Comparative evaluation of field samples using 2 in-clinic assays for heartworm antigen detection in dogs.

Dirofilaria immitis (heartworm) antigen testing is routinely performed in veterinary practices to detect canine heartworm infections. The purpose of this study was to evaluate the performance of two in-clinic assays to detect heartworm antigen on field samples from practices in heartworm endemic regions. Veterinary staff in 3 practices located in the Southern United States performed a side by side comparison of the SNAP® 4Dx® Plus Test (IDEXX) and the VETSCAN FLEX4® Test (Zoetis) on samples from canine patients presented for vector-borne disease screening. Assays were performed according to the manufacturer's instructions. The remaining plasma sample was submitted for confirmatory testing using the PetChek® Heartworm Test (IDEXX) including immune complex dissociation (ICD) by heat treatment. A total of 232 samples were evaluated by the two in-clinic assays and PetChek Test. SNAP 4Dx Plus was significantly more sensitive for the detection of heartworm antigen in this study; sensitivity was 97.4 % for the SNAP 4Dx Plus test and 76.9 % for VETSCAN FLEX4 test (p < 0.01). The specificity of both tests was 99.5 %. This study reveals significant difference in detecting canine heartworm antigen in field samples.

Isolation of mammalian cell mutants that are X-ray sensitive, impaired in DNA double-strand break repair and defective for V(D)J recombination.

The Chinese hamster lung V79-4 cell line was infected with a Moloney murine leukemia retrovirus and the infected cells were subsequently screened for mutants that were sensitive to X-rays using a toothpicking/96-well replica plating technique. Four independent mutants that were sensitive to X-irradiation (sxi-1 to sxi-4) were isolated from 9000 retrovirally infected colonies. A pulse-field gel electrophoresis (PFGE) assay demonstrated that all of the sxi mutants were impaired in DNA double-strand break (DSB) repair, thus providing a molecular explanation for the observed X-ray sensitivity. Interestingly, additional PFGE experiments demonstrated that for any given X-ray dose all of the mutants incurred more DNA DSBs than the parental V79-4 cell line indicating there may be some inherent fragility to sxi chromosomes. Cross-sensitivity to other DNA-damaging agents including bleomycin, mitomycin C and methyl methanesulfonate indicated that sxi-2, sxi-3 and sxi-4 appear to be specifically hypersensitive to genotoxic agents that cause DNA DSBs, whereas sxi-1 appeared to be hypersensitive to multiple types of DNA lesions. Lastly, in preliminary experiments all of the sxi mutants demonstrated an inability to carry out V(D)J recombination, a somatic DNA rearrangement process required for the assembly of lymphoid antigen receptor genes. Thus, the sxi cell lines have interesting phenotypes which should make them valuable tools for unraveling the mechanism(s) of DNA DSB repair and recombination in mammalian cells.

Complementation of the ionizing radiation sensitivity, DNA end binding, and V(D)J recombination defects of double-strand break repair mutants by the p86 Ku autoantigen.

Two ionizing radiation-sensitive (IRs) and DNA double-strand break (DSB) mutants, sxi-3 and sxi-2, were shown to be severely deficient in a DNA end binding activity, similar to a previously described activity of the Ku autoantigen, correlating with the xrs (XRCC5) mutations. Cell fusions with xrs-6, another IRs, DSB repair-deficient cell line, defined these sxi mutants in the XRCC5 group. sxi-3 cells have low expression levels of the p86Ku mRNA. Introduction of the Ku p86 gene, but not the p70 Ku gene, complemented the IRs, DNA end binding, and variable (diversity) joining [V(D)J] recombination signal and coding junction deficiencies of sxi-3. Thus, the p86 Ku gene product is essential for DSB repair and V(D)J recombination.