Detection of Leishmania (L.) infantum in stray dogs by molecular techniques with sensitive species-specific primers

Background: Canine visceral leishmaniasis (CVL) is a worldwide parasitic zoonosis caused by Leishmania (Leishmania) infantum around the world. Canids are the definitive hosts and sand flies the intermediate hosts.

Objective: To test the hypothesis that a new species-specific primers (Lch14:Lch15, targeting a multiple alignment for L. infantum kDNA minicircle) is an efficient diagnostic tool for L. infantum.

Methods: The presence of L. infantum DNA was assessed in blood samples of 69 stray dogs using the conventional PCR (cPCR) and quantitative PCR (qPCR). Additional 50 lymph nodes and 50 bone marrow samples (positive and negative samples for parasitological tests) from dogs from endemic and nonendemic areas for CVL were also used.

Results: L. infantum strains, and all positive lymph node and bone marrow samples for parasitological test gave positive results for cPCR and qPCR, presenting analytical sensitivity of ～10⁰ parasite mL^?1. For the blood samples, 40/69 (58%; CI 95%; 46%–69%) resulted positive for L. infantum in both tests. All positive samples were confirmed by sequencing.

Conclusion: This study showed the importance of the specific detection of L. infantum based on species-specific primers by molecular techniques, highlighting the application as a confirmation method in epidemiological studies and to adopt the best control measures.     

Characterization of symptoms of senescence and chilling injury on inflorescences of Heliconia bihai (L.) cv. Lobster Claw and cv. Halloween

Inadequate temperatures during the shipping and commercialization of cut tropical flowers may accelerate the senescence process and cause chilling injury, leading to symptoms that have not yet been described for Heliconia bihai. The aim of the present study was to evaluate physiological responses in cut inflorescences of H. bihai cv. Lobster Claw (LC) and cv. Halloween (HW) as well as symptoms of senescence and chilling injury. For such, changes in fresh weight, bract color (L*, a* and b*), percentage of absolute integrity (PAI) of cell membranes and leakage of potassium ions (LPI) were determined. The flowering stems were evaluated at five different intervals after harvest (0, 2, 4, 6 and 8 d). A refrigerated treatment (RT) with a temperature of 6.5 °C and 85% relative humidity was compared to a control treatment (CT) at room temperature of 24 °C and 66% relative humidity. Both cultivars stored at 6.5 °C exhibited dryness of bract tissue (symptom of senescence) and dark stains that became brownish and evolved to necrosis (symptom of chilling injury). The visual quality of inflorescences decreased with time in both cultivars maintained without refrigeration. The severity of chilling injury increased with the length of storage time in both cultivars. There was a significant reduction in the fresh weight of inflorescences in both treatments (RT and CT) and both cultivars (LC and HW). Bract color changed in both cultivars at 6.5 °C. There was no change in PAI throughout the evaluation period in the inflorescences stored at room temperature, whereas those stored at 6.5 °C for 6 and 8 d had lower PAI values. The inflorescences in the control treatment underwent no change in LPI values, whereas those stored under refrigeration had increased LPI values after the sixth day of storage. The physiological responses of cut Heliconia flowers were influenced by storage period and temperature, as demonstrated by visual symptoms of chilling injury and senescence.     

Detection of the root lesion nematode Pratylenchus penetrans in potato tubers

The root lesion nematode Pratylenchus penetrans parasitizes a wide range of economically important crops, including potato (Solanum tuberosum). Damage by P. penetrans impacts not only the potato yield but can also reduce the tuber quality. Detailed information on tuber infection by P. penetrans is scarce for most cultivars and molecular detection of nematodes from infected tubers is needed. The objective of this study was to assess tuber symptomatology due to P. penetrans infection in 10 potato cultivars and to provide an accurate molecular methodology for nematode detection using tuber peels. Sprouts of certified potato seed from cultivars Agata, Agria, Camel, Désirée, Dirosso, Kennebec, Laura, Picasso, Royata, and Stemster were planted in 2 L pots, and soil was inoculated with 4 P. penetrans/g of soil. Sixty days after inoculation, tubers were harvested, inspected for lesions, and the number of nematodes/g of potato peel assessed. Observations of tubers with symptoms showed the presence of P. penetrans in superficial layers of peels around the lenticels and injured necrotic tissue. Different nematode stages were detected in tubers of all inoculated cultivars, varying from 4 to 46 nematodes/g of potato peel. Species-specific primers showed suitable sensitivity and reproducibility for the detection of P. penetrans in tuber potato peel samples. The molecular detection of P. penetrans directly from tuber peels can facilitate routine nematode inspections of potato seed tubers or cull potatoes for nematode detection, and prevent further dissemination of this species.     

