Mining the genetic diversity of Ehrlichia ruminantium using map genes family

Understanding bacterial genetic diversity is crucial to comprehend pathogenesis. Ehrlichia ruminantium (E. ruminantium), a tick-transmitted intracellular bacterial pathogen, causes heartwater disease in ruminants. This model rickettsia, whose genome has been recently sequenced, is restricted to neutrophils and reticulo-endothelial cells of its mammalian host and to the midgut and salivary glands of its vector tick. E. ruminantium harbors a multigene family encoding for 16 outer membrane proteins including MAP1, a major antigenic protein. All the 16 map paralogs are expressed in bovine endothelial cells and some are specifically translated in the tick or in the mammalian host.In this study, we carried out phylogenetic analyses of E. ruminantium using sequences of 6 MAP proteins, MAP1, MAP1-2, MAP1-6, MAP1-5, MAP1+1 and MAP1-14, localized either in the center or at the borders of the map genes cluster.We show that (i) map1 gene is a good tool to characterize the genetic diversity among Africa, Caribbean islands and Madagascar strains including new emerging isolates of E. ruminantium; (ii) the different map paralogs define different genotypes showing divergent evolution; (iii) there is no correlation between all MAP genotypes and the geographic origins of the strains; (iv) The genetic diversity revealed by MAP proteins is conserved whatever is the scale of strains sampling (village, region, continent) and thus was not related to the different timing of strains introduction, i.e. continuous introduction of strains versus punctual introduction (Africa versus Caribbean islands).These results provide therefore a significant advance towards the management of E. ruminantium diversity. The differential evolution of these paralogs suggests specific roles of these proteins in host–vector–pathogen interactions that could be crucial for developing broad-spectrum vaccines.     

Assessment of PCR for routine identification of species of the Mycoplasma mycoides cluster in ruminants

DNA amplification techniques offer considerable promise for the identification of Mycoplasma mycoides cluster members. They avoid antigenic cross-reactivity and variability that hamper serological methods. Many sets of primers, specific of these different members and of Mycoplasma putrefaciens, have been proposed. To assess the reliability of some of these PCR tests in routine laboratory diagnostic use, 230 field strains supposed to belong to this group were simultaneously identified by PCR and an antigenic method. The results were well correlated to antigenic identification for M. putrefaciens, but PCR failed to identify respectively 74% and 52% of M. mycoides subsp. mycoides Large Colony type and M. capricolum subsp. capricolum strains. Any identification of M. mycoides subsp. mycoides Small Colony type must be confirmed by two different tests. Difficulties in defining the M. species bovine serogroup 7 were also encountered with both the PCR and immunological methods. The occurrence of putative variable antigen(s) on the mycoplasma surface may explain part of the identification difficulties encountered with the immunological methods.     

Canine visceral leishmaniasis: comparison of in vitro leishmanicidal activity of marbofloxacin, meglumine antimoniate and sodium stibogluconate

The control of canine leishmaniasis largely depends on the success of treatment. Drugs currently available to treat this disease are toxic and partially effective. The curative effect of marbofloxacin, a third-generation fluoroquinolone developed for veterinarian individual treatment, was evaluated in vitro in the presence of Leishmania infantum promastigotes and dog-monocyte-derived macrophages; meglumine antimoniate and sodium stibogluconate were used as comparative treatments. We observed that the killing of Leishmania promastigotes and intracellular amastigotes by marbofloxacin was dose-dependent. We demonstrated that successful treatment of canine infected macrophages for 48 h was possible with 500 microg/ml of marbofloxacin. Leishmanicidal activity acted through a TNF-alpha and nitric oxide pathway and correlated with the generation of nitric oxide (NO(2)) production by monocytes derived macrophages from infected (23+/-5 microM) or healthy (21+/-6 microM) dogs, in comparison with NO(2) concentration in infected/non-treated macrophages (< 3 microM, P<0.01). This significant induced parasiticidal effect correlated with extensive elimination of amastigotes by macrophages derived from infected (11+/-5) and healthy dogs (6+/-2), when compared to infected/non-treated macrophages (530+/-105 and 472+/-86 amastigotes, respectively, P< 0.01). Marbofloxacin was shown to be non-toxic at 500 microg/ml in vitro and no cell apoptosis was observed. The molecule was able to induce a parasitic process after significant elimination of amastigotes in leishmania-infected dog macrophages. We propose that marbofloxacin, compared to standard chemotherapeutic agents (meglumine antimoniate and sodium stibogluconate), could be an effective and pragmatic oral route alternative to treat canine leishmaniasis.     

