Genetic diversity of 3′ region of glycoprotein D gene of bovine herpesvirus 1 and 5

Bovine herpesviruses 1 (BoHV-1) and 5 (BoHV-5) are closely related alphaherpesviruses of cattle. While BoHV-1 is mainly associated with respiratory/genital disease and rarely associated with neurological disease, BoHV-5 is the primary agent of meningoencephalitis in cattle. The envelope glycoprotein D of alphaherpesviruses (BoHV-1/gD1 and BoHV-5/gD5) is involved in the early steps of virus infection and may influence virus tropism and neuropathogenesis. This study performed a sequence analysis of the 3′ region of gD gene (gD3′) of BoHV-1 isolates recovered from respiratory/genital disease (n = 6 and reference strain Cooper) or from neurological disease (n = 7); and from seven typical neurological BoHV-5 isolates. After PCR amplification, nucleotide (nt) sequencing, and aminoacid (aa) sequence prediction; gD3′ sequences were compared, identity levels were calculated, and selective pressure was analyzed. The phylogenetic reconstruction based on nt and aa sequences allowed for a clear differentiation of BoHV-1 (n = 14) and BoHV-5 (n = 7) clusters. The seven BoHV-1 isolates from neurological disease are grouped within the BoHV-1 branch. A consistent alignment of 346 nt revealed a high similarity within each viral species (gD1 = 98.3 % nt and aa; gD5 = 97.8 % nt and 85.8 % aa) and an expected lower similarity between gD1 and gD5 (73.7 and 64.1 %, nt and aa, respectively). The analysis of molecular evolution revealed an average negative selection at gD3′. Thus, the phylogeny and similarity levels allowed for differentiation of BoHV-1 and BoHV-5 species, but not further division in subspecies. Sequence analysis did not allow for the identification of genetic differences in gD3′ potentially associated with the respective clinical/pathological phenotypes, yet revealed a lower level of gD3′ conservation than previously reported.     

The apicomplexan parasite Eimeria arloingi induces caprine neutrophil extracellular traps

As a novel effector mechanism polymorphonuclear neutrophils (PMN) release neutrophil extracellular traps (NETs), which represent protein-labeled DNA matrices capable of extracellular trapping and killing of invasive pathogens. Here, we demonstrate for the first time NET formation performed by caprine PMN exposed to different stages (sporozoites and oocysts) of the goat apicomplexan protozoan parasite Eimeria arloingi. Scanning electron microscopy as well as fluorescence microscopy of sporozoites- and oocysts-PMN co-cultures revealed a fine network of DNA fibrils partially covering the parasites. Immunofluorescence analyses confirmed the co-localization of histones (H3), neutrophil elastase (NE), and myeloperoxidase (MPO) in extracellular traps released from caprine PMN. In addition, the enzymatic activity of NE was found significantly enhanced in sporozoite-exposed caprine PMN. The treatment of caprine NET structures with deoxyribonuclease (DNase) and the NADPH oxidase inhibitor diphenylene iodondium (DPI) significantly reduced NETosis confirming the classical characteristics of NETs. Caprine NETs efficiently trapped vital sporozoites of E. arloingi since 72 % of these stages were immobilized—but not killed—in NET structures. As a consequence, early infection rates were significantly reduced when PMN-pre-exposed sporozoites were allowed to infect adequate host cells. These findings suggest that NETs may play an important role in the early innate host response to E. arloingi infection in goats.     

Evaluation of the effect of miltefosine on Trichomonas vaginalis

Trichomonas vaginalis causes trichomoniasis in humans, a sexually transmitted disease commonly treated with metronidazole (MTZ). MTZ is known to cause undesirable side effects, and MTZ-resistant parasites have been reported. Thus, the development of an alternative treatment is desirable. Miltefosine (MLT) is an alkylphosphocholine synthetic lipid analogue that displays antiparasitic activity against Leishmania, Trypanosoma cruzi, Entamoeba histolytica, Acanthamoeba spp., Giardia lamblia, T. vaginalis and some fungi. Moreover, it has been used for oral treatment of visceral leishmaniosis in several countries. Here, we analysed the MLT-induced antiproliferative effect on T. vaginalis as well its effect on the fine structure and viability of the parasite. We observed a dose-dependent effect with an IC₅₀ of 14.5 and 20 μM after 24 and 48 h, respectively. Furthermore, reversibility assays demonstrated that new incubations were necessary in order to maintain the antiproliferative effect. Ultrastructural analyses demonstrated that MLT induced several alterations, including the appearance of wrinkled and rounded cells, membrane blebbing, intense vacuolization and nuclear condensation, all indicative of cell death by apoptosis. In addition, the quantitative analyses of the viability assays using combined markers of live and dead cells demonstrated that treatment with the IC₅₀ concentration of MLT significantly reduced the number of viable parasites compared with untreated cells. Taken together, these observations suggest that MLT is a promising compound for the treatment of trichomoniasis.     