Chemical and microbiological soil properties in organic and conventional management systems of Coffea arabica L.

The goal of this study was to evaluate the effect of organic (OS) and conventional (CS) farming of Arabica coffee on chemical and microbiological soil properties and to identify which attributes correlate and/or contribute to distinguishing these two management systems. We collected soil samples on a 100 × 100 m² grid in both, the OS and CS, in winter and summer and submitted chemical and microbiological properties to univariate and multivariate analyses (canonical discriminant analysis – CDA – and redundancy analysis – RDA). The best indicators to discriminate OS and CS were chemical and microbiological properties, as the elements calcium (Ca) and boron (B), besides microbial biomass carbon (MBC), metabolic quotient (qCO₂), acid phosphatase (ACP), and alkaline phosphatase (ALP). The RDA showed that, regardless of the season, MBC and ACP correlated with nitrogen (N), sulfur (S) and organic matter (OM), whereas basal respiration (C-CO₂) and ACP correlated with qCO₂ and with magnesium (Mg), calcium (Ca), phosphorus (P), potassium (K) and pH.     

Chronic toxicity of fenoxycarb to the midge Chironomus riparius after exposure in sediments of different composition

Background, aim, and scope  

Fenoxycarb is an insect growth regulator widely used to control a variety of pests. As a juvenile hormone analogue, it interacts with the natural hormones involved in insect development, inhibiting metamorphosis to the adult stage. Adverse effects of fenoxycarb on non-target organisms have been repeatedly observed and its rapid dissipation from water to the organic fraction of the sediment is known. The aim of this work was to investigate the impact of fenoxycarb on the development of sediment-dwelling larvae of the midge Chironomus riparius after exposure to spiked sediment.     

Effects of road transportation or droving on the weight and metabolism of young bulls

This study evaluated the effects of the mode of transportation on weight and metabolism of young bulls, which are generally transported by two systems: droving and truck. This experiment consisted of two phases: transportation and refeeding and involved transportation by droving and truck. Fifty young Nellore bulls were separated in equal numbers in both groups, droving group and truck group. In the transportation phase, animals were moved, in a 640-km journey. The refeeding phase consisted of 84 days and commenced after the animals arrived at the fattening farm. The effect of the transportation system on animals’ weight, metabolic hormones, and serum biochemistry was assessed after 28, 56, and 84 days. During the transportation phase, animals in the droving group lost 10.11 kg and animals in the truck group gained 13.1 kg. The truck group showed 20.5 kg of weight gain at the end of the refeeding phase. The highest triiodothyronine (T₃) and thyroxine (T₄) concentrations were observed on days 0 and 60 in the transportation phase in both droving and truck groups. The serum cortisol concentration was higher in the droving group than in the truck group on day 56 of the refeeding phase. The IGF-I concentrations were higher during the refeeding phase in the droving group than in the truck group. Our results showed that transportation by droving leads to increased weight loss and reduced further weight gain compared to transportation by truck and that serum concentrations of T₄, cortisol, and insulin-like growth factor (IGF-I) hormones are altered by the transportation system.     

Heat Shock Protein 70 and Sex Steroid Receptors in the Follicular Structures of Induced Ovarian Cysts

The purpose of this study was to estimate the expression and relative amounts of estrogen (ER) and progesterone receptors (PR) and their isoforms as well as heat shock protein 70 (HSP70) in ovaries of rats with induced cystic ovarian disease (COD). Primary, secondary, tertiary, atretic and cystic follicles were evaluated by immunohistochemistry and total ovarian proteins were analyzed by Western blot. In the granulosa layer, growing and cystic follicles in the treated group have a higher expression of ERα than growing follicles of control individuals. In the theca interna layer, tertiary follicles presented a significantly higher expression of ERα in the treated group. An increase in total ERα protein was detected in the treated group. Granulosa cells of all growing, atretic and cystic follicles show a lower expression of ERβ in animals with COD, and the total protein expression of ERβ was lower in this group. The expression of PR was lower in the granulosa cell layer of tertiary and cystic follicles in treated animals, and theca interna layer had less intense immunostaining in this group. Although there were no differences in the expression of PR-B by Western blotting, the expression of PR-A was higher and the expression of PR-C was smaller in the treated group. An intense HSP70 immunostaining was observed in the cells of cystic follicles. By Western blotting, higher protein expression of HSP70 was detected in the ovarian samples of the control group than those of the treated ones. Ovaries of animals with COD exhibited an altered steroid receptor expression and subtype balance as compared with control animals, and an increase in HSP70 immunoexpression.     

