Mouse models of aerosol-acquired tularemia caused by Francisella tularensis types A and B.

After preliminary assessment of virulence in AKR/J, DBA/1, BALB/c, and C57BL/6 mice, we investigated histopathologic changes in BALB/c and C57BL/6 mice infected with type A (strain SCHU S4) or type B (strain 425) Francisella tularensis by aerosol exposure. In mice exposed to type A infection, changes in histologic presentation were not apparent until day 3 after infection, when pyogranulomatous inflammation was detected in spleens and livers of BALB/c mice, and in lungs and spleens of C57BL/6 mice. Histopathologic changes were most severe and widespread in both mouse strains on day 5 after infection and seemed to completely resolve within 22 d of challenge. BALB/c mice were more resistant than C57BL/6 mice in lethal-dose calculations, but C57BL/6 mice cleared the infection more rapidly. Mice similarly challenged with type B F. tularensis also developed histopathologic signs of infection beginning on day 3. The most severe changes were noted on day 8 and were characterized by granulomatous or pyogranulomatous infiltrations of the lungs. Unlike type A infection, lesions due to type B did not resolve over time and remained 3 wk after infection. In type B, but not type A, infection we noted extensive inflammation of the heart muscle. Although no microorganisms were found in tissues of type A survivors beyond 9 d after infection, mice surviving strain 425 infection had a low level of residual infection at 3 wk after challenge. The histopathologic presentation of tularemia caused by F. tularensis types A and B in BALB/c and C57BL/6 mice bears distinct similarities to tularemia in humans.

The cluster 1 type VI secretion system is a major virulence determinant in Burkholderia pseudomallei.

The Burkholderia pseudomallei K96243 genome encodes six type VI secretion systems (T6SSs), but little is known about the role of these systems in the biology of B. pseudomallei. In this study, we purified recombinant Hcp proteins from each T6SS and tested them as vaccine candidates in the BALB/c mouse model of melioidosis. Recombinant Hcp2 protected 80% of mice against a lethal challenge with K96243, while recombinant Hcp1, Hcp3, and Hcp6 protected 50% of mice against challenge. Hcp6 was the only Hcp constitutively produced by B. pseudomallei in vitro; however, it was not exported to the extracellular milieu. Hcp1, on the other hand, was produced and exported in vitro when the VirAG two-component regulatory system was overexpressed in trans. We also constructed six hcp deletion mutants (Δhcp1 through Δhcp6) and tested them for virulence in the Syrian hamster model of infection. The 50% lethal doses (LD(50)s) for the Δhcp2 through Δhcp6 mutants were indistinguishable from K96243 (<10 bacteria), but the LD(50) for the Δhcp1 mutant was >10(3) bacteria. The hcp1 deletion mutant also exhibited a growth defect in RAW 264.7 macrophages and was unable to form multinucleated giant cells in this cell line. Unlike K96243, the Δhcp1 mutant was only weakly cytotoxic to RAW 264.7 macrophages 18 h after infection. The results suggest that the cluster 1 T6SS is essential for virulence and plays an important role in the intracellular lifestyle of B. pseudomallei.

Early events in the pathogenesis of eastern equine encephalitis virus in mice.

To elucidate the pathogenesis of eastern equine encephalitis (EEE) virus infections, we used histopathology, immunohistochemistry, and in situ hybridization to track the spread and early cellular targets of viral infection in mice. Young mice were inoculated with virulent EEE virus in their right rear footpad and were followed in a time-course study for 4 days. Virulent EEE virus produced a biphasic illness characterized by an early self-limiting replication phase in peripheral tissues followed by an invariably fatal central nervous system (CNS) phase. In the early extraneural phase, there was primary amplifying replication of virus within fibroblasts at the inoculation site and within osteoblasts in active growth areas of bone that resulted in a transient high-titer viremia. Pathological changes and viral infection were observed as early as 12 hours post-infection (PI) in osteoblasts, skeletal muscle myocytes, and in fibroblasts along fascial sheaths. The severity and extent of infection in peripheral tissues peaked at day 1 PI. In the neural phase of infection, virus was first detected in the brain on day 1 PI, with rapid interneuronal spread of infection leading to death by day 4 PI. EEE virus appeared to be directly cytopathic for neurons. The very rapid onset and apparently random and widely dispersed infection in the CNS, with concurrent sparing of olfactory neuroepithelium, strongly suggests that invasion of the CNS by EEE occurs by a vascular route, rather than via peripheral nerves or the olfactory neuroepithelium. Our finding that metaphyseal osteoblasts are an early site of amplifying viral replication may explain the higher-titer viremias and higher incidence of neuroinvasion and fulminant encephalitis seen in the young, and may also explain why mature animals become refractory to encephalitis after peripheral inoculation with EEE virus.

Characterization of experimental equine glanders.

Considerable advances in understanding of the disease caused by Burkholderia mallei have been made employing a combination of tools including genetic techniques and animal infection models. The development of small animal models has allowed us to assess the role of a number of putative virulence determinants in the pathogenesis of disease due to B. mallei. Due to the difficulties in performing active immunization studies in small animals, and due to the fact that the horse is the target mammalian species for glanders, we have initiated experimental studies on glanders in horses. Intratracheal deposition of B. mallei produced clinical glanders with organisms being recovered from tissues of infected horses. The model should prove to be of considerable value in our ongoing studies on the pathogenesis and vaccine development for glanders.

Burkholderia thailandensis E125 harbors a temperate bacteriophage specific for Burkholderia mallei.

