Analysis of resistance to Xylella fastidiosa within a hybrid population of Pera sweet orange × Murcott tangor

Resistance to Xylella fastidiosa was evaluated within a population of 20 interspecific hybrids of Pera sweet orange and Murcott tangor under greenhouse conditions. Efficiency of inoculation, multiplication of bacteria within the plants, xylem vessel morphology, and symptom expression were analysed. The rate of infection ranged from 40 to 100% (average 70%) for all genotypes analysed. Xylella fastidiosa populations ranged from log 0·59 to log 2·13 cells mg⁻¹ tissue for the resistant hybrids. These values were significantly different ( P  = 0·05) from those obtained for the tolerant (no symptoms but bacteria recovered) or susceptible (symptoms and bacteria recovered) hybrids (log 3·02 to log 4·06 cells mg⁻¹). Xylella fastidiosa was recovered from all hybrids (log 2·31 to 5·03 CFU mg⁻¹ tissue) except the resistant ones. The first foliar symptoms appeared at least 90 days post-inoculation, the time varying according to genotype. No correlation between xylem vessel morphology and disease expression was observed, indicating that the resistance was the result of a genetic response of the host. According to this hypothesis, a high broad-based heritability index for resistance was obtained (0·96) at 210 days from X. fastidiosa inoculations, using bacterial quantification by real-time PCR, which indicated that the influence of the number of bacteria was the result of genetic rather than environmental variations.     

A new peptide of melon seeds which shows sequence homology with vicilin: Partial characterization and antifungal activity

Plant resistance against pathogens involves a broad variety of proteins, which can be either constitutive or induced. Different types of antimicrobial proteins have been purified from plant seeds, including chitinases, β-1,3-glucanases, defensins, thionins, lipid transfer proteins, lectins and vicilins. The aims of this study were to isolate and characterize defense-related proteins, particularly antimicrobial peptides, present in melon (Cucumis melo L.) seeds. Seed proteins were extracted with phosphate buffer, pH 5.4 and subjected to precipitation with 90% relative ammonium sulfate saturation. The precipitate was re-dissolved in distilled water and heated at 80 °C for 15 min. A DEAE–Sepharose column equilibrated with 0.1 M Tris–HCl pH 8.0 was employed for further purification of the peptides with antifungal properties. Fractions with inhibitory activity were detected in the non-retained basic peak (P1). To test whether P1 peptides were able to permeabilize the yeast plasma membrane, we monitored their effects on glucose-stimulated acidification of the incubation medium by yeast Saccharomyces cerevisiae cells. This fraction strongly inhibited glucose-stimulated acidification of the medium by yeast cells in a dose-dependent manner, and inhibition was up to 60% for all concentrations tested. Effects of the P1 fraction upon the growth of the phytopathogenic fungus Fusarium oxysporum were also observed. Proteins in P1 fraction were also subjected to automated N-terminal amino acid sequencing. Partial N-terminal sequence of a major 8 kDa protein revealed homology with previous reported storage vicilins from different seeds.     

Effects of Storing Pig Ovaries at 4 or 20°C for Different Periods of Time on the Morphology and Viability of Pre-Antral Follicles

The purpose of this study was to evaluate the effect of cooling ovarian tissue on pig pre-antral follicles. Ovaries were maintained in saline solution (0.9%) at 4 or 20 degrees C for 6, 12 or 18 h. After storage, pre-antral follicles were morphologically evaluated. While primordial follicles were not affected by the storage, the percentage of morphologically normal growing follicles was significantly reduced in ovarian tissue stored at 20 degrees C for 12 or 18 h. To test the viability of stored follicles, growing follicles isolated from ovaries stored at 4 degrees C for 18 h and at 20 degrees C for 6 h were cultured for 3 days. Follicles stored in either condition presented the same growth pattern in vitro as fresh follicles. We conclude that storage of pig ovaries at 4 degrees C for up to 18 h or at 20 degrees C for up to 6 h does not affect the morphology of growing follicles or their ability to grow in vitro.     

Quantification of Xylella fastidiosa from Citrus Trees by Real-Time Polymerase Chain Reaction Assay

ABSTRACT Xylella fastidiosa is the causal agent of citrus variegated chlorosis (CVC), a destructive disease of sweet orange cultivars in Brazil. Polymerase chain reaction (PCR)-based assays constitute the principal diagnostic method for detection of these bacteria. In this work, we established a real-time quantitative PCR (QPCR) assay to quantify X. fastidiosa in naturally and artificially infected citrus. The X. fastidiosa cell number detected in the leaves increased according to the age of the leaf, and bacteria were not detected in the upper midrib section in young leaves, indicating temporal and spatial distribution patterns of bacteria, respectively. In addition, the X. fastidiosa cell number quantified in leaves of 'Pera' orange and 'Murcott' tangor reflected the susceptible and resistant status of these citrus cultivars. None of the 12 endophytic citrus bacteria or the four strains of X. fastidiosa nonpathogenic to citrus that were tested showed an increase in the fluorescence signal during QPCR. In contrast, all 10 CVC-causing strains exhibited an increase in fluorescence signal, thus indicating the specificity of this QPCR assay. Our QPCR provides a powerful tool for studies of different aspects of the Xylella-citrus interactions, and can be incorporated into breeding programs in order to select CVC-resistant plants more quickly.     

