ABSTRACT
The toluidine blue (TB) stain has been used in different species to evaluate the degree of chromatin condensation. The objectives of this study were as follows: simplify the TB stain to evaluate sperm in canine raw semen, verify the staining patterns for this species using this simplified technique and establish a protocol for using dithiothreitol (DTT) as a positive control for TB staining in dogs. Twenty-one ejaculates were collected from 7 adult male dogs; semen was extended, fixed with ethanol 96° and stained with TB using 2 staining times: 15 and 30 min. In addition, 3 incubation times with 1% DTT were assayed (2, 5 and 30 min). Three staining patterns were established: light blue colouring (TB negative, normal chromatin condensation), light violet (TB intermediate, some degree of chromatin decondensation) and dark blue-violet (TB positive, high degree of chromatin decondensation). No significant differences (p > 0.05) were observed between the staining times (15 and 30 min) for any of the TB patterns. All DTT incubation times (2, 5 and 30 min) showed 100% sperm positive to TB. To conclude, it was possible to simplify the TB stain and determine the different patterns in canine spermatozoa. Also, DTT can be used both as a positive control for the stain and to evaluate individual susceptibility to decondensation in vitro.
Subject(s)
Coloring Agents/chemistry , DNA/chemistry , Semen Analysis/veterinary , Tolonium Chloride/chemistry , Animals , Chromatin/ultrastructure , Dogs , Male , Semen/cytology , Semen Analysis/methods , Spermatozoa/cytology , Staining and Labeling/methods , Staining and Labeling/veterinaryABSTRACT
VirB proteins from Brucella spp. constitute the type IV secretion system, a key virulence factor mediating the intracellular survival of these bacteria. Here, we assessed whether a Th1-type immune response against VirB proteins may protect mice from Brucella infection and whether this response can be induced in the dog, a natural host for Brucella. Splenocytes from mice immunized with VirB7 or VirB9 responded to their respective antigens with significant and specific production of gamma interferon (IFN-γ), whereas interleukin-4 (IL-4) was not detected. Thirty days after an intraperitoneal challenge with live Brucella abortus, the spleen load of bacteria was almost 1 log lower in mice immunized with VirB proteins than in unvaccinated animals. As colonization reduction seemed to correlate with a Th1-type immune response against VirB proteins, we decided to assess whether such a response could be elicited in the dog. Peripheral blood mononuclear cells (PBMCs) from dogs immunized with VirB proteins (three subcutaneous doses in QuilA adjuvant) produced significantly higher levels of IFN-γ than cells from control animals upon in vitro stimulation with VirB proteins. A skin test to assess specific delayed-type hypersensitivity was positive in 4 out of 5 dogs immunized with either VirB7 or VirB9. As both proteins are predicted to locate in the outer membrane of Brucella organisms, the ability of anti-VirB antibodies to mediate complement-dependent bacteriolysis of B. canis was assessed in vitro. Sera from dogs immunized with either VirB7 or VirB9, but not from those receiving phosphate-buffered saline (PBS), produced significant bacteriolysis. These results suggest that VirB-specific responses that reduce organ colonization by Brucella in mice can be also elicited in dogs.