Karen Kelley

Alterations in Ultrastructural Morphology of Two-Cell Bovine Embryos Produced In Vitro and In Vivo Following a Physiologically Relevant Heat Shock

Abstract Exposure of cultured preimplantation embryos to temperatures similar to those experienced by heat-stressed cows inhibits subsequent development. In this study, the effects of heat shock on the ultrastructure of two-cell bovine embryos were examined to determine mechanisms for inhibition of development. Two-cell embryos produced in vitro were harvested at ∼28 h postinsemination and cultured for 6 h at one of three temperatures: 38.5°C (cow body temperature), 41.0°C (characteristic temperature for heat-stressed cows), or 43.0°C (severe heat shock). Ultrastructural examinations revealed that both heat shocks resulted in the movement of organelles towards the center of the blastomere. In addition, heat shock increased the percentage of mitochondria exhibiting a swollen morphology. Distance between the membranes comprising the nuclear envelope was increased but only when embryos were treated at 43.0°C. To determine whether ultrastructural responses to heat shock in culture were similar for embryos produced in vitro and in vivo, two-cell embryos were collected from superovulated Angus cows 48 h postinsemination and treated ex vivo for 6 h at 38.5°C or 41.0°C. Again, heat shock caused an increase in number of swollen mitochondria and movement of organelles away from the periphery of the blastomere. Exposure of two-cell bovine embryos to physiologically relevant elevated temperatures causes disruption in ultrastructural morphology that is inimical to development. The observation that overall morphology and response to heat was similar for embryos produced in vitro and in vivo implies that the former can be a good model for understanding embryonic responses to heat shock.

Reorganization of Microfilaments and Microtubules by Thermal Stress in Two-Cell Bovine Embryos

Abstract Two-cell bovine embryos become arrested in development when exposed to a physiologically relevant heat shock. One of the major ultrastructural modifications caused by heat shock is translocation of organelles toward the center of the blastomere. The objective of the present study was to determine if heat- shock-induced movement of organelles is a result of cytoskeletal rearrangement. Two-cell bovine embryos were cultured at 38.5°C (homeothermic temperature of the cow), 41.0°C (physiologically relevant heat shock), or 43.0°C (severe heat shock) for 6 h in the presence of either vehicle, latrunculin B (a microfilament depolymerizer), rhizoxin (a microtubule depolymerizer), or paclitaxel (a microtubule stabilizer). Heat shock caused a rearrangement of actin-containing filaments as detected by staining with phalloidin. Moreover, latrunculin B reduced the heat-shock-induced movement of organelles at 41.0°C but not at 43.0°C. In contrast, movement of organelles caused by heat shock was inhibited by rhizoxin at both temperatures. Furthermore, rhizoxin, but not latrunculin B, reduced the swelling of mitochondria caused by heat shock. Paclitaxel, while causing major changes in ultrastructure, did not prevent the movement of organelles or mitochondrial swelling. It is concluded that heat shock disrupts microtubule and microfilaments in the two-cell bovine embryo and that these changes are responsible for movement of organelles away from the periphery. In addition, intact microtubules are a requirement for heat-shock-induced swelling of mitochondria. Differences in response to rhizoxin and paclitaxel are interpreted to mean that deformation of microtubules can occur through a mechanism independent of microtubule depolymerization.

Ball Python Nidovirus: a Candidate Etiologic Agent for Severe Respiratory Disease in Python regius

