Genetic variability of cloned Cytauxzoon felis ribosomal RNA ITS1 and ITS2 genomic regions from domestic cats with varied clinical outcomes from five states.

Cytauxzoon felis is a tick-borne hemoparasite that causes cytauxzoonosis in domestic cats in the United States. Historically, feline cytauxzoonosis was reported to be nearly always fatal. However, increasing evidence of cats surviving acute infection and/or harboring a chronic, subclinical infection has suggested the existence of different C. felis strains that may vary in pathogenicity. In this study, the intraspecific variation of the C. felis first and second ribosomal RNA internal transcribed spacer (ITS1, ITS2) regions was assessed for any clinical outcome or geographic associations. Sequence data were obtained for 122C. felis ITS1 and ITS2 clones from 41 domestic cat blood samples from Arkansas, Kansas, Missouri, Oklahoma, and Texas. Seven previously reported ITS1 region sequences were found, and a previously undescribed 23-bp insert was detected in cloned ITS1 sequences from a domestic cat in Missouri and two cats in Oklahoma. Four previously reported ITS2 region sequences were identified, and a 40-bp insert similar to that previously reported in C. felis of a domestic cat from Arkansas and pumas was detected in 18 cloned C. felis sequences from 12 domestic cats. One clone contained both the 23-bp insert and 40-bp insert within the ITS1 and ITS2 regions, respectively. Combined ITS1 and ITS2 sequence genotypes revealed that C. felis sequences from 27 cats (72/122 clones) corresponded to four previously described genotypes, ITSa, ITSc, ITSd, and ITSn. Five clones with the novel 23-bp insert from three cat isolates represented two new genotypes, ITSaa and ITSbb. Genotypes ITScc, ITSdd, ITSee, ITSff, ITSgg, and ITShh denoted 13 clones that matched prior sequences but had no previously assigned genotype. Genotypes ITSii through ITStt comprised 32 clones that were similar to, but did not exactly match, previously described genotypes. Twenty-five cats had C. felis infections with multiple ITS genotypes. Considerable C. felis genetic diversity was revealed with no significant geographic or clinical outcome associations.

The hyaloid vasculature facilitates basement membrane breakdown during choroid fissure closure in the zebrafish eye.

A critical aspect of vertebrate eye development is closure of the choroid fissure (CF). Defects in CF closure result in colobomas, which are a significant cause of childhood blindness worldwide. Despite the growing number of mutated loci associated with colobomas, we have a limited understanding of the cell biological underpinnings of CF closure. Here, we utilize the zebrafish embryo to identify key phases of CF closure and regulators of the process. Utilizing Laminin-111 as a marker for the basement membrane (BM) lining the CF, we determine the spatial and temporal patterns of BM breakdown in the CF, a prerequisite for CF closure. Similarly, utilizing a combination of in vivo time-lapse imaging, ß-catenin immunohistochemistry and F-actin staining, we determine that tissue fusion, which serves to close the fissure, follows BM breakdown closely. Periocular mesenchyme (POM)-derived endothelial cells, which migrate through the CF to give rise to the hyaloid vasculature, possess distinct actin foci that correlate with regions of BM breakdown. Disruption of talin1, which encodes a regulator of the actin cytoskeleton, results in colobomas and these correlate with structural defects in the hyaloid vasculature and defects in BM breakdown. cloche mutants, which entirely lack a hyaloid vasculature, also possess defects in BM breakdown in the CF. Taken together, these data support a model in which the hyaloid vasculature and/or the POM-derived endothelial cells that give rise to the hyaloid vasculature contribute to BM breakdown during CF closure.

Molecular characterization of Trypanosoma (Megatrypanum) spp. infecting cattle (Bos taurus), white-tailed deer (Odocoileus virginianus), and elk (Cervus elaphus canadensis) in the United States.

In the United States, the generally non-pathogenic trypanosome of cattle is designated Trypanosoma (Megatrypanum) theileri and is distinguished morphologically from Trypanosoma (M.) cervi, a trypanosome originally described in mule deer and elk. Phylogenetic studies of the Megatrypanum trypanosomes using various molecular markers reveal two lineages, designated TthI and TthII, with several genotypes within each. However, to date there is very limited genetic data for T. theileri, and none for the Megatrypanum trypanosomes found in wild ungulates, in the U.S. In this study U.S. isolates from cattle (Bos taurus), white-tailed deer (Odocoileus virginianus) (WTD), and elk (Cervus elaphus canadensis) were compared by ribosomal DNA (rDNA) sequence analysis and their incidence in cattle and WTD in south Texas counties was investigated. Phylogenetic analyses showed clear separation of the bovine and cervine trypanosomes. Both lineages I and II were represented in the U.S. cattle and WTD parasites. Lineage I cattle isolates were of a previously described genotype, whereas WTD and elk isolates were of two new genotypes distinct from the cattle trypanosomes. The cattle isolate of lineage II was of a previously reported genotype and was divergent from the WTD isolate, which was of a new genotype. In La Salle, Starr, Webb, and Zapata counties in south Texas a total of 51.8% of white-tailed deer were positive for trypanosomes by 18S rDNA PCR. Of the cattle screened in Webb County, 35.4% were positive. Drought conditions prevailing in south Texas when the animals were screened suggest the possibility of a vector for Trypanosoma other than the ked (Lipoptena mazamae) and tabanid flies (Tabanus spp. and Haematopota spp.).