CD86 molecule is a specific marker for canine monocyte-derived dendritic cells

In this study, canine monocyte-derived dendritic cells (cMo-DC) were produced in presence of canine GM-CSF (cGM-CSF) and canine IL-4 (cIL-4), and they were characterized by their dendritic morphology, MLR functionality and phenotype. We noticed that cMo-DC were labelled with three anti-human CD86 (FUN-1, BU63 and IT2.2 clones), whereas resting and activated lymphocytes or monocytes were not stained. CD86 expression was induced by cIL-4 and was up-regulated during the differentiation of the cMo-DC, with a maximum at day 7. Furthermore, cMo-DC were very potent even in low numbers as stimulator cells in allogeneic MLR, and BU63 mAb was able to completely block the cMo-DC-induced proliferation in MLR. We also observed that cMo-DC highly expressed MHC Class II and CD32, but we failed to determine their maturation state since the lack of commercially available canine markers. Moreover, cMo-DC contained cytoplasmic periodic microstructures, potentially new ultrastructural markers of canine DC recently described. In conclusion, this work demonstrates that the CD86 costimulatory marker is now usable for a better characterization of in vitro canine DC.     

HIGH-RESOLUTION PARATHYROID SONOGRAPHY

The purpose of this study was to assess the diagnostic utility of parathyroid ultrasonography to differentiate causes of hypercalcemia in dogs. We analyzed qualitative and quantitative ultrasound imaging findings and clinical pathology data from 33 dogs that underwent parathyroid ultrasound examination as part of the diagnostic evaluation for hypercalcemia. Diagnoses of the diseases causing hypercalcemia included parathyroid carcinoma (n = S), parathyroid adenoma (n = 15), parathyroid adenomatous hyperplasia (n = 6), chronic renal insufficiency (n = 3), and hypercalcemia of malignancy (n = 4). All parathyroid lesions were round or oval and hypoechoic compared with surrounding thyroid parenchyma. Adenomatous and adenocarcinomatous glands were 4 mm or larger in longest linear measurement on US examination and were statistically significantly larger than hyperplastic glands. (p < 0.001) Linear measurements of parathyroid glands acquired at the time of ultrasound examination correlated well with direct siz determination after surgical excision. (r²= 0.9, p < 0.0001) Parathyroid lesions <4 mm are highly suspicious for parathyroid adenoma or carcinoma, while US lesions <4 mm most likely represent primary adenomatous hyperplasia or secondary parathyroid hyperplasia. Parathyroid size estimation from ultrasound examination is an accurate predictor of true size.     

Atmospheric methane and nitrous oxide of the Late Pleistocene from Antarctic ice cores

The European Project for Ice Coring in Antarctica Dome C ice core enables us to extend existing records of atmospheric methane (CH4) and nitrous oxide (N2O) back to 650,000 years before the present. A combined record of CH4 measured along the Dome C and the Vostok ice cores demonstrates, within the resolution of our measurements, that preindustrial concentrations over Antarctica have not exceeded 773 +/- 15 ppbv (parts per billion by volume) during the past 650,000 years. Before 420,000 years ago, when interglacials were cooler, maximum CH4 concentrations were only about 600 ppbv, similar to lower Holocene values. In contrast, the N2O record shows maximum concentrations of 278 +/- 7 ppbv, slightly higher than early Holocene values.     

Disseminated histoplasmosis in a Bengal tiger (Panthera tigris)

Disseminated infection with Histoplasma capsulatum was diagnosed in a 7-yr-old female Bengal tiger (Panthera tigris). Clinical signs were nonspecific with the exception of brief periods of tachypnea for 5 days prior to death. H. capsulatum organisms were found in the lungs, tracheobronchial lymph nodes, and liver. Diagnosis was confirmed by tracheal wash, urine H. capsulatum enzyme immunoassay, and necropsy results. This report represents the first published account of disseminated histoplasmosis in a tiger.     

Minimally invasive patent ductus arteriosus occlusion in 5 dogs

OBJECTIVE: To report a technique for minimally invasive occlusion of patent ductus arteriosus (PDA) and outcome in 5 dogs. STUDY DESIGN: Clinical cases. Animals: Five, 4-6-month-old, dogs with PDA. MATERIALS AND METHODS: Titanium ligating clips were used for PDA closure in all dogs. Three dogs had video-enhanced mini-thoracotomy PDA occlusion. Two other dogs had thoracoscopic PDA occlusion using a custom-designed thoracoscopy clip applicator. RESULTS: Thoracoscopic PDA occlusion was successful in both dogs in which it was attempted. Complete PDA closure was achieved in 4 dogs. Three months after surgery, the largest dog had residual ductal flow that hemodynamically was insignificant. CONCLUSIONS: Although technically demanding, minimally invasive PDA occlusion is a safe and reliable technique in dogs. Preoperative measurement of the diameter of the PDA is crucial to determine if complete closure with metal clips can be achieved. CLINICAL RELEVANCE: Minimally invasive PDA occlusion should be considered as an alternative to occlusion via conventional thoracotomy.