Roraimine: a bisbenzylisoquinoline alkaloid from Cissampelos sympodialis roots

The roots of Cissampelos sympodialis yielded Roraimine (1), a novel bisbenzylisoquinoline alkaloid and liriodenine (2), a known oxoaporphine alkaloid.     

Reactions between Cr(VI) and wood and its model compounds

Hexavalent chromium solutions at two pH levels (about 1.3 and 7.8) were reacted with wood, gum ghatti, lignin, cellulose and simple model compounds representing wood chemical structures (guaiacol, vanillin, vanillyl alcohol, homovanillyl alcohol, methyl-(β-D-glucoside, and methyl-β-cellobioside). Reaction products were isolated and characterised by elemental analysis, magnetic susceptibility, and Fourier-transform infrared spectroscopy (FTIR). Data suggest that all the chromium present in the reaction products is in its trivalent oxidation state. After reduction reactions, wood and macromolecular substances generally fixed a relatively low level of chromium, which contrasts with the high level in the products isolated from the reaction with the model compounds. It appears that the reaction of the simple model compounds with Cr(VI) did not produce complex adducts of Cr(III) but inorganic substances, of the hydrated chromium oxide type. Therefore, under the experimental conditions applied, simple model compounds do not seem to behave chemically as wood or as the macromolecular substances investigated. Hence, their significance as model compounds must be object of further assessment. An exception is the product from the reaction of vanillyl alcohol and K₂CrO₄ aq. This contains a relatively small amount of chromium and its FTIR spectrum presents rather well defined bands indicating reaction of the phenolic hydroxyl and secondary alcohol groups. This system must be worthy of further investigation as a model for lignin.     

Effect of imidocarb and levamisole on the experimental infection of BALB/c mice by Leishmania (Leishmania) amazonensis

The adverse effects from using currently available drugs for the treatment of leishmaniasis have motivated the search for new therapeutical agents. The aim of this work was to evaluate the effect of imidocarb and levamisole on the treatment of BALB/c mice experimentally infected by Leishmania (Leishmania) amazonensis. BALB/c mice were infected with 10(6) promastigotes of L. (L.) amazonensis (IFLA/BR/67/PH8) and, starting on day 51, mice were treated subcutaneously with imidocarb (IMD, 34 mg/kg), imidocarb plus levamisole (IMD+LVS, 34 and 12 mg/kg, respectively), only levamisole (LVS, 12 mg/kg) or without treatment (control). Lesion size and swelling were weekly monitored for 10 weeks after the beginning of the treatment. On day 121 post-infection, serum levels of specific IgG from infected mice were evaluated, as well as histopathological and morphometric alterations in the footpad, lymph nodes and spleen of these animals. The data obtained in this study demonstrated that, when compared to controls, mice treated with IMD had lower levels of IgG anti-L. (L.) amazonensis (34.45%), smaller vacuolar area in macrophages (3.75%), lower number of megakaryocytes in spleen (63.19%) and lower parasite burden in the footpad (30.2%). Thus, the evaluated parameters suggest the use of imidocarb as a potential drug in the treatment of tegumentary leishmaniasis.     

Polyarthritis associated with visceral leishmaniasis in a juvenile dog

Canine visceral leishmaniasis (CL) is a zoonosis and a chronic systemic disease characterized by a wide spectrum of clinical signs, including, in rare occasions, polyarthritis. This report describes a case of CL in an 8-month old male boxer dog with a history of lameness, fever and lymphadenopathy. A definitive diagnosis of CL was based on the observation of the Leishmania amastigotes seen concomitantly, and for the first time, in the lymph nodes aspiration smears (in macrophages), synovial fluid (in macrophages and neutrophils) and blood (in neutrophils). Despite this extensive dissemination of the parasite, the animal was successfully treated with a multi-step combination of meglumine antimoniate, aminosidine and allopurinol.     

Parasite density and impaired biochemical/hematological status are associated with severe clinical aspects of canine visceral leishmaniasis

We have performed a detailed investigation in 40 dogs naturally infected with Leishmania infantum (syn. chagasi), subdivided into three groups: asymptomatic (AD = 12), oligosymptomatic (OD = 12) and symptomatic (SD = 16), based on their clinical features. Twenty non-infected dogs (CD) were included as control group. Serological analysis, performed by IFAT and ELISA, demonstrated higher antibodies titers in SD in comparison to the AD. A positive correlation was found between parasite density in the spleen and skin smears as well as the bone marrow parasitism with clinical status of the infection. We observed that the progression of the disease from asymptomatic to symptomatic clinical form was accompanied by intense parasitism in the bone marrow. It is likely that this led to the impaired biochemical/hematological status observed. Finally, we believe that the follow-up of these parameters could be a relevant approach to be used as markers during therapeutic and vaccine evaluations.     

A femorotibial joint swelling with popliteal lymph node enlargement in a Rottweiler

A 9-year-old male Rottweiler was presented to the Veterinary Medical Hospital Montenegro, Porto, Portugal with a large mass medial to the left stifle and radiographic signs of bone lysis involving the proximal tibia, fibula, and distal femur. A fine-needle aspiration was obtained from left popliteal lymph node, which was markedly enlarged. Cytologic examination revealed a highly cellular sample consisting of pleomorphic cells with marked anisocytosis and anisokaryosis and other criteria of malignancy. Some cells contained intracytoplasmic, granular, dark brown material, consistent with hemosiderin. Histologic evaluation of the surgically-excised lymph node revealed a neoplastic proliferation of histiocytic cells, with marked pleomorphism; occasional cells were erythrophagocytic. Mitotic figures were frequently observed, and many were atypical. Histologic findings were consistent with malignant histiocytosis. Necropsy examination confirmed the diagnosis of malignant histiocytosis with systemic involvement affecting most organs examined. This case of malignant histiocytosis case had an atypical clinical presentation, mimicking a musculoskeletal disorder. It underlines the importance of cytology as a simple, inexpensive, rapid and noninvasive complementary exam in routine clinical practice that can permit early diagnosis of the disease and timely selection of the most adequate therapy.     

