James B. Stanton

Characterization of "Candidatus Piscichlamydia salmonis" (Order Chlamydiales), a Chlamydia-Like Bacterium Associated With Epitheliocystis in Farmed Atlantic Salmon (Salmo salar)

ABSTRACT To characterize intracellular gram-negative bacteria associated with epitheliocystis in farmed Atlantic salmon (Salmo salar), gills with proliferative lesions were collected for histopathology, conventional transmission and immunoelectron microscopy, in situ hybridization, and DNA extraction during epitheliocystis outbreaks in Ireland and Norway in 1999 and 2000, respectively, and compared by ultrastructure and immunoreactivity to nonproliferative gills from Ireland archived in 1995. Genomic DNA from proliferative gills was used to amplify 16S ribosomal DNA (rDNA) for molecular phylogenetic analyses. Epitheliocystis inclusions from proliferative gills possessed variably elongate reticulate bodies, examples of binary fission, and vacuolated and nonvacuolated intermediate bodies, whereas inclusions in nonproliferative gills had typical chlamydial developmental stages plus distinctive head-and-tail cells. Immunogold processing using anti-chlamydial lipopolysaccharide antibody labeled reticulate bodies from proliferative and nonproliferative gills. 16S rDNA amplified directly from Irish (1999) and Norwegian (2000) gill samples demonstrated 99% nucleotide identity, and riboprobes transcribed from cloned near-full-length 16S rDNA amplicons from Norwegian gills hybridized with inclusions in proliferative lesions from Irish (1999) and Norwegian (2000) sections. A 1,487-bp consensus 16S rRNA gene sequence representing the chlamydia-like bacterium (CLB) from proliferative gills had the highest percent nucleotide identity with endosymbionts of Acanthamoeba spp. (order Chlamydiales). Molecular phylogenetic relationships inferred from 16S rRNA gene sequences using distance and parsimony indicated that the CLB from proliferative gills branched with members of the order Chlamydiales. “Candidatus Piscichlamydia salmonis” is proposed for the CLB associated with epitheliocystis from proliferative gills of Atlantic salmon, which exhibits developmental stages different from those identified in nonproliferative gills.

Coxiella burnetii Infection of a Steller Sea Lion (Eumetopias jubatus) Found in Washington State

ABSTRACT A pregnant sea lion stranded in the State of Washington was found to have placentitis caused by a unique strain of Coxiella burnetii. This is the first description of coxiellosis in a sea lion and suggests that exposure to sea lions may be a risk factor for contracting Q fever.

Small-Ruminant Lentivirus Enhances PrPSc Accumulation in Cultured Sheep Microglial Cells

ABSTRACT Sheep scrapie is the prototypical transmissible spongiform encephalopathy (prion disease), which has a fundamental pathogenesis involving conversion of normal cellular prion protein (PrPC [C superscript stands for cellular]) to disease-associated prion protein (PrPSc [Sc superscript stands for sheep scrapie]). Sheep microglial cell cultures, derived from a prnp 136VV/171QQ near-term fetal brain, were developed to study sheep scrapie in the natural host and to investigate potential cofactors in the prion conversion process. Two culture systems, a primary cell culture and a cell line transformed with the large T antigen of simian virus 40, were developed, and both were identified as microglial in origin as indicated by expression of several microglial phenotype markers. Following exposure to PrPSc, sheep microglial cells demonstrated relatively low levels (transformed cell line) to high levels (primary cell line) of PrPSc accumulation over time. The accumulated PrPSc demonstrated protease resistance, an inferred beta-sheet conformation (as determined by a commercial enzyme-linked immunosorbent assay), specific inhibition by anti-PrP antibodies, and was transmissible in a dose-dependent manner. Primary microglia coinfected with a small-ruminant lentivirus (caprine arthritis encephalitis virus-Cork strain) and PrPSc demonstrated an approximately twofold increase in PrPSc accumulation compared to that of primary microglia infected with PrPSc alone. The results demonstrate the in vitro utility of PrPSc-permissive sheep microglial cells in investigating the biology of natural prion diseases and show that small-ruminant lentiviruses enhance prion conversion in cultured sheep microglia.