Inheritance of the stay-green trait in tropical maize

Stay-green maize genotypes have been associated with tolerance to biotic and abiotic stresses, including tolerance to drought, and to stalk and root lodging, but there is limited information on its inheritance. Thus, this research was conducted to study the inheritance of the stay-green trait using both conventional analysis and QTL mapping of the Design III in a tropical maize population developed from two inbred lines genetically divergent for this trait. Two-hundred and fifty F₂ plants were genotyped with 177 microsatellite markers, and their backcrossed progenies to both parental inbreds were evaluated at three locations. Ten plants per plot were assessed 120 days after sowing and the plot means scores for stay-green, adjusted for days to silking emergence, were used for analysis. The additive variance was larger than the dominance variance, the genetic by location interaction variance presented a high magnitude, and the heritability coefficient on a plant-basis a low magnitude. Seventeen QTL were mapped, most of them were clustered on four chromosomes and accounted for by 73.08 % of the genetic variance. About half of the QTL interacted with location, and the average level of dominance was partial dominance. The additive effects were larger than the dominance effects; the latter were not unidirectional, so that heterosis could not be exploited in crosses. Procedures for marker-assisted selection to increase the level of stay-green are discussed and an approach is suggested for using both stable and non-stable QTL in a marker-assisted backcross program.     

Effect of oil-based formulations of acaripathogenic fungi to control Rhipicephalus microplus ticks under laboratory conditions

The formulations of acaripathogenic fungi to control ticks have been widely studied. The present study evaluated the efficacy of oil-based formulations of Metarhizium anisopliae sensu lato (s.l.), isolate Ma 959, and Beauveria bassiana, isolate Bb 986, on different Rhipicephalus microplus stages, comparing the efficacy between aqueous suspensions and 10, 15 and 20% mineral oil formulations. Twelve groups were formed: one aqueous control group; three mineral oil control groups, at 10, 15 or 20%; two aqueous fungal suspensions of M. anisopliae s.l. or B. bassiana; and three formulations of M. anisopliae (s.l.) or B. bassiana containing 10, 15, and 20% mineral oil. To prepare aqueous suspensions and oily formulations, fungal isolates were cultivated on rice grains in polypropylene bags. The conidial suspensions and formulations had a concentration of 10(8)conidia/mL. Bioassays were repeated twice. After treatment, the following biological parameters of engorged females were evaluated: hatching percentage, egg production index, nutritional index, and percentage of tick control. The following parameters were evaluated in the bioassays with eggs: period of incubation, period of hatch, and hatching percentage. Mortality was evaluated in bioassays with larvae. M. anisopliae s.l. and B. bassiana oil-based formulations were more effective than aqueous suspensions against R. microplus eggs, larvae and engorged females, however, there was no significant difference between the three oil concentrations used. M. anisopliae s.l. and B. bassiana formulated in mineral oil reached 93.69% and 21.67% efficacy, respectively, while M. anisopliae s.l. and B. bassiana aqueous suspensions attained 18.70% and 1.72% efficacy, respectively. M. anisopliae s.l. oil-based formulations caused significant effects in all biological parameters of engorged females while B. bassiana oil-based formulations modified significantly the nutritional index only. Eggs treated with M. anisopliae s.l. and B. bassiana oil-based formulations showed hatching rates that decreased 102.5 and 3.65 times, respectively. In the bioassay with larvae, M. anisopliae s.l. oil-based formulations caused nearly 100% mortality five days after treatment, while larva treated with B. bassiana oil-based formulations reached 100% mortality at day 20 after treatment. Larva from oil-based control groups showed mortality at day 15 after treatment, which indicated a possible toxic effect of the oil for this R. microplus stage. The results showed that the fungal mineral oil formulations tested were more effective than the aqueous suspension. Oil-based formulations at 10, 15 and 20% enhanced the activity of M. anisopliae s.l. Ma 959, and B. bassiana Bb 986, isolates against R. microplus eggs, larvae, and engorged females tick. Mineral oil was effective as an adjuvant in formulations of M. anisopliae s.l., Ma 959, and B. bassiana, Bb 986, for the control of R. microplus under laboratory conditions.