ABSTRACT A severe, sometimes fatal respiratory disease has been observed in captive ball pythons (Python regius) since the late 1990s. In order to better understand this disease and its etiology, we collected case and control samples and performed pathological and diagnostic analyses. Electron micrographs revealed filamentous virus-like particles in lung epithelial cells of sick animals. Diagnostic testing for known pathogens did not identify an etiologic agent, so unbiased metagenomic sequencing was performed. Abundant nidovirus-like sequences were identified in cases and were used to assemble the genome of a previously unknown virus in the order Nidovirales. The nidoviruses, which were not previously known to infect nonavian reptiles, are a diverse order that includes important human and veterinary pathogens. The presence of the viral RNA was confirmed in all diseased animals (n = 8) but was not detected in healthy pythons or other snakes (n = 57). Viral RNA levels were generally highest in the lung and other respiratory tract tissues. The 33.5-kb viral genome is the largest RNA genome yet described and shares canonical characteristics with other nidovirus genomes, although several features distinguish this from related viruses. This virus, which we named ball python nidovirus (BPNV), will likely establish a new genus in Torovirinae subfamily. The identification of a novel nidovirus in reptiles contributes to our understanding of the biology and evolution of related viruses, and its association with lung disease in pythons is a promising step toward elucidating an etiology for this long-standing veterinary disease. IMPORTANCE Ball pythons are popular pets because of their diverse coloration, generally nonaggressive behavior, and relatively small size. Since the 1990s, veterinarians have been aware of an infectious respiratory disease of unknown cause in ball pythons that can be fatal. We used unbiased shotgun sequencing to discover a novel virus in the order Nidovirales that was present in cases but not controls. While nidoviruses are known to infect a variety of animals, this is the first report of a nidovirus recovered from any reptile. This report will enable diagnostics that will assist in determining the role of this virus in the causation of disease, which would allow control of the disease in zoos and private collections. Given its evolutionary divergence from known nidoviruses and its unique host, the study of reptile nidoviruses may further our understanding of related diseases and the viruses that cause them in humans and other animals. Ball pythons are popular pets because of their diverse coloration, generally nonaggressive behavior, and relatively small size. Since the 1990s, veterinarians have been aware of an infectious respiratory disease of unknown cause in ball pythons that can be fatal. We used unbiased shotgun sequencing to discover a novel virus in the order Nidovirales that was present in cases but not controls. While nidoviruses are known to infect a variety of animals, this is the first report of a nidovirus recovered from any reptile. This report will enable diagnostics that will assist in determining the role of this virus in the causation of disease, which would allow control of the disease in zoos and private collections. Given its evolutionary divergence from known nidoviruses and its unique host, the study of reptile nidoviruses may further our understanding of related diseases and the viruses that cause them in humans and other animals.

Transgenerational Transmission of the Glossina pallidipes Hytrosavirus Depends on the Presence of a Functional Symbiome

The vertically transmitted endosymbionts (Sodalis glossinidius and Wigglesworthia glossinidia) of the tsetse fly (Diptera: Glossinidae) are known to supplement dietary deficiencies and modulate the reproductive fitness and the defense system of the fly. Some tsetse fly species are also infected with the bacterium, Wolbachia and with the Glossina hytrosavirus (GpSGHV). Laboratory-bred G. pallidipes exhibit chronic asymptomatic and acute symptomatic GpSGHV infection, with the former being the most common in these colonies. However, under as yet undefined conditions, the asymptomatic state can convert to the symptomatic state, leading to detectable salivary gland hypertrophy (SGH+) syndrome. In this study, we investigated the interplay between the bacterial symbiome and GpSGHV during development of G. pallidipes by knocking down the symbionts with antibiotic. Intrahaemocoelic injection of GpSGHV led to high virus titre (109 virus copies), but was not accompanied by either the onset of detectable SGH+, or release of detectable virus particles into the blood meals during feeding events. When the F1 generations of GpSGHV-challenged mothers were dissected within 24 h post-eclosion, SGH+ was observed to increase from 4.5% in the first larviposition cycle to >95% in the fourth cycle. Despite being sterile, these F1 SGH+ progeny mated readily. Removal of the tsetse symbiome, however, suppressed transgenerational transfer of the virus via milk secretions and blocked the ability of GpSGHV to infect salivary glands of the F1 progeny. Whereas GpSGHV infects and replicates in salivary glands of developing pupa, the virus is unable to induce SGH+ within fully differentiated adult salivary glands. The F1 SGH+ adults are responsible for the GpSGHV-induced colony collapse in tsetse factories. Our data suggest that GpSGHV has co-evolved with the tsetse symbiome and that the symbionts play key roles in the virus transmission from mother to progeny.