Ovine trophoblast is a primary source of TNFα during Chlamydophila abortus infection

Chlamydophila abortus is a Gram-negative obligate intracellular bacterium that causes infectious abortion in sheep (ovine enzootic abortion, OEA) and humans. Infected placentas recovered from sheep that experience OEA have thickened membranes, contain dense inflammatory cellular infiltrates and show evidence of intravascular thrombosis. Despite widespread inflammation, chlamydial multiplication is restricted to the chorionic trophoblast cells. To investigate the potential role of trophoblast in the initiation and propagation of placental inflammation during OEA, the AH-1 ovine trophoblast cell line was experimentally infected with C. abortus and analysed for the release of pro-inflammatory mediators. C. abortus was found to induce the release of both tumour necrosis factor-alpha (TNFalpha) and CXCL8 (interleukin-8) from AH-1 cells in a dose- and time-dependent manner. Ultra-violet (UV)-killed organisms did not elicit this profile, indicating that intracellular multiplication of C. abortus was required for release of these pro-inflammatory mediators. Exposure of AH-1 cells to recombinant ovine TNFalpha alone resulted in the release of CXCL8, suggestive of a self-propagating inflammatory cytokine and chemokine cascade. These data indicate a primary role for trophoblast in the initiation and propagation of placental inflammation during chlamydial abortion.

RETROSPECTIVE DIFFERENTIATION OF CANINE DISTEMPER VIRUS AND PHOCINE DISTEMPER VIRUS IN PHOCIDS

Formalin-fixed paraffin-embedded tissues from one Caspian seal (Phoca caspica), one harp seal (Phoca groenlandica), one hooded seal (Cystophora cristata), and one harbor seal (Phoca vitulina vitulina) were used to compare the utility of immunohistochemistry (IHC) versus that of a novel seminested reverse transcriptase polymerase chain reaction (RT-PCR) to detect and differentiate canine distemper virus (CDV) and phocine distemper virus (PDV). Four antibodies made against PDV were able to detect both viruses. Two antibodies made against cetacean morbillivirus (CMV) did not label antigens from either CDV or PDV. A third anti-CMV antibody inconsistently stained CDV antigens but did not label PDV antigens. The seminested RT-PCR was able to detect RNA of the phosphoprotein gene in all positive cases. Nucleotide sequence analyses of seminested RT-PCR products were used to differentiate CDV RNA from PDV RNA. From these data, it was determined that IHC using antibodies generated against PDV provided a rapid means of detection for both CDV and PDV antigens; however, differentiation between CDV and PDV was achieved only with the RT-PCR assay.

Development of a semi-nested reverse transcription polymerase chain reaction assay for the retrospective diagnosis of canine distemper virus infection.

Formalin-fixed, paraffin-embedded tissue blocks from 14 dogs were used to test the utility of a newly developed semi-nested reverse transcription polymerase chain reaction (RT-PCR) assay for canine distemper virus (CDV). The results from this new test were compared with those of histopathologic examination, fluorescent antibody detection (FA), and immunohistochemistry (IHC). The semi-nested RT-PCR protocol was used to detect CDV RNA in 9 of the 10 cases that were positive by at least 1 of the immunologic (FA and IHC) methods. Sequence data indicated that the amplified strains of CDV are more closely related to a naturally occurring strain than to a vaccine strain. Thus, the semi-nested RT-PCR assay is a useful diagnostic method applicable to the retrospective diagnosis of CDV infection. Sequence determination may yield molecular epidemiologic information regarding vaccine efficacy.

Use of immunohistochemistry to diagnose chytridiomycosis in Dyeing poison dart frogs (Dendrobates tinctorius)

Chytridiomycosis, caused by Batrachochytrium dendrobatidis, is an emerging disease of both wild and captive amphibians, posing a threat to their survival in many parts of the world. As the disease can be difficult to diagnose on routine pathologic sections, the purpose of this study was to develop an additional method for visualization. To accomplish this, immunohistochemical staining was applied to histologic skin sections from four experimentally infected Dyeing poison dart frogs (Dendrobates tinctorius). Staining of the positive tissue sections was distinct and readily visualized, making this technique a valuable ancillary diagnostic test for this important disease.