Influence of the stage for anther excision and heterostyly in embryogenesis induction from eggplant anther cultures

In this work we address two aspects of eggplant flower biology potentially involved on the efficiency of anther culture: the selection of the best floral stage to extract anthers for culture, and the effect of heterostyly in the identification of suitable buds and anthers. For 12 different accessions, we determined morphological criteria (length ranges) to identify buds and anthers enriched in vacuolate microspores and young bicellular pollen, the stages most responsive to embryogenesis induction. While these microspore/pollen stages were the most responsive when isolated and cultured in liquid medium, we observed that culture of anthers containing these stages is not the best choice. Instead, the highest response was found for younger anthers, containing mostly young and mid microspores. We analyzed eggplant anther walls and found that their particular thickness may be behind this apparent discrepancy, since they may delay the diffusion of inducing factors to the anther locule, reducing their effect over inducible microspores. Thus, the culture of younger anthers would allow for younger microspores to grow up to the inducible stages while factors are entering the locule. We also analyzed the embryogenic response of short and long-styled buds present in Cristal, a heterostylic cultivar. Our results demonstrated that each floral morph produced buds and anthers of different lengths, but equally useful for anther culture, since similar amounts of embryos were produced. The practical application of these results may improve the efficiency of anther culture not only in these cultivars, but in others also presenting thick anther walls and heterostyly.     

Qualitative and quantitative polymerase chain reaction (PCR) for detection of Leishmania in spleen samples from naturally infected dogs

Because infected dogs are widely considered to be the main domestic reservoir for Leishmania infantum (syn Leishmania chagasi) parasites in Brazil, the diagnosis of canine visceral leishmaniasis (CVL) must be made both accurately and promptly. The present study attempted to standardize a conventional polymerase chain reaction (cPCR) protocol for the detection of L. infantum DNA in canine spleen samples. Quantitative PCR (qPCR) technique was used to confirm the presence of Leishmania DNA in the canine spleen fragments. A comparison was made between the efficacies of these molecular diagnostic techniques and conventional parasitological and serological methods. cPCR protocols for spleen samples were standardized using primers that amplify a 145 bp fragment, located at the parasite kinetoplast minicircle. The genus specificity of the cPCR protocol was assessed by its inability to amplify the DNA of other common canine pathogens, such as Ehrlichia canis, Babesia canis, Toxoplasma gondii and Trypanosoma cruzi. cPCR protocol sensitivity was tested by assessing the reaction detection limit, determined to be 10 fg of L. infantum reference strain DNA, which corresponds to a range of 0.03-0.1 parasites per fragment. Standardized cPCR protocol was used to detect the presence of Leishmania in 45 dog spleen samples. Our results showed that 40% of the spleen fragment cultures were positive for Leishmania parasites, 58% of the dog serum samples tested positive using ELISA, and parasite DNA was detected in 44% using qPCR, while 47% of the spleen samples using cPCR. Diagnostic methods performance was assessed and revealed a better degree of ascertainment for cPCR when compared to other diagnostic methods. The sensitivity of ELISA was 83.3%, qPCR was 83.3%, and cPCR was 88.9%; PPV for ELISA was 57.7%, qPCR was 75% and cPCR was 76.2%; the Kappa coefficients were found to be 0.40 (fair) for ELISA, 0.64 (substantial) for qPCR and 0.68 (substantial) for cPCR. In both oligosymptomatic and polysymptomatic dogs, cPCR revealed the better performance analysis when compared to other diagnostic methods. The findings presented herein establish cPCR as the most indicated test to detect Leishmania when compared to the other two diagnostic methods evaluated. Despite the fact that the qPCR protocol provides a highly accurate quantification of parasites when targeting the SSU rRNA gene, this technique does not significantly improve the diagnosis of CVL when compared with the performance of the cPCR protocol, which focused on the kinetoplast minicircle.     

Humoral and cellular immune responses in pigs immunized intranasally with crude rhoptry proteins of Toxoplasma gondii plus Quil-A

We evaluated the humoral and cellular immune responses in pigs immunized intranasally with crude rhoptry proteins of Toxoplasma gondii plus Quil-A. The experiment used 13 mixed-breed pigs divided into the following three groups: G1 (vaccinated-challenged, n=6), which received the rhoptry vaccine (200(g/dose); G2 (adjuvant-challenged, n=4), which received PBS plus Quil-A; and G3 (unvaccinated-challenged, n=3), which was the control group. The treatments were performed intranasally at days 0, 21, and 42. Three pigs from G1 produced IgG and IgM antibody levels above the cut-off in the ELISA on the challenge day. Partial protection was observed in G1 at the chronic phase of infection when compared with G3. The preventable fractions were 41.6% and 6.5%, in G1 and G2, respectively. The results of this study suggest that rhoptry proteins plus Quil-A stimulated humoral, local, and systemic immune responses, which were able to partially protect the brain from cyst formation.