ABSTRACT
Germ-line transformation of a major agricultural pest, the Mexican fruit fly (Anastrepha ludens Loew, Mexfly), was achieved using composite piggyBac transposable elements marked with green, yellow and red fluorescent proteins (CopGreen, PhiYFP and J-Red). We also investigated the possibility of generating transposon-free insertions, in order to address potential concerns relating to proposed field use of transgenic Mexfly. We describe a highly efficient method for transforming Mexfly, compare efficiency of piggyBac terminal sequences for transformation and also describe the derivation of a transposon-free insertion line. The development of an efficient transformation system for Mexfly holds great promise for improved applications of the sterile insect technique, a major component of the present control measures for this economically important pest species.
Subject(s)
DNA Transposable Elements , Genetic Engineering/methods , Germ Cells , Tephritidae/genetics , Transformation, Genetic , Animals , Embryo, Nonmammalian , Female , Genetic Markers , Microinjections , Plasmids , Tephritidae/embryologyABSTRACT
Studies on cytokine (IFN-gamma, IL-2, IL-4, IL-5, IL-10) and inducible NO-synthase (iNOS) gene transcription in mesenteric lymph nodes (MLN) and the caecum wall were performed 0, 48, and 72h after primary and challenge infections of rats with Eimeria separata using RT-PCR. The amount of IFN-gamma mRNA was elevated in MLN and caeca 72h after primary and 48-72h after challenge infection when compared with uninfected controls. Increased amounts of IL-2 mRNA were only found in MLN of infected rats 72h post-infection (p.i.). In case of IL-10, infections did not affect the amount of mRNA in MLN, but led to markedly increased levels in the caecum wall of both infected groups 48 and 72h p.i. Levels of IL-4 mRNA remained unchanged after infections and IL-5 gene transcripts were undetectable. Amounts of iNOS mRNA (not investigated in MLN) were found strongly enhanced 48 and 72h p.i. in the caecum walls of all infected animals when compared with naive controls. The data are discussed in regard of the cellular source of the cytokines and their immunological role.
Subject(s)
Coccidiosis/veterinary , Cytokines/biosynthesis , Eimeria , Nitric Oxide Synthase/biosynthesis , RNA, Messenger/metabolism , Rodent Diseases/metabolism , Animals , Cecum/parasitology , Coccidiosis/metabolism , Cytokines/genetics , Interferon-gamma/biosynthesis , Interferon-gamma/genetics , Interleukin-10/biosynthesis , Interleukin-10/genetics , Interleukin-2/biosynthesis , Interleukin-2/genetics , Interleukin-4/biosynthesis , Interleukin-4/genetics , Interleukin-5/biosynthesis , Interleukin-5/genetics , Male , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase Type II , Rats , Rats, Inbred Lew , Reverse Transcriptase Polymerase Chain Reaction/veterinary , Rodent Diseases/parasitologyABSTRACT
A cDNA clone Ls110 was isolated from a female Litomosoides sigmodontis expression library using an antiserum raised against the microfilarial sheath. The complete cDNA encodes a protein (Ls110) of 382 amino acids. Southern and PCR analyses revealed the presence of Ls110 in L. sigmodontis as a single copy gene. The transcription of the Ls110 gene was limited to female worms. In these worms the transcription was confined to the epithelial cells of the uterus. The protein Ls110 was detected not only in the epithelial layer of the uterus but also secreted in the lumen of the uterus. All the intra-uterine embryonic stages showed the protein bound to their egg shell/sheath, except the early multicellular embryonic stages and fully developed microfilariae. The transient occurrence of Ls110 on these structures of intra-uterine stages besides the presence of a cysteine-rich N-terminal region (SXC-like domain) suggest that the protein may play a role in the formation of the microfilarial sheath during embryogenesis.
Subject(s)
Filarioidea/embryology , Helminth Proteins/genetics , Microfilariae/embryology , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Blotting, Southern , Blotting, Western , Cloning, Molecular , DNA, Complementary/analysis , DNA, Helminth/analysis , Female , Filarioidea/genetics , Helminth Proteins/chemistry , Helminth Proteins/metabolism , Immunohistochemistry , In Situ Hybridization , Male , Microfilariae/genetics , Molecular Sequence Data , Polymerase Chain Reaction , RNA, Helminth/analysis , Transcription, Genetic/geneticsABSTRACT
In this study Onchocerca gutturosa was compared with O. volvulus in an ELISA test to detect Onchocerca-specific IgG and IgG subclasses. The test was developed and standardized to detect Onchocerca-specific IgG and IgG subclasses in sera of onchocerciasis patients and endemic controls. Onchocerca volvulus and O. gutturosa crude water-soluble antigens showed no significant difference in detecting onchocerca-specific IgG antibody (T = 1.88, P greater than 0.05). The levels of IgG subclasses varied greatly. IgG4 showed the highest detected mean level (0.84 +/- 0.59) and the other three subclasses showed considerably lower mean levels (IgG1 = 0.27 +/- 0.16, IgG2 = 0.24 +/- 0.17, IgG3 = 0.28 +/- 0.12). The status and score of skin lesions were found to have significant effect on the IgG and IgG subclasses levels (all P less than 0.001). IgG4 showed a positive correlation with the microfilarial (Mf) load (r = 0.21, P less than 0.03). IgG3 levels have a significant negative correlation with the Mf load (r = -0.23, P less than 0.02). The biological significance of these IgG and IgG subclasses in onchocerciasis is discussed.