Towards a rapid molecular identification of the common phlebotomine sand flies in the Mediterranean region

The present study reports two simple molecular approaches allowing a rapid identification of the most prevalent species of phlebotomine sand flies in the Mediterranean region. A PCR protocol for the amplification of ITS2 ribosomal region and a PCR-RFLP on a mitochondrial DNA fragment (cytb-nd1) were settled in order to identify and discriminate among Phlebotomus perniciosus, Phlebotomus neglectus, Phlebotomus perfiliewi, Phlebotomus papatasi and Sergentomyia minuta. The ITS2 regions showed a certain degree of interspecific variability, which led to PCR amplicons of different sizes, i.e., 450, 490, 460, 480 and 530 bp for P. perniciosus, P. neglectus, P. perfiliewi, P. papatasi, and S. minuta, respectively. Analogously, the digestion of a mitochondrial DNA amplicon with Ase I enzyme showed five different restriction profiles, which allowed the unequivocal differentiation of the sand fly species examined. These methods might represent useful tools for a molecular large scale screening of phlebotomine sand fly species caught in areas where leishmaniasis is endemic, in order to plan appropriate epidemiological surveillance programs for both Leishmania spp. and their vectors.     

Effects of oxypolygelatin and dextran 70 on hemostatic variables in dogs

Objective To evaluate and compare coagulation variables following the administration of oxypolygelatin and dextran 70 to clinically healthy dogs. Study design Randomized cross‐over experimental study. Animals A total of eight healthy adult female Beagles aged 2–4 years old and weighing 11.8 ± 2.7 kg. Methods The dogs received a 15‐minute intravenous (IV) infusion of 5 mL kg^?1 oxypolygelatin or 10 mL kg^?1 6% dextran 70. Before (PRE) and at 2, 5, and 24 hours after administration, packed cell volume (PCV), total solids concentration (TS), prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen concentration (FIB), platelet numbers (Plat), factor VIII coagulant activity (VIII:C), von Willebrand factor antigen concentration (vWf:Ag) and platelet function and buccal mucosal bleeding time (BMBT) were measured. Platelet function was assessed using aggregation and by measuring ATP release from aggregating platelets over 6 minutes, with 20, 10, and 5 µm ADP and 5 and 10 µg of collagen mL^?1 as platelet activation agonists. Results All baseline values were within our normal ranges, except for one dog that had low vWf:Ag PRE values prior to both dextran and oxypolygelatin administration. Following dextran and oxypolygelatin administration, the PCV and TP were significantly (p < 0.05) decreased. Plat, FIB, and vWf:Ag decreased, while BMBT and VIII:C increased following dextran administration. Dextran also caused a significant decrease in platelet aggregation in response to ADP. Oxypolygelatin caused a significant decrease in vWf:Ag, Plat, and FIB compared to PRE values. The total amount of ATP released, standardized to platelet number, did not vary significantly for either group at any sampling time from PRE values. No significant changes from PRE values were noted at any time in either group for PT or APTT. Conclusion At the doses administered, both dextran and oxypolygelatin can interfere with hemostatic variables in healthy dogs, but dextran's effect is more profound and prolonged when compared to oxypolygelatin. Clinical relevance Oxypolygelatin causes fewer hemostatic abnormalities when compared to dextran, making it a superior colloid for administration at the doses tested.     

Transcranial Doppler sonographic findings in granulomatous meningoencephalitis in small breed dogs

Granulomatous meningoencephalitis (GME) is an acute, progressive, and often fatal inflammatory disease of the central nervous system, affecting mainly small and toy dog breeds. A definitive diagnosis of GME can only be achieved through histopathologic examination of samples collected after death. This retrospective study describes transcranial Doppler ultrasonography (TDS) findings in dogs with confirmed clinical histopathology of GME. Eleven dogs were selected for this study. Sonographic findings in B-mode demonstrated diffuse decreased brain parenchyma echogenicity in 9 dogs, ventriculomegaly in 8 dogs, brain atrophy in 4 dogs, and hyperechoic focal lesions in 6 dogs. Color Doppler imaging revealed more obvious vessels of the arterial circle in 10 dogs. Spectral Doppler examination was performed in 10 dogs to detect the 6 major cerebral arteries of interest. The examination showed normal and high resistive index (RI) values in the outlined arteries. The TDS findings were consistent with pathology found on postmortem examination.     