Ocular syphilis in HIV-positive patients receiving highly active antiretroviral therapy.

BACKGROUND: From October 2001 to October 2002, we have observed a surprisingly high incidence of ocular syphilis in human immunodeficiency virus-positive (HIV+) patients receiving highly active antiretroviral therapy at our clinic. METHODS: We conducted a retrospective chart and patient database review. RESULTS: From 1997 to 2002, 455 patients in our clinic were screened for syphilis; 320 were screened from 2001 to 2002; 7.3% of patients (33/455) were diagnosed with syphilis. During the past year, syphilis was diagnosed in 7.5% of patients (24/320), of whom 13% (3/24) had ocular syphilis. We estimate the prevalence of ocular syphilis in HIV+ patients on highly active antiretroviral therapy screened for syphilis to be 9% (3/33). Presenting symptoms included blurred vision, loss of vision, central scotomas, and bilateral ocular involvement. The most common ocular manifestation of syphilis was posterior chamber uveitis; one patient also had a retinal detachment. All patients demonstrated reactive rapid plasma reagin and fluorescent treponemal antibody absorption test results, cerebrospinal fluid pleocytosis, and elevated total protein. Each patient received a 21-day course of intravenous penicillin G (24 million units daily) with improvement of visual symptoms. CONCLUSION: Our data demonstrate an unexpectedly high incidence of ocular syphilis in our HIV+ patients receiving highly active antiretroviral therapy during the past year. A diagnosis of ocular syphilis should be considered in any HIV+ patient who presents with visual symptoms, irrespective of the patient's CD4 count.

Reporting of deaths during pre-approval clinical trials for advanced HIV-infected populations.

The Division of Antiviral Drug Products of the US FDA has regulatory authority over the investigational new drugs under development by various sponsors to treat HIV-infected populations. The FDA and the sponsors of investigational new drugs use the Code of Federal Regulations to guide the entire drug development process, in order to ensure that safe and efficacious drugs are brought to market. To achieve this goal, diligent monitoring for safety during the pre-approval phase of new drug development is particularly crucial. When deciding what adverse experiences on clinical trials should be expeditiously reported, the Division recommends a conservative interpretation of the Code of Federal Regulations, where an adverse experience in a clinical trial of advanced HIV-infected patients is considered to be 'associated with the use of the drug' when the relationship cannot be ruled out with objective evidence. Fatal adverse experiences for subjects on clinical trials should be especially scrutinised. Safety reporting should be expedited when death occurs during clinical trials of advanced HIV-infected populations. The three components of an expedited reportable death occurrence, namely 'serious', 'unexpected' and 'associated with the drug use' as they relate to advanced HIV-infected populations, are discussed in this article. An occurrence of death is by definition serious. Unexpected experiences are unlisted adverse experiences, but need to be put into the context of specificity and severity. 'Associated with the drug use' has been clarified as 'relationship to the drug cannot be ruled out'. Because death in the advanced HIV-infected/AIDS population is usually a complex event, the possible contribution of the study drug is difficult to rule out. Thus, if the three components of the reporting requirement are met or insufficient information is available to make a firm determination of causality by the seventh day of the reporting period, the Division of Antiviral Drug Products expects expedited death reports on subjects participating in investigational new drug clinical studies.

Newly characterized species-specific immunogenic Chlamydophila pneumoniae peptide reactive with murine monoclonal and human serum antibodies.

A monoclonal antibody (MAb) directed against an unknown Chlamydophila pneumoniae epitope has been characterized, and the respective peptide mimotope has been identified. A murine MAb specific for C. pneumoniae was used to select peptides from phage display libraries. The peptides identified from the phage display library clones reacted specifically with the respective target murine MAb and with human sera previously identified as having antibody titers to C. pneumoniae. The selected peptide mimotope sequences tended to be composed of charged residues surrounding a core of hydrophobic residues. The peptide with the best binding could inhibit >95% of binding to the MAb, suggesting that the selected peptide binds the paratope of the respective MAb. The peptide reacted with human sera previously determined by microimmunofluorescence to have anti-C. pneumoniae antibodies. The peptide was competitively competed with the MAb against Renografin-purified, sonicated C. pneumoniae in an enzyme-linked immunosorbent assay and with whole-cell C. pneumoniae in an indirect fluorescence assay format, demonstrating its potential utility in the development of diagnostics. The use of this novel peptide may allow investigators to establish standardized assays free from cross-reactive Chlamydia trachomatis and Chlamydophila psittaci epitopes and immunoreactivity.