VACCINIA H5 IS A MULTIFUNCTIONAL PROTEIN INVOLVED IN VIRAL DNA REPLICATION, POSTREPLICATIVE GENE TRANSCRIPTION, AND VIRION MORPHOGENESIS

The vaccinia H5 protein has been implicated in several steps of virus replication including DNA synthesis, postreplicative gene transcription, and virion morphogenesis. Our recent mapping of mutants in the consolidated Condit-Dales collection identified a temperature-sensitive vaccinia mutant in the H5R gene (Dts57). We demonstrate here that Dts57 has a DNA negative phenotype, strongly suggesting a direct role for H5 in DNA replication. We used a temperature shift protocol to determine the impact of H5 temperature sensitivity on postreplicative gene expression and observed changes in the pattern of postreplicative viral mRNA metabolism consistent with a role of H5 in postreplicative transcription. Finally, using a rifampicin release temperature shift protocol, we show that H5 is involved in multiple steps of virion morphogenesis. These data demonstrate directly that H5 plays roles in DNA replication, transcription and morphogenesis in vivo.

Immunohistochemical Detection of a Unique Protein within Cells of Snakes Having Inclusion Body Disease, a World-Wide Disease Seen in Members of the Families Boidae and Pythonidae

Inclusion body disease (IBD) is a worldwide disease in captive boa constrictors (boa constrictor) and occasionally in other snakes of the families Boidae and Pythonidae. The exact causative agent(s) and pathogenesis are not yet fully understood. Currently, diagnosis of IBD is based on the light microscopic identification of eosinophilic intracytoplasmic inclusion bodies in hematoxylin and eosin stained tissues or blood smears. An antigenically unique 68 KDa protein was identified within the IBD inclusion bodies, called IBD protein. A validated immuno-based ante-mortem diagnostic test is needed for screening snakes that are at risk of having IBD. In this study, despite difficulties in solubilizing semi-purified inclusion bodies, utilizing hybridoma technology a mouse anti-IBD protein monoclonal antibody (MAB) was produced. The antigenic specificity of the antibody was confirmed and validated by western blots, enzyme-linked immunosorbent assay, immuno-transmission electron microscopy, and immunohistochemical staining. Paraffin embedded tissues of IBD positive and negative boa constrictors (n=94) collected from 1990 to 2011 were tested with immunohistochemical staining. In boa constrictors, the anti-IBDP MAB had a sensitivity of 83% and specificity of 100% in detecting IBD. The antibody also cross-reacted with IBD inclusion bodies in carpet pythons (Morelia spilota) and a ball python (python regius). This validated antibody can serve as a tool for the development of ante-mortem immunodiagnostic tests for IBD.

Megalocytivirus infection in orbiculate batfish Platax orbicularis

Megalocytiviruses cause systemic disease in both marine and freshwater fishes, negatively impacting ornamental and food fish aquaculture. In this report, we characterize a megalocytivirus infection in a captive marine ornamental fish, the orbiculate batfish Platax orbicularis. Histologic examination revealed cytomegalic cells characterized by strongly basophilic granular intracytoplasmic inclusions within various organs. Transmission electron microscopy revealed icosahedral virus particles within the cytoplasm of cytomegalic cells consistent with an iridovirus infection. Analysis of the major capsid protein gene sequence confirmed that the orbiculate batfish virus is a member of the family Iridoviridae and is identical to the only other megalocytivirus reported from a marine ornamental fish, the Banggai cardinalfish Pterapogon kauderni iridovirus.