The innate antiviral immune system of the cat: molecular tools for the measurement of its state of activation

The innate immune system plays a central role in host defence against viruses. While many studies portray mechanisms in early antiviral immune responses of humans and mice, much remains to be discovered about these mechanisms in the cat. With the objective of shedding light on early host-virus interactions in felids, we have developed 12 real-time TaqMan(?) qPCR systems for feline genes relevant to innate responses to viral infection, including those encoding for various IFNα and IFNω subtypes, IFNβ, intracellular antiviral factor Mx, NK cell stimulator IL-15 and effectors perforin and granzyme B, as well as Toll-like receptors (TLRs) 3 and 8. Using these newly developed assays and others previously described, we measured the relative expression of selected markers at early time points after viral infection in vitro and in vivo. Feline embryonic fibroblasts (FEA) inoculated with feline leukemia virus (FeLV) indicated peak levels of IFNα, IFNβ and Mx expression already 6h after infection. In contrast, Crandell-Rees feline kidney (CrFK) cells inoculated with feline herpes virus (FHV) responded to infection with high levels of IFNα and IFNβ only after 24h, and no induction of Mx could be detected. In feline PBMCs challenged in vitro with feline immunodeficiency virus (FIV), maximal expression levels of IFNα, β and ω subtype genes as well as IL-15 and TLRs 3, 7 and 8 were measured between 12 and 24h after infection, whereas expression levels of proinflammatory cytokine gene IL-6 were consistently downregulated until 48h post inoculation. A marginal upregulation of granzyme B was also observed within 3h after infection. In an in vivo experiment, cats challenged with FIV exhibited a 2.4-fold increase in IFNα expression in blood 1 week post infection. We furthermore demonstrate the possibility of stimulating feline immune cells in vitro with various immune response modifiers (IRMs) already known for their immunostimulatory properties in mice and humans, namely Poly IC, Resiquimod (R-848) and dSLIM?, a synthetic oligonucleotide containing several unmethylated CpG motifs. Stimulation of feline PBMCs with dSLIM? and R-848 effectively enhanced expression of IFNα within 12h by factors of 6 and 12, respectively, and Poly IC induced an increase in Mx mRNA expression of 28-fold. Altogether, we describe new molecular tools and their successful use for the characterization of innate immune responses against viruses in the cat and provide evidence that feline cells can be stimulated by synthetic molecules to enhance their antiviral defence mechanisms.     

In vitro inhibitory effect of trichostatin A on canine grade 3 mast cell tumor

Mast cell tumor (MCT) is one of the most prevalent neoplasms that affect skin and soft tissue in dogs. Because mast cell tumors present a great variety of clinical appearance and behavior, their treatment becomes a challenge. Trichostatin A (TSA), an antifungal antibiotic, has shown inhibitory effects on the proliferation and induction of apoptosis in various types of cancer cells. In order to evaluate the potential of trichostatin A as a therapeutic drug, cells of grade 3 MCT were cultured and treated with concentrations of 1 nM to 400 nM of TSA. MTT assay and trypan blue exclusion assays were performed to estimate cell growth and cell viability, and cell cycle analysis was evaluated. TSA treatment showed a reduction in numbers of viable cells and an increase of cell death by apoptosis. The cell cycle analysis showed an increase of hypodiploid cells and a reduction of G0/G1 and G2/M –phases. According to these results, trichostatin A may be an interesting potential chemotherapeutic agent for the treatment of canine MCT.     

Clinical evaluation of random skin flaps based on the subdermal plexus secured with sutures or sutures and cyanoacrylate adhesive for reconstructive surgery in dogs

OBJECTIVE: To evaluate the use of subdermal plexus skin flaps for closing defects after excision of cutaneous and subcutaneous tumors in dogs and to compare outcome of flaps secured with sutures and those secured with butyl-cyanoacrylate and intermittent sutures. STUDY DESIGN: Clinical study. ANIMALS: Fifteen dogs. METHODS: After excision of cutaneous or subcutaneous tumors the skin defect was reconstructed by random flaps based on the subdermal plexus. Flap skin edges were apposed with simple interrupted 4-0 monofilament nylon sutures (group 1; 5 dogs) or nylon sutures alternated with butyl-cyanoacrylate adhesive (group 2; 10 dogs). Flaps were evaluated every 48 hours when bandages were changed, until complete healing. RESULTS: Random flaps based on the subdermal plexus were effectively used to close wound defects; mean flap survival was 89%. Partial flap necrosis occurred in 4 dogs. Wound margins apposed with butyl-cyanoacrylate had thinner and more esthetic scars than sutured margins. CONCLUSION: Random flaps based on the subdermal plexus proved to be versatile for covering limb wounds after excision of cutaneous or subcutaneous tumors. Mean survival rate was comparable to that reported for axial pattern flaps. Butyl-cyanoacrylate adhesive was easy to apply, allowed accurate margin apposition with good cosmetic outcome and reduced sutures needed. CLINICAL RELEVANCE: Cyanoacrylate adhesive should be considered in lieu of suture closure to secure random skin flaps based on the subdermal plexus in dogs.     

