Visceral leishmaniasis: a practical strategy for quantitative molecular diagnosis in naturally infected dogs.

The diagnosis of canine visceral leishmaniasis (CVL) has been a problem for public health services due to the variety of clinical signs similar to other diseases and low sensitivity and specificity of available tests. In this sense, our main objective was to develop a simple, rapid, and accurate quantitative real-time PCR (qPCR) diagnosis for CVL. Thus, low-invasive samples from bone marrow (BM), popliteal lymph nodes (PLN), and conjunctival swabs (CS) were selected from negative and VL-positive dogs, using as gold standard, immunological and parasitological tests performed with different tissues. Oligonucleotides for Leishmania infantum kDNA were designed and the limit of quantification and amplification efficiency of the qPCR were determined using tissue-specific standards produced with DNA from those different tissues, mixed with DNA from a known amount of L. infantum promastigotes. Endogenous control was used to validate a comparative Ct method, and tissue parasite concentrations were estimated by comparison with tissue-specific reference standard samples. The overall analysis of the qPCR data suggests the following ranking for tissue choice: PLN > BM > CS. Finally, we have concluded that this molecular approach simplifies and accelerates the quantitative diagnostic process because it is easy to perform, requiring no DNA dosing or standard curve application, and it shows good diagnostic parameters, especially when using popliteal lymph node samples.

Thymol and eugenol microemulsion for Rhiphicephalus sanguineus sensu lato control: Formulation development, field efficacy, and safety on dogs.

The present study aimed to develop a microemulsion formulation containing thymol and eugenol for field control of Rhipicephalus sanguineus sensu lato on dogs, as well to evaluate its safety and the physical characteristics of the formulation. The microemulsion using thymol and eugenol (5.0 + 5.0 mg/mL) had as vehicles water, propylene glycol, polysorbate 80 and canola oil. On the next day the preparation (formulation freshly prepared) and after 24 months, the size of the microemulsion droplets, polydispersion index (PdI), organoleptic properties (color, viscosity), and presence of precipitate in the microemulsion were evaluated. For the field assay, on day -1, 10 English Cocker Spaniel dogs were experimentally infested with 200 larvae, 100 nymphs and 30 adults of R. sanguineus s.l. On day 0, after tick counts, the animals were divided into two groups: treated with the freshly prepared microemulsion (10 mL/kg), and control, which received the vehicle (10 mL/kg). Tick counts on dogs were performed daily for three more days. Engorged females were recovered from the dogs and their biological and reproductive parameters were monitored. The dogs' clinical parameters (temperature, mucosa color, and general physical condition) were evaluated daily. In addition, blood samples were collected before infestation to verify hematological (packed cell volume) and biochemical parameters (total serum protein, albumin, globulins, creatinine, urea, alanine transaminase, aspartate aminotransferase, and alkaline phosphatase). Freshly prepared and 24-month aged microemulsions had droplets with mean sizes of 30.94 nm and 27.93 nm, and PdI values of 0.214 and 0.161, respectively. In addition, no difference in the organoleptic properties and no precipitation formation were observed, indicating physical stability. Treatment with the microemulsion resulted in reduction of larvae (p < 0.05) parasitizing the dogs on day 1 while the number of nymphs and adults was not reduced (P> 0.05). In the evaluation of the reproductive biology of engorged females, the larval hatchability (%) was compromised (p < 0.05), and the microemulsion had control rate of 85.5 %. The microemulsion and its vehicles did not change the clinical, hematological and biochemical parameters of the dogs. We concluded that the microemulsion was efficient against R. sanguineus s.l. by reducing the number of larvae and affecting the reproductive parameters of engorged females, safe for dogs, and stable (physical stability) during a two-year interval.

Attract or repel Amblyomma sculptum ticks: Screening of semiochemicals.