Identity of Two Sympatric Species of Orius (Hemiptera: Heteroptera: Anthocoridae)

Abstract The minute pirate bugs, Orius insidiosus (Say) and Orius pumilio (Champion) (Hemiptera: Heteroptera: Anthocoridae), are closely related species known to be sympatric in north Florida. Here, male and female genitalia, DNA sequences, and the effects of within- and between-species pairings on egg production and egg development were examined to develop a better understanding of the relationship between these two species. Interspecific matings between the two species did not result in viable progeny. Although there were gross similarities in the morphology of the male parameres (external genitalia) between the two species, the cone in O. pumilio was much broader with a greater spiral twist and the flagellum was longer than in O. insidiosus. Correspondingly, there were differences in the morphology of the copulatory tubes of the females of the two species. In O. insidiosus, the organ was somewhat longer than in O. pumilio and oriented parallel to the abdominal midline, while the copulatory tube in O. pumilio tilted slightly towards the midline. Additionally, the copulatory tube for O. pumilio included a sclerotized basal mound that was not present in O. insidiosus. These morphological differences suggest that successful copulation between these species could be difficult. In contrast to conspecific matings, interspecific matings resulted in few or no eggs laid over a period of two weeks and no viable progeny. Comparison of the 18S ribosomal gene ITS-1 sequences between the two species demonstrated only 91% homology. When yolk protein contents were examined to determine whether reproductive physiology had shifted to full egg production, interspecifically mated females contained amounts of yolk protein comparable to that in fed, but unmated females; this was less than 10% of the yolk protein previously found in fed and conspecifically mated females. These findings together confirm that O. insidiosus and O. pumilio are indeed two separate species.

Megalocytivirus infection in cultured Nile tilapia Oreochromis niloticus.

Megalocytiviruses, such as infectious spleen and kidney necrosis virus (ISKNV), induce lethal systemic diseases in both ornamental and food fish species. In this study, we investigated an epizootic affecting Nile tilapia Oreochromis niloticus cultured in the US Midwest. Diseased fish displayed lethargy, gill pallor, and distension of the coelomic cavity due to ascites. Histopathological examination revealed a severe systemic abundance of intravascular megalocytes that were especially prominent in the gills, kidney, spleen, liver, and intestinal submucosa. Transmission electron microscopic examination revealed abundant intracytoplasmic polygonal virions consistent with iridovirus infection. Comparison of the full-length major capsid protein nucleotide sequences from a recent outbreak with a remarkably similar case that occurred at the same facility many years earlier revealed that both epizootics were caused by ISKNV. A comparison of this case with previous reports suggests that ISKNV may represent a greater threat to tilapia aquaculture than previously realized.

Somatic embryogenesis in perennial statice Limonium bellidifolium, Plumbaginaceae

Cotyledon explants from perennial statice Limonium bellidifolium (Statice caspia Willd.) were cultured on Murashige and Skoogs basal medium (MS) supplemented with various levels of 2,4-D, kinetin and sucrose. Embryogenic calluses developed over a period of 10 days with the highest response at 4.5 μM (1 mg l−1) 2,4-D, 0.5 μM (0.1 mg l−1) kinetin and 117 mM sucrose. Following induction, the calluses were transferred to MS media supplemented with 88 or 117 mM sucrose and 0 or 0.5 μM kinetin. Somatic embryos at the globular, heart-shaped, torpedo, and cotyledonary stages developed. Fully germinated plantlets developed with the best response in medium supplemented with 117 mM sucrose and 0.5 μM kinetin. Direct somatic embryogenesis without a callus phase was observed with some of the cotyledon explants. Induction, maturation and germination of somatic embryos on the optimized media were equally effective using cotyledon, hypocotyl and root explants. Serial sections of L. bellidifolium cotyledon explants cultured for two weeks indicated that pro-embryogenic masses originated from parenchyma cells below the epidermis. Further histological observations of embryogenic calluses confirmed the initiation and development of globular and heart-shaped embryos and repetitive somatic embryogenesis. Ultrastructural observations indicated that the embryogenic cells were less vacuolate with abundant organelles compared to the cells of the explant. This is the first report of somatic embryogenesis in the Plumbaginaceae.