Development of miniaturized fluorimetric device for caffeine determination using a smartphone

Caffeine is an element that is consumed worldwide. It is present in many products such as beverages, chocolate, coffee, tea, energy drinks and medicines. Portable 3D devices working together with colorimetric and fluorimetric reactions have been able to determine the presence of caffeine in different kinds of samples. Also, digital image-based methods using smartphones have conferred portability and accessibility to miniaturized devices that are innovative and promising options for quick and low cost analyses. This study proposes a miniaturized fluorimetric device to determine caffeine by digital image using a smartphone. The OpenCamera app was used to capture images that were processed using ImageJ software to obtain RGB channels values. The red (R) channel signal intensity was selected as the analytical response. The device developed was applied to determine caffeine in an energy drink and medicines. The method developed presented a linear range from 100 to 600 mg L⁻¹ of caffeine, and quantification (LOQ) and detection (LOD) limits of 100 mg L⁻¹ and 30.0 mg L⁻¹, respectively. The caffeine concentration found in the products analyzed was 328 mg L⁻¹ (±2.5%) for the energy drink, 345 mg L⁻¹ (±15%) for medicine A and 322 mg L−¹ (±7.3%) for medicine B. The proposed device presented important characteristics such as low cost, required small volumes of reagents and samples, quick analysis, portability and suitable to be applied in complex matrices.

This study proposes a miniaturized fluorimetric device to determine caffeine in an energy drink and medicines by digital image using a smartphone.     

The morphological and molecular characterization of Henneguya rotunda n. sp., a parasite of the gill arch and fins of Salminus brasiliensis from the Mogi Guaçu River,Brazil

A new species of myxosporea (Henneguya rotunda n. sp.) was found in the membrane of the gill arch and the fins of Salminus brasiliensis in the Mogi Guaçu River, municipality of Pirassununga, São Paulo state, Brazil. Morphological and morphometric analyses using light microscopy revealed parasites with similar characteristics at both infection sites. The mature spores found infecting the fins had oval spore body with 7.1?±?0.2 μm in length, 5.6?±?0.2 μm in width, 3.7?±?0.1 μm in thickness, 16.4?±?1.2 μm in length of the caudal process, and 23.6?±?1.1 μm in total length of the spore. In a frontal view, the polar capsule was observed to be symmetrical with 3.4?±?0.2 μm in length and 1.8?±?0.1 μm in width. Mature spores contain six to seven turns of the polar filaments. The morphometric data concerning the spores obtained from plasmodia from the membrane of the gill arch were similar to those from the fins. Ultrastructure analysis revealed that the plasmodial wall was formed by a single membrane and had numerous pinocytotic canals connecting the outside of the plasmodia to the ectoplasm zone. Beyond that, various electron-translucent vesicles also were observed at the periphery of the plasmodium. The molecular analyses of the 18S rDNA gene from the spores obtained from the gill arch membrane and fin membrane showed that these sequences shared 100 % similarity. Phylogenetic studies using maximum parsimony and maximum likelihood methods demonstrated the polyphyletic clustering of the myxosporean parasites of characiform fishes. H. rotunda n. sp. clustered as a sister species of Myxobolus pantanalis, also a parasite of S. brasiliensis.     

Acaricidal properties of the formulations based on essential oils from Cymbopogon winterianus and Syzygium aromaticum plants

The cattle tick, Rhipicephalus (Boophilus) microplus, has caused serious harm to livestock raising in Brazil, considering the costs of controlling it, loss of revenue due to smaller production of milk and meat, and damage to leather, in addition to transmitting diseases. The use of medicinal plants is considered an alternative to the recurring resistance to chemicals. Due to the need for efficient alternatives with less environmental impact, this study aimed to develop contact formulations with essential oils from the Java citronella (Cymbopogon winterianus) and clove (Syzygium aromaticum) plants and to assess in vitro the effects in different stages of the tick cycle. In the present study, concentrations from 0.5–15.0 % of the essential oils incorporated in the formulations were used. The ticks from different geographical areas were treated with those formulations, and their effects on the production levels of eggs, on the larvae hatching, and their efficiency on ticks were assessed. The obtained results were compared with other commercial acaricidal products. After the 20th day of treatment, the formulations with citronella essential oil had 2.09–55.51 % efficiency, depending on the concentration of the oil incorporated. The efficiency of the treatment with formulations containing clove essential oil was higher, from 92.47–100 %. The results showed the acaricidal effects of the formulations tested when compared to commercial chemical products. In vivo studies should be performed in order to assess the efficiency of those formulations in the fields, aiming to use these products as an alternative for controlling cattle ticks.     

