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.     

Microplate technique to determine hemolytic activity for routine typing of Listeria strains.

Because the hemolysis produced by Listeria monocytogenes and Listeria seeligeri on blood agar is frequently difficult to interpret, we developed a microplate technique for the routine determination of hemolytic activity with erythrocyte suspensions. This microtechnique is a simple and reliable test for distinguishing clearly between hemolytic and nonhemolytic strains and could be used instead of the CAMP (Christie-Atkins-Munch-Petersen) test with Staphylococcus aureus in the routine typing of Listeria strains. Furthermore, our results suggest that the quantitation of the hemolytic activity of the Listeria strains, along with the D-xylose, L-rhamnose, and alpha-methyl-D-mannoside acidification tests, allows the differentiation of L. monocytogenes, L. seeligeri, and Listeria ivanovii. We also observed that the treatment of erythrocytes with crude exosubstances of rhodococcus equi, Pseudomonas fluorescens, Acinetobacter calcoaceticus, and S. aureus enhanced the hemolytic activity of all Listeria strains with this characteristic.     

Expression of “early” and “late” viral functions in a somatic cell hybrid between a mouse cell and a spontaneous yielder SV 40-transformed Chinese hamster cell

Summary A somatic cell hybrid (Cl. 6d) was originated from the fusion of mouse (3T3-4E) and spontaneous yielder SV 40-transformed Chinese hamster (CHK/SVLP AG) cells. During the early stages of its history, the Cl. 6d hybrid underwent a rapid chromosome loss, preferentially loosing hamster chromosomes. This was not a constant tendency of the hybrid cells.As the parental CHK/SVLP AG cells, the hybrid cells were always found 100 per cent SV 40 T-antigen positive. While in CHK/SVLP AG cells infectious SV 40 DNA, V-antigen and virus were regularly detected, in the hybrid cells only infectious DNA was occasionally detected. This was not due either to the loss of an essential Chinese hamster gene(s) or to the presence of an inhibiting mouse cell component(s); it was apparently the consequence of inability of the cells to properly activate the resident SV 40 genome(s).After superinfection with SV 40 DNA, the hybrid cells—though capable of synthesizing SV 40 V-antigen—were unable to ensure virus assembly. Experimental evidence was obtained suggesting that SV 40 maturation is dependent on a cellular function(s).With 1 Figure     

Coxsackievirus B3-associated myocardial pathology and viral load reduced by recombinant soluble human decay-accelerating factor in mice

Coxsackievirus B3 (CVB3) infection can result in myocarditis, which in turn may lead to a protracted immune response and subsequent dilated cardiomyopathy. Human decay-accelerating factor (DAF), a binding receptor for CVB3, was synthesized as a soluble IgG1-Fc fusion protein (DAF-Fc). In vitro, DAF-Fc was able to inhibit complement activity and block infection by CVB3, although blockade of infection varied widely among strains of CVB3. To determine the effects of DAF-Fc in vivo, 40 adolescent A/J mice were infected with a myopathic strain of CVB3 and given DAF-Fc treatment 3 days before infection, during infection, or 3 days after infection; the mice were compared with virus alone and sham-infected animals. Sections of heart, spleen, kidney, pancreas, and liver were stained with hematoxylin and eosin and submitted to in situ hybridization for both positive-strand and negative-strand viral RNA to determine the extent of myocarditis and viral infection, respectively. Salient histopathologic features, including myocardial lesion area, cell death, calcification and inflammatory cell infiltration, pancreatitis, and hepatitis were scored without knowledge of the experimental groups. DAF-Fc treatment of mice either preceding or concurrent with CVB3 infection resulted in a significant decrease in myocardial lesion area and cell death and a reduction in the presence of viral RNA. All DAF-Fc treatment groups had reduced infectious CVB3 recoverable from the heart after infection. DAF-Fc may be a novel therapeutic agent for active myocarditis and acute dilated cardiomyopathy if given early in the infectious period, although more studies are needed to determine its mechanism and efficacy.     

Nucleolar organizer regions in transitional cell tumors of the bladder

The traditional silver staining technique that identifies nucleolar organizer region-associated proteins has recently been adapted and applied to paraffin sections, and used to examine changes in nucleolar organizer region numbers in a variety of benign and malignant conditions. We describe herein the enumeration of nucleolar organizer regions in a variety of benign and malignant conditions of the transitional epithelium of the urinary bladder. While there is a statistical separation between normal and neoplastic groups, there is a degree of overlap between normal, inflammatory, dysplastic, and neoplastic urothelium.