Amblyomma sculptum is a tick of medical-veterinary importance. Areas with high infestations need to be monitored, and parasitized hosts treated accordingly. Carbon dioxide (CO2) traps and acaricides are commonly deployed as control measures, although with some disadvantages such as high costs, challenging logistics and vertebrate intoxication. Semiochemicals have the potential to improve tick attraction to traps and monitoring devices and alleviate the burden of A. sculptum infestations. Four concentrations (10, 5, 2.5 and 1.25 %) of 13 semiochemical candidates (CO2 only at 5 % as the standard, benzaldehyde, benzoic acid, salicylic acid, 2,6 diclorophenol, R-limonene, S-limonene, methyl salicylate, 1-octen-3-ol, acetone, ammonium hydroxide, isobutyric acid and lactic acid) were tested on unfed A. sculptum nymphs and adults using a Y-tube olfactometer to evaluated repellence and attraction behaviors. All stages tested were attracted to CO2, whereas nymphs were repelled by benzaldehyde and R-limonene, both at 10 %, and isobutyric acid at 5 and 10 %. Nymphs were attracted by methyl salicylate, benzoic acid and salicylic acid, all at 1.25 %, and by ammonium hydroxide at 2.5 %. Males were attracted by benzoic acid at 2.5 %, while females were repelled by benzaldehyde at 5 %. Mixtures with the attractive compounds achieved no attraction response. The compounds that caused attractiveness in the olfactometer assay (CO2, methyl salicylate, benzoic acid, salicylic acid and ammonium hydroxide) were placed randomly in traps in a grassland plot naturally infested with A. sculptum in triplicate. Notably, dry ice (CO2) remained the best at luring ticks in the field (P < 0.001). Benzoic acid should be further investigated since attractant activity was strongly confirmed in both laboratory and field tests. On the other hand, isobutyric acid and R-limonene could be better exploited due to their repellent role revealed by the lab assay, which makes them worthwhile molecules as natural repellents for the management of this tick.

Tetracycline hydrochloride-loaded electrospun nanofibers mats based on PVA and chitosan for wound dressing.

Fibrous mats built from biopolymer have been extensively explored for tissue engineering due mainly to their mimic structure to the extracellular matrix. The incorporation of drug in such scaffolds represents a growing interest for control drug delivery system in order to promote the tissue repair. In the present work, we present an experimental investigation of morphological, thermal, mechanical, drug release, antibacterial and cytotoxicity properties of electrospun PVA/Chitosan and PVA/Chitosan/Tetracycline hydrochloride (TCH) mats for wound dressing. Fibrous mats with cross-linked three-dimensional nanofibers were formed from the polymer blends. A uniform incorporation of drug was achieved along the nanofibers with not significant change on the morphological and thermal properties of the mats. Furthermore, the TCH release profile with a burst delivery during the first 2h allows an effective antibacterial activity on the Gram-negative Escherichia coli as well as on the Gram-positive Staphylococci epidermidis and Staphylococcus aureus. In vitro indirect MTT assay also showed that the developed drug-loaded nanofibrous scaffolds have good cytocompatibility, which was confirmed by scratch assay, indicating that the investigated scaffold may be used as antibacterial wound dressing for healing promotion.

In vitro schistosomicidal and antiviral activities of Arctium lappa L. (Asteraceae) against Schistosoma mansoni and Herpes simplex virus-1.