Discovery of a Novel, Monocationic, Small-Molecule Inhibitor of Scrapie Prion Accumulation in Cultured Sheep Microglia and Rov Cells

Prion diseases, including sheep scrapie, are neurodegenerative diseases with the fundamental pathogenesis involving conversion of normal cellular prion protein (PrPC) to disease-associated prion protein (PrPSc). Chemical inhibition of prion accumulation is widely investigated, often using rodent-adapted prion cell culture models. Using a PrPSc-specific ELISA we discovered a monocationic phenyl-furan-benzimidazole (DB772), which has previously demonstrated anti-pestiviral activity and represents a chemical category previously untested for anti-prion activity, that inhibited PrPSc accumulation and prion infectivity in primary sheep microglial cell cultures (PRNP 136VV/154RR/171QQ) and Rov9 cultures (VRQ-ovinized RK13 cells). We investigated potential mechanisms of this anti-prion activity by evaluating PrPC expression with quantitative RT-PCR and PrP ELISA, comparing the concentration-dependent anti-prion and anti-pestiviral effects of DB772, and determining the selectivity index. Results demonstrate at least an approximate two-log inhibition of PrPSc accumulation in the two cell systems and confirmed that the inhibition of PrPSc accumulation correlates with inhibition of prion infectivity. PRNP transcripts and total PrP protein concentrations within cell lysates were not decreased; thus, decreased PrPC expression is not the mechanism of PrPSc inhibition. PrPSc accumulation was multiple logs more resistant than pestivirus to DB772, suggesting that the anti-PrPSc activity was independent of anti-pestivirus activity. The anti-PrPSc selectivity index in cell culture was approximately 4.6 in microglia and 5.5 in Rov9 cells. The results describe a new chemical category that inhibits ovine PrPSc accumulation in primary sheep microglia and Rov9 cells, and can be used for future studies into the treatment and mechanism of prion diseases.

hTERT-immortalized ovine microglia propagate natural scrapie isolates

Ex vivo propagation of natural prion isolates (i.e., propagated solely in the natural host) is crucial for the characterization and study of transmissible spongiform encephalopathies (TSEs). Several well-established, prion-permissive cell culture systems are available; however, only a few cell lines are permissive to natural prion isolates and these cells are not pathophysiologically relevant (e.g., renal epithelium and fibroblast-like cells). Therefore, a pathophysiologically relevant cell line derived from a natural TSE host could be used for propagation of natural prion isolates. In this study, ovine brain macrophages (microglia) were immortalized by transfection with the human telomerase reverse transcriptase (hTERT) gene to identify cell lines (hTERT-microglia) permissive to natural scrapie prion isolates. Following transfection, hTERT-microglia were passaged up to 100 times and their lifespan was significantly longer compared to parental cells (Fishers exact test, P<0.001). Multiple sublines were permissive to cell culture-adapted prions; two sublines were also permissive to natural scrapie isolates (i.e., derived from brain homogenates of sheep infected with scrapie). Prion infectivity and partial protease resistance of the prion protein were maintained in hTERT-microglia. Comparisons between scrapie-permissive and non-permissive hTERT-microglia sublines revealed that overall quantity of the normal cellular prion protein was not associated with prion permissiveness. The use of hTERT-microglia in future TSE studies may be more germane to the characterization of the cellular and subcellular pathophysiology of natural scrapie prion isolates and to investigate host-specific factors involved in prion replication.

Limited transcriptional response of ovine microglia to prion accumulation

The conversion of normal cellular prion protein to disease-associated prion protein (PrP(Sc)) is a fundamental component of prion disease pathogenesis. The molecular mechanisms contributing to prion conversion and the impact of PrP(Sc) accumulation on cellular biology are not fully understood. To further define the molecular changes associated with PrP(Sc) accumulation in cultured cells, the transcriptional profile of PrP(Sc)-accumulating primary ovine microglia was compared to the profile of PrP(Sc)-lacking microglia using the Affymetrix Bovine Genome Array. The experimental design included three biological replicates, each with three technical replicates, and samples that were collected at the point of near maximal PrP(Sc) accumulation levels as measured by ELISA. The array analysis revealed only 19 upregulated genes and 30 downregulated genes in PrP(Sc)-accumulating microglia. The results support the hypothesis that chronic PrP(Sc) accumulation in cultured microglia results in a limited transcriptional response.