Evaluation of recombinant proteins of Neospora caninum as vaccine candidates (in a mouse model)

Abortion, resulting from infections by the parasite Neospora caninum, is a major cause of economic loss to both the dairy and beef industries of cattle-producing countries of the world. Vaccination as a means of preventing abortion and/or infection represents a viable control strategy; indeed a commercial vaccine is available in some countries, albeit of unknown efficacy. The commercial vaccine is based on inactivated tachyzoites of N. caninum but other approaches based on lysates and recombinant antigens of N. caninum may also be feasible. In this study we have used an immunisation/challenge model of transplacental transmission, based on the Qs mouse with an Nc-Liverpool challenge, to investigate the vaccine potential of a number of formulations based on four recombinant proteins of N. caninum (GRA1, GRA2, MIC10, and p24B). All formulations studied were immunogenic in the mouse when assessed by ELISA using sonicated tachyzoite antigen as the target antigen. In one experiment, a mixture of MIC10 and p24B produced partial protection against transplacental transmission of N. caninum in this mouse model; in contrast a live infection of tachyzoites of NC-Nowra given before pregnancy always induces very high levels of protective immunity. The field of vaccines against Neospora-associated abortion in cattle is discussed. © 2008 Elsevier Ltd. All rights reserved

Design and Performance of the Advanced-Light-Source Double-crystal Monochromator

A new “Cowan type” double-crystal monochromator, based on the boomerang design used at National Synchrotron Light Source (NSLS) beamline X-24A, has been developed for beamline 9.3.1 at the Advanced Light Source (ALS), a windowless ultrahigh vacuum beamline covering the 1-6 keV photon-energy range. Beamline 9.3.1 is designed to simultaneously achieve the goals of high energy resolution, high flux, and high brightness at the sample. The mechanical design of the monochromator has been simplified, and recent developments in technology have been included. Measured mechanical precision of the monochromator shows significant improvement over existing designs. In tests with x-rays at NSLS beamline X-23Ʌ2, maximum deviations in the intensity of monochromatic light were just 7% during scans of several hundred eV in the vicinity of the Cr K edge (6 keV) with the monochromator operating without intensity feedback. Such precision is essential because of the high brightness of the ALS radiation and the overall length of beamline 9.3.1 (26 m)

Microsatellite documentation of male-mediated outcrossing between inbred laboratory strains of the self-fertilizing mangrove killifish (Kryptolebias marmoratus