Schistosomiasis and herpes diseases represent serious issues to the healthcare systems, infecting a large number of people worldwide, mainly in developing countries. Arctium lappa L. (Asteraceae), known as "bardana" and "burdock", is a medicinal plant popularly used for several purposes, including as antiseptic. In this study, we evaluated the in vitro schistosomicidal and antiherpes activities of the crude extract of A. lappa, which have not yet been described. Fruits of A. lappa L. were extracted by maceration with ethanol: H2O (96:4 v/v) in order to obtain the hydroalcoholic extract of A. lappa (AL). In vitro schistosomicidal assays were assessed against adult worms of Schistosoma mansoni, while the in vitro antiviral activity of AL was evaluated on replication of Herpes simplex virus type-1 (HSV-1). Cell viability was measured by MTT assay, using Vero cells and chemical composition of AL was determined by qualitative UPLC-ESI-QTOF-MS analysis. UPLC-ESI-QTOF-MS analysis of AL revealed the presence of dibenzylbutyrolactone lignans, such as arctiin and arctigenin. Results showed that AL was not cytotoxic to Vero cells even when tested at 400µg/mL. qPCR results indicated a significant viral load decreased for all tested concentrations of AL (400, 50, and 3.125µg/mL), which showed similar antiviral effect to acyclovir (50µg/mL) when tested at 400µg/mL. Also, AL (400, 200, and 100µg/mL) caused 100% mortality and significantly reduction on motor activity of all adult worms of S. mansoni. Confocal laser scanning microscopy showed tegumental morphological alterations and changes on the numbers of tubercles of S. mansoni worms in a dose-dependent manner after treatment with AL. This report provides the first evidence for the in vitro schistosomicidal and antiherpes activities of AL, opening the route to further schistosomicidal and antiviral studies with AL and their compounds, especially lignans.

Activity of a paromomycin hydrophilic formulation for topical treatment of infections by Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis.

Studies on in vitro skin permeation and in vivo anti-leishmanial activity in mice experimentally infected with Leishmania (Leishmania) major pointed out to the potential of a new paromomycin (PA) formulation (hydrophilic gel) for treatment of cutaneous leishmaniasis (CL). In this study, the activity of this formulation was evaluated in animals experimentally infected by Leishmania species that prevail in the New World. PA gel activity was compared to antimony treatment, since it is still the first choice treatment to the different clinical forms of leishmaniasis. The topical treatment activity with 10% PA gel in BALB/c mice infected by Leishmania (Leishmania) amazonensis was higher than that observed for parenteral antimony treatment, while the efficacy of these two regimes in hamsters infected by Leishmania (Viannia) braziliensis was similar. These results suggest that this formulation could be suitable for clinical studies and may represent an alternative novel formulation for topical treatment of CL.

Embryonic development of chicken (Gallus Gallus Domesticus) from 1st to 19th day-ectodermal structures.

Birds occupy a prominent place in the Brazilian economy not only in the poultry industry but also as an animal model in many areas of scientific research. Thus the aim of this study was to provide a description of macro and microscopic aspects of the ectoderm-derived structures in chicken embryos / fetuses poultry (Gallus gallus domesticus) from 1st to 19th day of incubation. 40 fertilized eggs, from a strain of domestic chickens, with an incubation period of 2-19 days were subjected to macroscopic description, biometrics, light, and scanning microscopy. All changes observed during the development were described. The nervous system, skin and appendages and organs related to vision and hearing began to be identified, both macro and microscopically, from the second day of incubation. The vesicles from the primitive central nervous system-forebrain, midbrain, and hindbrain-were identified on the third day of incubation. On the sixth day of incubation, there was a clear vascularization of the skin. The optic vesicle was first observed fourth day of development and on the fifth day there was the beginning of the lens formation. Although embryonic development is influenced by animal line as well as external factors such as incubation temperature, this paper provides a chronological description for chicken (Gallus gallus domesticus) during its embryonic development.

Antileishmanial activity of azithromycin against Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis, and Leishmania (Leishmania) chagasi.

Azithromycin, an azalide antibiotic, is highly concentrated within different phagocytic cells, especially macrophages. The potential antileishmanial activity of azithromycin against three species of Leishmania from the New World was assessed using in vitro models. Azithromycin decreased viability of promastigote cultures of Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis, and Leishmania (Leishmania) chagasi as determined by the colorimetric Alamar blue assay. In amastigote intracellular cultures, a significant decrease in infected macrophages counts was observed for all three species with IC(50) of 20.83 (27 micromol/L), 2.18 (2.7 micromol/L), and 6.12 (7.8 micromol/L) microg/mL, respectively. Azithromycin showed in vitro activity against L. (L.) amazonensis, L. (V.) braziliensis, and L. (L.) chagasi and may offer an alternative to current leishmaniasis treatment.