Electron microscopic observation of the respiratory tract of SPF piglets inoculated with Mycoplasma hyopneumoniae.

Seven hysterectomy derived piglets were repeatedly challenged with Mycoplasma hyoneumoniae during the first week of life. Samples of trachea, bronchi and lung tissue collected 2–11 weeks post-inoculation (p.i.) were examined using light and electron microscopy. Autoradiography was used to study in more detail the site of M. hyopneumoniae multiplication. Gross lesions were observed in lung tissue and were characterized by hyperplasia of the epithelium and an increased mononuclear cell accumulation in perivascular and peribronchiolar areas. Mild lesions of the trachea and the bronchi, including epithelial hyperplasia and infiltration of the lamina propria by inflammatory cells, were noted.

Electron microscopy showed that, 2–6 weeks p.i., changes in the mid-trachea and bronchi surface consisted of the loss of cilia. Mycoplasmas covered tufts of cilia remaining on the epithelial cell surface. Scanning and transmission electron micrographs showed that they were predominantly found closely associated with the top of cilia. No specialized terminal structure could be seen and no mycoplasma cells were identified lying free in the lumen nor in close contact with the plasma membrane of cells of microvilli. Some fine fibrils radiating from one mycoplasma to another or to cilia were seen at higher magnification by scanning electron microscopy. Six to eleven weeks p.i., a disrupted epithelial surface lacking cilia was observed. Cells were desquamated and shed into the lumen with cellular remains containing droplets of mucus.

Autoradiography revealed that label corresponded to the observed mycoplasma distribution. At the top of cilia, a high density of labeling was visible in the zone of high mycoplasma concentration. Therefore, incorporation of the label in the mycoplasma is proof or their multiplication in the trachea.

The intimate association between the mycoplasma and cilia may be an important factor in the pathogenesis of the disease caused by M. hyopneumoniae (swine enzootic pneumonia).     

Squash-prep cytology in the diagnosis of canine and feline nervous system lesions: a study of 42 cases

BACKGROUND: The increased sophistication of imaging techniques in veterinary medicine allows the detection of a wide variety of intracranial and intraspinal lesions; however, imaging often does not provide a definitive diagnosis for nervous system (NS) lesions. Cytology is emerging as a useful diagnostic tool for obtaining a fast and accurate assessment of NS lesions, but little information is available for dogs and cats. OBJECTIVES: The purpose of this study was to assess the accuracy of cytologic evaluation of squash samples from NS lesions in dogs and cats and to consider cytology-based diagnostic guidelines and sources of misdiagnosis. METHODS: Cytologic specimens from masses localized in the central and peripheral NS taken during surgery or postmortem examination were classified into 3 groups according to the final histopathologic diagnosis: Group 1 = completely correct diagnosis, when the cytologic diagnosis and final histologic diagnosis were exactly correlated; Group 2 = partial correlation, when the cytologic diagnosis only partially correlated with the final histologic diagnosis, and Group 3 = no correlation, when the cytologic diagnosis was incorrect and there was no correlation with the general histologic type of lesion. The diagnostic accuracy of cytopathology was calculated by considering the histopathologic diagnosis as the "gold standard," and calculating a 95% confidence interval (CI). RESULTS: A total of 42 animals (33 dogs and 9 cats) were included in the study. The cytologic diagnoses were classified in Group 1 for 32 cases (76%; 95% CI 0.63-0.89), in Group 2 for 6 cases (14%; 95% CI 0.04-0.25), and in Group 3 for 4 cases (10%; 95% CI 0.006-0.18). Considering both complete and partial correlation as an adequate result, cytologic diagnosis was satisfactory in 90% of biopsies. CONCLUSIONS: Although the current series of cases is relatively small, cytologic evaluation of squash preparations can be considered a fairly accurate and reliable tool in the diagnosis of NS lesions.