Molecular epidemiology of adenoviruses isolated from hospitalized children with severe lower acute respiratory infection in Santiago, Chile

Genome analysis was performed on 69 adenovirus isolates from pharyngeal secretions of young children hospitalized with severe lower acute respiratory disease in Santiago, Chile, between 1984 and 1986. As expected, most isolated strains belonged to subgenus B (68.1%). Among the isolates of 1984, Ad7c was the dominant genotype (12 out of 23). The majority of isolates of 1986 were of the recently described genomic variant 3f.     

Helicobacter pylori binds to CD74 on gastric epithelial cells and stimulates interleukin-8 production

The pathogenesis associated with Helicobacter pylori infection requires consistent contact with the gastric epithelium. Although several cell surface receptors have been suggested to play a role in adhesion, the bacterium-host interactions that elicit host responses are not well defined. This study investigated the interaction of H. pylori with the class II major histocompatibility complex (MHC)-associated invariant chain (Ii; CD74), which was found to be highly expressed by gastric epithelial cells. Bacterial binding was increased when CD74 surface expression was increased by gamma interferon (IFN-gamma) treatment or by fibroblast cells transfected with CD74, while binding was decreased by CD74 blocking antibodies, enzyme cleavage of CD74, and CD74-coated bacteria. H. pylori was also shown to bind directly to affinity-purified CD74 in the absence of class II MHC. Cross-linking of CD74 and the engagement of CD74 were verified to stimulate IL-8 production by unrelated cell lines expressing CD74 in the absence of class II MHC. Increased CD74 expression by cells increased IL-8 production in response to H. pylori, and agents that block CD74 decreased these responses. The binding of H. pylori to CD74 presents a novel insight into an initial interaction of H. pylori with the gastric epithelium that leads to upregulation of inflammatory responses.     

Detection of Cattle Naturally Infected with Anaplasma marginale in a Region of Endemicity by Nested PCR and a Competitive Enzyme-Linked Immunosorbent Assay Using Recombinant Major Surface Protein 5

A competitive enzyme-linked immunosorbent assay using recombinant major surface protein 5 (rMSP5-cELISA) of Anaplasma marginale was validated in a naturally infected cattle herd in an area of eastern Oregon where A. marginale is endemic. The true positive and negative A. marginale infection status of 235 randomly selected cattle was determined by using a nested PCR (nPCR) coupled with msp5 sequence analysis and hybridization. Judgment of the reliability of the nPCR and hybridization for detection of persistent infections was based on three observations. First, the nPCR was able to detect as few as 30 infected erythrocytes per ml. Second, the nPCR was able to consistently detect low levels of rickettsemia in seven carrier cattle experimentally infected with A. marginale. Third, msp5 sequence analysis showed >95% identity among 30 nPCR amplicons from cattle naturally infected with field strains of A. marginale. The nPCR and hybridization identified 151 infected and 84 uninfected cattle among the 235 animals tested. With a cutoff point of 28%, the rMSP5-cELISA showed a sensitivity of 96% and a specificity of 95%. These results indicate that the rMSP5-cELISA can sensitively and specifically detect cattle with naturally acquired persistent A. marginale infections and suggest that it is an excellent assay for epidemiological studies, eradication programs, and regulation of international cattle movement.     

Simple and rapid detection of Mycobacterium tuberculosis complex organisms in bovine tissue samples by PCR.

Mycobacterium bovis is a slowly growing microorganism, and confirmation of the diagnosis by conventional culture is a lengthy process. A simple, rapid method for the extraction of DNA from bovine tissue samples was developed and used in a PCR designed for the diagnosis of tuberculosis. Tissues from 81 cattle from tuberculosis-infected herds (group 1) and 19 cattle from tuberculosis-free herds (group 2) were tested in this PCR, and the results were compared with those of conventional culture. The PCR assay detected 71.4% of the culture-positive animals from group 1. Tissue from all animals in group 2 were negative in the PCR assay and by culture. The described method could be used as a rapid screening technique which would be complementary to culture of tissue specimens for the routine diagnosis of bovine tuberculosis. The PCR technique is much faster than culture and reduces the time for diagnosis from several months to 2 days. It also provides for the detection of M. bovis when rapidly growing Mycobacterium spp. are present in the sample and may be able to detect the presence of M. bovis in samples even when organisms have become nonviable.     