Histopathological study of ovaries of Rhipicephalus sanguineus (Acari: Ixodidae) exposed to different thymol concentrations

Thymol is a monoterpene with proven acaricide action for several tick species. In addition to killing these ectoparasites, thymol can also reduce oviposition and egg hatch rate. However, the effects of thymol on the morphophysiology of tick ovaries are still unknown. Thus, the aim of this study was to evaluate the morphophysiological changes caused by this active principle in ovaries of Rhipicephalus sanguineus after a 6-day feeding period, through the application of morphohistochemical techniques. After the feeding period, a total of 50 females were divided into five groups and immersed in the following solutions: (I) distilled water (control), (II) 30 % ethanol (control), (III) 1.25 mg/mL thymol, (IV) 2.5 mg/mL thymol, and (V) 5.0 mg/mL thymol. The experimental groups were kept in a climatic chamber (27?±?1 °C; RH 80?±?10 %) for 5 days. After this period, morphological (hematoxylin/eosin) and histochemical (von Kossa) techniques were applied after remotion of the ovaries. The morphological results revealed large vacuoles in germ cells at different developmental stages and invaginations that represent deformations in the chorionic membrane. From the results obtained in this study, it was concluded that thymol interfered with the development of oocytes, which showed degeneration signs. The treatment containing 5.0 mg/mL thymol affected more accentuately the morphological development. Moreover, thymol also altered the calcium content of yolk granules, which generally showed an intense staining for this element.     

The crowding effect in Ancylostoma ceylanicum: density-dependent effects on an experimental model of infection

This study compared the course of Ancylostoma ceylanicum infection in hamsters infected with different inocula and the consequences for the host and helminth populations. The average of adult worms recovered, according to the number of third stage larva used, were 28.0, 24.8, 24.6, and 24.8 % to inocula size of 25 L3, 75 L3, 125 L3, and 250 L3, respectively. The size of the inoculum did not affect the establishment, survival, or fecundity of adult helminths. Reductions in the red blood cell and hemoglobin levels in the infected group were inversely proportional to the number of white blood cells. Moreover, differential cell counting revealed a positive correlation between the worm load and leucocyte numbers. The humoral response against excretion-secretion antigens was more robust and sensitive compared with the response against crude extract, with no direct linear correlation with the number of worms. The effect of the population density was more evident in females.     

Improved nanocomposite of montmorillonite and hydroxyapatite for defluoridation of water

A novel hydroxyapatite montmorillonite (HAP-MMT) nanocomposite system was synthesized using a simple wet chemical in situ precipitation method. Neat nano hydroxyapatite (HAP) was also synthesized for comparison. The characterization of the materials was carried out using Fourier Transform Infrared Spectroscopy (FT-IR), Scanning Electron Microscopy (SEM), X-ray diffraction (XRD) and Brunauer–Emmett–Teller (BET) isotherms to study the functional groups, morphology, crystallinity and the surface area respectively. Batch adsorption studies and kinetic studies on fluoride adsorption were conducted for the HAP-MMT system and for neat HAP. The effect of parameters such as contact time, pH, initial concentration, temperature, and thermodynamic parameters and the effect of coexisting ions on fluoride adsorption by HAP-MMT were studied. Results of the isotherm experiments were fitted to four adsorption isotherm models namely Langmuir, Freundlich, Temkin and Dubinin Radushkevich. Fluoride adsorption over HAP-MMT fitted to the Freundlich adsorption isotherm model and showed more than two-fold improved adsorption capacity (16.7 mg g⁻¹) compared to neat HAP. The best-fitting kinetic model for both adsorbents was found to be pseudo second order. Calculated thermodynamic parameters indicated that the fluoride adsorption by HAP-MMT is more favorable compared to that on HAP within the temperature range of 27 °C–60 °C. Improved fluoride adsorption by HAP-MMT is attributed to the exfoliated nature of HAP-MMT. Gravity filtration studies carried out using a 1.5 ppm fluoride solution, which is closer to the ground water fluoride concentrations of Chronic Kidney Disease of unknown etiology (CKDu) affected areas in Sri Lanka, resulted in a 1600 ml g⁻¹ break through volume indicating the potential of HAP-MMT to be used in real applications.

A novel hydroxyapatite montmorillonite (HAP-MMT) nanocomposite was synthesized using a simple wet chemical in situ precipitation method. This nanocomposite showed improved adsorption properties towards fluoride ions in water.     

Haemogregarina spp. in a wild population from Podocnemis unifilis Troschel, 1848 in the Brazilian Amazonia

The study objectives were to characterize the morphology of the parasitic forms and describe the prevalence and intensity of Haemogregarina spp. in a population of the turtle Podocnemis unifilis as well as to examine the relationships between parasitism and turtle variables such as gender, size, and weight. Samples were taken in the Tapajós and Jamanxim rivers, Itaituba, Pará state, Brazil. Blood was collected from the tail vein of 72 P. unifilis specimens, including 35 males, 36 females, and one unsexed juvenile. The prevalence of Haemogregarina spp. was 98 % (n?=?71). The mean parasite intensity of Haemogregarina spp. was 118 (1–582) parasites/2000 blood cells (6 %). There was no significant difference in the mean parasite intensity between male (137.68?±?121.8, n?=?35) and female turtles (101.42?±?123.59, n?=?35). There was no relationship between parasite intensity and carapace length. Although the relationship between parasite intensity and host body weight was significant, the relationship was weak. This is the first study on Haemogregarina parasitism with a relatively high number of turtles in Brazil.