however, although these dogs were ophthalmoscopically "atapetal," they had scattered tapetal cells visible by electron microscopy, and these tapetal cells had ultrastructural changes indistinguishable from those that occurred in treated normal-eyed dogs. Tapetal degeneration caused by 1192U90 resembled that described in hereditary tapetal degeneration in beagles. That 1192U90 caused no ocular changes in nontapetal species suggests that the ocular changes in dogs do not imply a risk for humans, whose eyes also lack a tapetum lucidum.