Abstract Primers for 36 microsatellite loci were developed and employed to characterize genetic stocks and detect possible outcrossing between highly inbred laboratory strainsof the self-fertilizing mangrove killifish, Kryptolebias marmoratus. From attempted crosses involving hermaphrodites from particular geographic strains and gonochoristic males from others, 2 among a total of 32 surveyed progenies (6.2%) displayed multilocus heterozygosity clearly indicative of interstrain gametic syngamy. One of these outcross hybrids was allowed to resume self-fertilization, and microsatellite assays of progeny showed that heterozygosity decreased by approximately 50% after one generation, as expected. Although populations of K. marmoratus consist mostly of synchronous hermaphrodites with efficient mechanisms of internal self-fertilization, these laboratory findings experimentally confirm that conspecific males can mediate occasional outcross events and that this process can release extensive genic heterozygosity. The mangrovekillifish,Kryptolebias(formerlyRivulus)marmoratus, is the only vertebrate species known to reproduce uniparentally by internal self-fertilization Earlier molecular techniques including multilocus DNA fingerprinting have revealed extensive genetic variation in some natural populations of K. marmoratus Materials and Methods Microsatellite Development To enrich for microsatellites in a genomic library for K. marmoratus, we employed a modified version of a hybridization The hybridization solution was mixed with magnetic streptavidin beads (Invitrogen, Carlsbad, CA), and hybridized fragments were captured on a magnetic block. Enriched DNA, recovered by precipitation, was polymerase chain reaction (PCR) amplified (25 ll total reaction volume) under the following conditions: 10 mM Tris-HCl pH 9.0, 50 mM KCl, 0.1% Triton X-100, 2.0 mM MgCl 2 , 25.0 lg/ml bovine serum albumin, 0.2 mM each dNTP, 0.5 lM SuperSNX24 forward primer, and 0.5 units Taq DNA Polymerase (Promega, Madison, WI). The PCR product was ligated into a PCR 2.1-TOPO vector and transformed into One Shot Top10 Chemically Competent Escherichia coli cells. Positive colonies were screened for b-galactosidase activity using materials in the TOPO TA cloning kit (Invitrogen; Carlsbad, CA). Insert sizes were verified from positive colonies by PCR amplification with the M13 forward (À20) and reverse (À27) primers (0.5 lM final concentration). Inserts !500 bp were purified using ExoSAP-IT (United States Biochemicals, Cleveland, OH) and sequenced with Big Dye chemistry (version 3.1, Applied Biosystems, Foster City, CA) using M13 forward or reverse primer on an ABI 3100 Genetic Analyzer equipped with 80-cm capillaries. Sequences were edited using Sequencher version 4.1.4 (Gene Codes Corporation, Ann Arbor, MI), and primers flanking microsatellite regions were developed using OligoAnalyzer 3.0 (Integrated DNA Technologies, Coralville, IA; http://www. idtdna.com/Scitools/Applications/Primerquest/). Primers were tested by amplifying genomic DNA from adult K. marmoratus specimens in laboratory strains initiated many generations ago from single hermaphroditic specimens from Florida, Belize, or Honduras. PCR conditions were optimized for each primer pair, and one primer from each pair was labeled at the 5#-end in either of 2 ways: directly with a 6-FAM or HEX fluorophore (Integrated DNA Technologies) or by attaching a reverse tag (5#-GGAAACAGCTATGACCATG-3#) for tailed PCR with an M13 primer labeled with a 6-FAM, HEX, or NED (Applied Biosystems) fluorophore. The 36 sequences from which primers were developed to amplify microsatellite loci were deposited to GenBank (accession numbers DQ335412-DQ335447). Laboratory Crosses Crossing experiments involved highly inbred laboratory lines each propagated generation after generation by a single selffertilizing hermaphrodite (herm) and each tracing back to a single wild herm originally collected at Utila Island, Honduras (strains Hon2 and Hon7), Belize (Bel50.91), Florida's Everglades National Park (ENP12), or Brevard County, Florida (CCHA). Each attempted cross involved placing an old (nearly senescent) herm with a secondary male in a small culture dish containing 13% saltwater solution DNA Extractions High molecular weight genomic DNA samples were isolated using a standard guanidinium isothiocyanate (GIT) chaotropic salt procedure Genotypic Assays Amplifications of microsatellite loci with fluorescently labeled PCR primers were performed in a 12.5-ll reaction volume containing 1 ll of purified genomic DNA, PCR Master Mix (Promega), and 0.5 lM of both forward and reverse primers. Tailed PCRs were performed in a 10-ll volume containing 1 ll of purified genomic DNA, 10 mM Tris-HCl pH 9.0, 50 mM KCl, 0.1% Triton X-100, 1.5 mM MgCl 2 , 25.0 lg/ml bovine serum albumin, 0.2 mM each dNTP, 0.25 lM M13-labeled primer, 0.25 lM locus-specific primer, 0.025 lM tailed locus-specific primer, and 0.4 units Taq DNA Polymerase (Promega). PCR products (1.5 ll) were mixed with 2.5 ll deionized formamide, 0.5 ll of either GeneScan-400HD [ROX] or GeneScan-500[ROX] internal lane standard, and 0.5 ll loading dye. Samples were denatured in a 95°C heating block for 3-5 min and chilled on ice before being loaded onto 5% acrylamide gels. Samples were electrophoresed on an ABI 377 DNA Sequencer at 3000 V for 2 h at 55°C. Alleles were sized using the software packages GeneScan version

Characterization of an alpha tubulin gene sequence from Neospora caninum and Hammondia heydorni, and their comparison to homologous genes from Apicomplexa

The gene coding for α tubulin has been isolated by the polymerase chain reaction and sequenced from 2 isolates of Neospora caninum (Nc-Liverpool and Nc-SweB1). The data show that the gene, as in Toxoplasma gondii, is single copy and contains 3 exons and 2 introns and is identical in sequence in the 2 isolates studied. Comparison of the predicted protein sequence shows it to be identical to the α tubulin protein encoded by the T. gondii gene. The majority of the nucleotide substitutions that have occurred during the evolution of the T. gondii and N. caninum genes from their common ancestor have occurred in the third codon position. A partial coding sequence for α tubulin was also obtained from Hammondia heydorni and compared to other α tubulin sequences from Apicomplexa. The results show the sequences of the T. gondii, N. caninum and H. heydorni α tubulin genes to be similar but not identical in sequence, thereby providing new evidence that N. caninum and H. heydorni are genetically distinct species

Reduction in transplacental transmission of Neospora caninum in outbred mice by vaccination

Infection with the protozoan parasite Neospora caninum is an important cause of abortion in cattle. A major source of infection is transplacental transfer of the parasite from mother to offspring during pregnancy. This study describes investigations on the immunisation of outbred Qs mice before pregnancy with live or a crude lysate of N. caninum (NC-Nowra isolate) to prevent transplacental transfer of a challenge infection administered during pregnancy. Parasites present in the brains of pups from mice challenged with N. caninum (NC-Liverpool) were detected by PCR. Injection of live NC-Nowra tachyzoites before pregnancy dramatically reduced transplacental transfer from 75 to 0.8% in one experiment and from 76 to 8% in a second experiment. Injection of a crude lysate of NC-Nowra tachyzoites reduced transplacental transfer from 67 to 53% in one experiment and from 76 to 63% in a second experiment. Analysis of N. caninum-specific IgG1 and IgG2a antibody levels prior to pregnancy and challenge showed that NC-Nowra lysate induced a response skewed towards IgG1 whereas live parasites induced both IgG1 and IgG2a antibodies. After pregnancy and a challenge infection, a similar IgG1/IgG2a response was seen in all challenged groups. These results provide further positive support for the hypothesis that transplacental transmission of this parasite is preventable by vaccination