Identification of vaccine candidate peptides in the NcSRS2 surface protein of Neospora caninum by using CD4+ cytotoxic T lymphocytes and gamma interferon-secreting T lymphocytes of infected holstein cattle

Previously, our laboratory showed that Holstein cattle experimentally infected with Neospora caninum develop parasite-specific CD4+ cytotoxic T lymphocytes (CTL) that lyse infected, autologous target cells through a perforin-granzyme pathway. To identify specific parasite antigens inducing bovine CTL and helper T-lymphocyte responses for vaccine development against bovine neosporosis, the tachyzoite major surface proteins NcSAG1 and NcSRS2 were targeted. In whole tachyzoite antigen-expanded bovine T-lymphocyte lines, recombinant NcSRS2 induced potent memory CD4+- and CD8+-T-lymphocyte activation, as indicated by proliferation and gamma interferon (IFN-gamma) secretion, while recombinant NcSAG1 induced a minimal memory response. Subsequently, T-lymphocyte epitope-bearing peptides of NcSRS2 were mapped by using overlapping peptides covering the entire NcSRS2 sequence. Four experimentally infected cattle with six different major histocompatibility complex (MHC) class II haplotypes were the source of immune cells used to identify NcSRS2 peptides presented by Holstein MHC haplotypes. NcSRS2 peptides were mapped by using IFN-gamma secretion by rNcSRS2-stimulated, short-term T-lymphocyte cell lines, IFN-gamma enzyme-linked immunospot (ELISPOT) assay with peripheral blood mononuclear cells, and 51Cr release cytotoxicity assay of rNcSRS2-stimulated effector cells. Four N. caninum-infected Holstein cattle developed NcSRS2 peptide-specific T lymphocytes detected ex vivo in peripheral blood by IFN-gamma ELISPOT and in vitro by measuring T-lymphocyte IFN-gamma production and cytotoxicity. An immunodominant region of NcSRS2 spanning amino acids 133 to 155 was recognized by CD4+ T lymphocytes from the four cattle. These findings support investigation of subunit N. caninum vaccines incorporating NcSRS2 gene sequences or peptides for induction of NcSRS2 peptide-specific CTL and IFN-gamma-secreting T lymphocytes in cattle with varied MHC genotypes.     

Stimulation of T-helper cell gamma interferon and immunoglobulin G responses specific for Babesia bovis rhoptry-associated protein 1 (RAP-1) or a RAP-1 protein lacking the carboxy-terminal repeat region is insufficient to provide protective immunity against virulent B. bovis challenge

Rhoptry-associated protein 1 (RAP-1) is a targeted vaccine antigen for Babesia bovis and Babesia bigemina infections of cattle. The 60-kDa B. bovis RAP-1 is recognized by antibodies and T lymphocytes from cattle that recovered from infection and were immune to subsequent challenge. Immunization with native or recombinant protein was reported to reduce parasitemias in challenged animals. We recently reported that the NT domain of B. bovis RAP-1 contained immunodominant T-cell epitopes, whereas the repeat-rich CT domain was less immunostimulatory for T lymphocytes from cattle immune to B. bovis. The present study was therefore designed to test the hypothesis that the NT region of RAP-1, used as a vaccine with interleukin-12 and RIBI (catalog no. R-730; RIBI Immunochem Research, Inc., Hamilton, Mont. [now Corixa, Seattle, Wash.]) adjuvant to induce a type 1 response, would prime calves for antibody and T-helper cell responses comparable to or greater than those induced by full-length RAP-1 containing the C-terminal repeats. Furthermore, a type 1 immune response to RAP-1 was hypothesized to induce protection against challenge. Following four inoculations of either recombinant full-length RAP-1 or RAP-1 NT protein, RAP-1-specific immunoglobulin G (IgG) titers, T-lymphocyte proliferation, and gamma interferon production were similar. Similar numbers of NT region peptides were recognized. However, in spite of the presence of strong RAP-1-specific IgG and CD4(+)-T-lymphocyte responses that were recalled upon challenge, neither antigen stimulated a protective immune response. We conclude that successful priming of calves with recombinant RAP-1 and adjuvants that elicit strong Th1 cell and IgG responses is insufficient to protect calves against virulent B. bovis challenge.     

A double mutant [N543H+2393del9] allele in the LDL receptor gene in familial hypercholesterolemia: effect on plasma cholesterol levels and cardiovascular disease

Familial hypercholesterolemia is a genetic disorder caused by mutations in the LDL receptor gene. During a survey of mutations of LDL receptor gene in Spanish FH patients we found two mutations in the same allele: a missense N543H mutation in exon 11 and a 9bp inframe deletion (2393del9) located in exon 17. This double mutant allele was founded in 10 out of 458 unrelated patients: one homozygous FH [N543H+2393del9] + [N543H+2393del9], one compound heterozygote [N543H+2393del9] + [W-18X+E256K] and 8 heterozygotes. Flow cytometric analysis showed a defective LDL binding (20% of normal value) and internalization (23%) in lymphocytes from the homozygous patient; furthermore, studies of mitogen-stimulated lymphocytes demonstrated that the ability of LDL to support cell proliferation was impaired. Unexpectedly, not all carriers of the double mutant allele develop hypercholesterolemia and, furthermore, cholesterol-lowering treatment of the homozygous patient resulted in a 58% LDL cholesterol reduction. In conclusion, the phenotypic expression in the homozygous and heterozygous patients presented here, as well as the LDL-receptor residual activity, allowed the classification of this mutation as mild extending the group of mild mutations found at homozygosity.