Burkholderia thailandensis is a nonpathogenic gram-negative bacillus that is closely related to Burkholderia mallei and Burkholderia pseudomallei. We found that B. thailandensis E125 spontaneously produced a bacteriophage, termed phiE125, which formed turbid plaques in top agar containing B. mallei ATCC 23344. We examined the host range of phiE125 and found that it formed plaques on B. mallei but not on any other bacterial species tested, including B. thailandensis and B. pseudomallei. Examination of the bacteriophage by transmission electron microscopy revealed an isometric head and a long noncontractile tail. B. mallei NCTC 120 and B. mallei DB110795 were resistant to infection with phiE125 and did not produce lipopolysaccharide (LPS) O antigen due to IS407A insertions in wbiE and wbiG, respectively. wbiE was provided in trans on a broad-host-range plasmid to B. mallei NCTC 120, and it restored LPS O-antigen production and susceptibility to phiE125. The 53,373-bp phiE125 genome contained 70 genes, an IS3 family insertion sequence (ISBt3), and an attachment site (attP) encompassing the 3' end of a proline tRNA (UGG) gene. While the overall genetic organization of the phiE125 genome was similar to lambda-like bacteriophages and prophages, it also possessed a novel cluster of putative replication and lysogeny genes. The phiE125 genome encoded an adenine and a cytosine methyltransferase, and purified bacteriophage DNA contained both N6-methyladenine and N4-methylcytosine. The results presented here demonstrate that phiE125 is a new member of the lambda supergroup of Siphoviridae that may be useful as a diagnostic tool for B. mallei.

Canine Cutaneous Neosporosis: Clinical Improvement with Clindamycin.

Résumé- Une dermite pyogranulomateuse provoquée par le protozoaire Neospora caninum a été diagnostiquée chez un Golden Retriever de 12 ans. Le nombreux nodules fistulisés étaient localisés au niveau de la tête et du thorax. De nombreux tachyzoites de N. caninum ont été retrouvés dans les biopsies des nodules et le diagnostic a été confirmé pas immunohistologie et examen en microscopie électronique. Le chien avait un titre de sérologie Neospora caninum de 3200 par immunofluorescence indirecte. Après un traitement à base de clindamycine pendant 45 jours, les lésions cutanées ont disparu. Le chien est probablement mort à cause d'un lymphosarcome. Il existait aussi une infection latente àToxoplasma gondii. les Neospora caninum n'ont pas pu être retrouvés par des techniques biologiques ni en culture ou par inoculation de souris à partir de prélèvements nécropsiques. Seuls des tachyzoites dégenérés ont pu être observés histologiquement. Ces observations montrent que la néosporose peut être envisagée dans le diagnostic différentiel des dermites pyogranulomateuses du chien et que la clindamycine est un médicament efficace pour traiter la néosporoe canine. [Dubey, J. P., Metzger, F. L., Hattel, A. L., Lindsay, D. S., Fritz, D. L. Canine cutaneous neosporosis: clinical improvement with clindamycin (Néosporose cutanée canine: amélioration clinique par la clindamycine). Resumen- Se diagnosticó una dermatitis piogranulomatosa causada por el protozoo parásito Neospora caninum en un perro de raza Golden Retriever de 12 años. El animal presentaba varios nódulos en la piel de la cabeza y tórax. Se observaron numerosos taquizoitos de N. caninum en los cortes histológicos de tejido obtenido mediante biopsia de dichos nódulos y el diagnóstico fue confirmado por tinción inmunohistológica y por microscopia electrónica. El perro mostró un titulo de anticuerpos contra N. caninum de 1:3,200 en la prueba de fluorescencia indirecta. Las lesiones cutáneas se resolvieron tras un tratamiento con hidroclorido de clindamicina durante 45 dias. El perro murió posteriormente a causa de un linfoma y presentaba también una infestación latente por Toxoplasma gondii. No se pudo demostrar la presencia de Neospora caninum mediante bioensayos en cultivos celulares ni en ratones inoculados con tejido canino obtenido en la necrospia. Tan solo se pudieron observar taquizoitos degenerados de N. caninum en tejido cutáneo obtenido en la necrospia. Estos hallazgos indican que se debe incluir neosporosis en el diagnóstico diferencial de dermatitis piogranulomatosas en el perro y que la clindamicina puede ser un fármaco eficaz para el tratamiento de la neosporosis cutánea. [Canine cutaneous neosporosis: clinical improvement with clindamycin (Neosporosis cutánea canina: mejora clinica con clindamicina). Abstract- Pyogranulomatous dermatitis caused by the protozoan parasite Neospora caninum was diagnosed in a 12-year-old Golden Retriever dog. Multiple draining nodules were located in the skin of the head and thorax. Numerous tachyzoites of N. caninum were found in histologic sections of the biopsy tissue from the cutaneous nodules and the diagnosis was confirmed by immunohistochemieal staining and by electron microscopic examination. The dog had a 1:3200 serum antibody titer to N. caninum in the indirect fluorescent antibody test. The cutaneous lesions resolved after a 45-day treatment with clindamycin hydrochloride. The dog eventually died because of lymphosarcoma and also had a latent infection with Toxoplasma gondii. Neospora caninum could not be demonstrated by bioassays in cell culture or mice inoculated with canine tissue obtained at necropsy. Only degenerating N. caninum tachyzoites were seen in skin tissue taken at necropsy. These observations indicate that neosporosis should be considered in the differential diagnosis of pyogranulomatous dermatitis in dogs and that clindamycin may be an effective drug for treating cutaneous neosporosis.