Arterial supply of the penis in agoutis (Dasyprocta prymnolpha, Wagler, 1831)

The arterial vascularization of agoutis’ penis (Dasyprocta prymnolopha) were analysed using ten male adults from ‘Núcleo de Estudos e Preservação de Animais Silvestres da Universidade Federal do Piauí’ (FUFPI/IBAMA n° 02/99). Among the total number of specimens, six animals had natural death and were members of the research collection of the Laboratory of Anatomy, and four were killed after anaesthesia. Stained bi‐centrifugated‐Cis‐I‐4 latex was injected in arterial vessels responsible for penis vascularization throughout the abdominal portion of aorta. The samples were fixed in 10% formaldehyde solution and arteries were dissected. The penile artery is originated as a branch of internal pudendal artery. At the level of ischiatic arch, the penile artery project two branches, the penile dorsal and the deep arteries; those arteries irrigates the penile dorsal surface and the corpus cavernosum penis. The penile dorsal arteries have an independent course up to the glans penis. Based on the conditions of this work a remarkable similarity regarding the distribution of vessels destined to the agouti penis when compared to other domestic, wild and lagomorph rodents as rabbits.     

Pectus excavatum in two littermate dogs

One male and 1 female, 8-week-old, schnauzer littermates were presented with moderate and mild pectus excavatum, respectively. External application of a coaptation splint to the ventral aspect of the thorax was used for correction of the sternal deformity in the male; conservative treatment was used in the female.     

Evaluation of IFA, MAT, ELISAs and immunoblotting for the detection of anti-Toxoplasma gondii antibodies in paired serum and aqueous humour samples from experimentally infected pigs

The study evaluated the efficiency of diagnostic laboratory methods to detect anti-Toxoplasma gondii antibodies in paired serum and aqueous humour samples from experimentally infected pigs. 18-mixed breed pigs were used during the experiment; these were divided into two groups, G1 (infected group, n=10) and G2 (uninfected group, n=8). Infection was performed with 4 x 10(4) VEG strain oocysts at day 0 by the oral route in G1 animals. All pigs were euthanized at day 60, when retina, aqueous humour, and blood samples were collected. Anti-T. gondii antibody levels were assessed in serum (s) and aqueous humour (ah) by indirect immunofluorescence assay (IFA), modified agglutination test (MAT), m-ELISA (using crude membranes from T. gondii tachyzoites as antigen) and r-ELISA (using rhoptries from T. gondii tachyzoites as antigen). Polymerase chain reactions (PCR) of samples from the retina were performed by using Tox4 and Tox5 primers. Antibody titers of G1 animals ranged from 128 to 1024 and from 16 to 256 in serum and aqueous humour, respectively. There were differences in the correlation coefficients between IFA(s) x IFA (ah) (r=0.62, P=0.05), MAT(s) x MAT (ah) (r=0.97, P<0.0001); however, there was no significant difference between r-ELISA(s) x r-ELISA (ah) (r= 0.14, P=0.7). Antibodies present in serum and aqueous humour recognized similar antigens. Samples of retina were positive by PCR in 30% (3/10) of infected pigs. G2 animals remained without antibody levels and were PCR negative throughout the experiment. These results suggest that the use of a combination of tests and immunoblotting for paired aqueous humour and serum samples could improve the sensitivity and specificity for the diagnosis of ocular toxoplasmosis.     

Pregnancy rate after fixed‐time transfer of cryopreserved embryos collected by non‐surgical route in Lacaune sheep

This study investigated the feasibility of applying fixed‐time (cryopreserved) embryo transfer in ewes. Embryos (n = 106) were non‐surgically recovered from superovulated donors (n = 39) on day 6–7 after oestrus. Straws containing one or two embryos (morulae and/or blastocysts) subjected to either slow freezing (SF, n = 62) or vitrification (VT, n = 44) were randomly used within fixed‐time embryo transfer on Day 8.5. Recipient ewes were nulliparous (n = 58) bearing corpora lutea after synchronous oestrous induction protocol. The pregnancy rate was higher (p = .03) in SF (39.4%) than VT (16.9%) and survival rate tended (p = .08) to be higher in SF than in VT (25.8% vs. 15.9%). