Participation of Rhipicephalus sanguineus (Acari: Ixodidae) in the epidemiology of canine visceral leishmaniasis

The vectorial competence of the tick Rhipicephalus sanguineus is discussed in relation to the epidemiology of canine visceral leishmaniasis, taking into account its strict association with dogs and the low indices of natural infection presented by its known vector, the phlebotomine sand fly Lutzomyia longipalpis. In order to evaluate natural infection by Leishmania chagasi and the infectivity of these parasites in the tick, 39 specimens (6 females, 11 males and 22 nymphs) of R. sanguineus were removed from 21 dogs showing diverse symptoms of zoonotic visceral leishmaniasis (ZVL). Six ticks (15.4%) gave positive results for the genus Leishmania using the PCR technique. To determine the infectivity of the parasites, 36 hamsters were inoculated orally and peritoneally with macerates of ticks removed from nine dogs symptomatic for visceral leishmaniasis. After 6 months the hamsters were sacrificed and necropsied. Serum was removed for IFAT, as well as spleen and liver fragments to make imprint smears and for PCR. Eight (88.9%) of these dogs presented ticks that were infective for 14 hamsters (41.2%), 12 (85.7%) of them infected peritoneally and two (14.3%) orally. PCR revealed 27 smears (40.9%) to be positive, 20 (62.5%) of them infected peritoneally and seven (20.6%) orally. IFAT showed 14 positive animals (41.2%). Based on these findings, we suggest that the vectorial capacity of R. sanguineus for L. chagasi should be evaluated further, opening new perspectives in the epidemiology of ZVL.     

Characterization of Ehrlichia ruminantium replication and release kinetics in endothelial cell cultures

Ehrlichia ruminantium is the causative agent of Heartwater, a fatal tick-borne disease affecting ruminants in African countries and West Indies and can be used as an inactivated vaccine for wild and domestic animals. In order to improve E. ruminantium production yields we characterize E. ruminantium growth kinetics in terms of duplication time, maximum production yield, and peak of infectivity. After a 24 h period for E. ruminantium attachment/internalization and a lag phase of 12 h, the exponential growth occurred within 36-108 h post-infection (hpi) with a net increase of up to 2.2 orders of magnitude. Maximum E. ruminantium infectivity was observed at 120 hpi and was defined as the best time of harvesting (TOH) for propagation of E. ruminantium cultures. This study showed that considering the quality constraint of the final product (E. ruminantium vaccine), the E. ruminantium suspension should be harvested at 113 hpi. Overall, the characterization of E. ruminantium progression through the average infection cycle, not only can contribute to the maximization of E. ruminantium production yield, with important consequences for the large scale production and utilization of an inactivated Heartwater vaccine, but also to elucidate growth mechanisms of some of the other ehrlichial species, with emerging impact in human and animal health.     

Attaching and effacing Escherichia coli isolated from dogs in Brazil: characteristics and serotypic relationship to human enteropathogenic E. coli (EPEC)

Escherichia coli isolates recovered from 182 fecal specimens from dogs up to five months old from the cities of S?o Paulo and Campinas, SP, Brazil, were examined by polymerase chain reaction (PCR) for several virulence factors and properties. The eae gene was found in 23 isolates of E. coli from 22 dogs, 19 of 146 (13%) from dogs with diarrhea and 3 of 36 (8.3%) from dogs with no diarrhea. Two different eae+ isolates were recovered from one dog with diarrhea. Isolates from two dogs with diarrhea harbored the bfpA gene, and none of the isolates possessed genes for enterotoxins, the EAF plasmid or Shiga toxins. PCR showed that, among the 23 isolates, eight were positive for beta intimin, six for gamma, two for, one for alpha, one for kappa, and five showed no amplification with any of the nine pairs of specific intimin primers used. PCR also showed that the LEE (locus of enterocyte effacement) was inserted in selC in four isolates, likely in pheU in seven isolates, and in undetermined sites in twelve isolates. Fifteen isolates adhered to HEp-2 cells and were fluorescence actin staining (FAS) positive. The predominant adherence pattern was the localized adherence-like (LAL) pattern. The eae-positive isolates belonged to a wide diversity of serotypes, including O111:H25, O119:H2 and O142:H6, which are serotypes that are common among human EPEC. These results confirmed the presence of EPEC in dogs (DEPEC) with and without diarrhea. The virulence factors found in these strains were similar to those in human EPEC, leading to the possibility that EPEC may move back and forth among human and canine populations.     

Morphometric analysis of the corneal endothelium of Yacare caiman (Caiman yacare) using scanning electron microscopy

The objective of this study was to examine the endothelial surface morphology and to perform morphometric analysis of the corneal endothelial cells of Yacare caiman (Caiman yacare) using scanning electron microscopy. Morphometric analysis with regard to polygonality, mean cell area, cell density and coefficient of variation of mean cell area was performed. Cell areas were measured using image analysis software. The normal corneal endothelium of Yacare caiman consisted of polygonal cells of uniform size and shape with interdigitations of the cell borders. Microvilli appeared as protrusions on the cellular surface. The average cell area was 270 +/- 24 microm(2) and the endothelial cell density was 3704 +/- 324 cells/mm(2). The coefficient of variation of cell area was 0.22. This study demonstrates that the Yacare caiman corneal endothelium is similar to those described in other vertebrates.     

HPLC separation and determination of 12 cholesterol oxidation products in fish: comparative study of RI, UV, and APCI-MS detectors

A simple, fast, and sensitive method for the extraction through direct saponification, separation, quantification, and identification of 12 cholesterol oxidation products (COPs) and cholesterol in a single isocratic, normal-phase, high-performance liquid chromatography (HPLC) was developed. Three detectors were compared for determination of COPs and cholesterol in fish samples: refractive index (RI), ultraviolet (UV), and atmospheric pressure chemical ionization mass spectrometry (APCI-MS). The results did not show significant differences (p > 0.05) between the concentration of the cholesterol oxides and cholesterol obtained with these detectors. The present study demonstrated the presence of 19-hydroxycholesterol, 22"R"-hydroxycholesterol, 22"S"-hydroxycholesterol, 24"S"-hydroxycholesterol, and 25"R"-hydroxycholesterol for the first time in fish samples.     

Biometrical, biological, biochemical and molecular characteristics of Meloidogyne incognita isolates and related species

Meloidogyne incognita is one of the most polyphagous species of root-knot nematodes occurring in Brazil and worldwide. Eight M. incognita isolates were studied, representing two enzymatic phenotypes (esterase and malate desydrogenase: I1/N1, I2/N1) and four cryptic Meloidogyne sp.1 (S2/N1) isolates, representing one cytological type (3n?=?40–46). Three M. hispanica isolates (Hi3/N1, 2n?=?32–36) and two of an atypical Meloidogyne sp.2 (S2a/N3, 3n?=?40–44) were included in this study for comparison. All isolates were tested with three M. incognita-specific molecular markers. The primer pairs B06F/R, miF/R and incK14F/R amplified three species-specific fragments of 1,200?bp, 955?bp and 399?bp, respectively for M. incognita and Meloidogyne sp.1 isolates. No amplification occurred in the M. hispanica and Meloidogyne sp.2 isolates, except with primers miF/R (1,650?bp). The genetic variability of the Meloidogyne spp. isolates was evaluated, using RAPD and ISSR markers. The phylogenetic analyses revealed two strongly supported monophyletic clades: clade I, consisting of M. hispanica and the atypical Meloidogyne sp.2 isolates, and clade II, clustering together all M. incognita and the Meloidogyne sp.1 isolates. Considering the biometrical, cytological and molecular approaches, it was possible to conclude that the isolates with three enzymatic phenotypes (I1/N1, I2/N1 and S2/N1) presented the characteristics described for M. incognita. Some correlations were detected between the isozymatic phenotypes and the tree topology (S2a/N3, Hi3/N1, I1/N1, S2/N1), but no strict correlation could be observed for the phenotype I2/N1 and one isolate of S2/N1. Morphologically, the Msp.2 isolates differ from M. incognita and M. hispanica by the female stylet features presenting straight cone tip and round pear shaped knobs, posteriorly sloping. The results of this study suggested that the Msp.2 isolates with phenotypes S2aN3 belong to a new or an unidentified species closely related to M. hispanica.     

Sensitivity of Lasiodiplodia theobromae from Brazilian papaya orchards to MBC and DMI fungicides

Stem rot caused by Lasiodiplodia theobromae is an important postharvest disease of papaya in Brazil, responsible for reducing the quality and quantity of fruits. Fungicide use is one of the main disease management measures. However, there are no estimates available of pathogen sensitivity to commonly employed fungicides. Therefore, the EC₅₀ from 120 isolates of L. theobromae from northeastern Brazil, representative of six populations of the pathogen, was estimated in vitro for fungicides of the methyl benzimidazole carbamates—MBC (benomyl and thiabendazole) and demethylation-inhibiting—DMI (imazalil, prochloraz, tebuconazole) groups. Mycelial growth on fungicide-free media and virulence on papaya fruits of the MBC-sensitive and non-sensitive isolates were compared. For MBCs, 8.4% of isolates were non-sensitive to fungicides. For the remaining 91.6%, the mean EC₅₀ ranged from 0.002 to 0.13 μg ml⁻¹ and 0.36 to 1.27 μg ml⁻¹ for benomyl and thiabendazole, respectively. For DMIs, the mean EC₅₀ range for imazalil was 0.001 to 2.27 μg ml⁻¹, 0.04 to 1.75 μg ml⁻¹ for prochloraz, and 0.14 to 4.05 μg ml⁻¹ for tebuconazole. The EC₅₀ values of non-sensitive isolates were significantly (P≤0.05) higher those for the sensitive isolates for each of the DMI fungicides. Differences (P≤0.05) were found in the levels of sensitivity to DMI fungicides among the isolate populations associated with orchards. The populations from two orchards were less sensitive to DMIs. No solid evidence was found for fitness costs relating to MBC non-sensitive isolates because mycelial growth in fungicide-free media and virulence on papaya fruits were similar to those of sensitive isolates.     

Combining ability for grain chemistry quality traits in a white oat diallelic cross

There has been a strong demand for oat genotypes that contain caryopsis with high chemical quality which can suit the different market niches. Therefore, the objectives of this study were to assess the general (GCA) and specific combining ability (SCA) of white oat cultivars through diallelic crosses providing information about the genetic effects on expression of grain chemical quality components. Also, it was aimed to estimate the heterosis on F₁ and F₂ generations and the vigor loss due to inbreeding. During 2008, 21 hybrid populations F₁ and F₂ were obtained from artificial crossing among seven Brazilian white oat cultivars, following the complete diallel design, without considering the reciprocals. These populations and their parents were evaluated in the 2009 season in the experimental field in Capão do Leão, RS, Brazil. The higher values of mean squares associated to GCA indicates a strong contribution of additive genetic effects to the expression of grain chemical components. The parents tested showed a tendency to develop progeny with negative heterosis regarding protein, lipid, β-glucan and soluble dietary fiber in the grain, and positive for the content of nitrogen-free extract, total and insoluble dietary fiber. IAC 7 features a potential parent for obtaining grains with high protein and dietary fiber content, and low caloric content, fit to human diet. Meanwhile, UPF 15 and FAPA Louise can represent donors of alleles to increase lipid contents, while FAPA Louise and URS Guapa can be used to raise the grain nitrogen-free extract contents of lines intended for animal feeding.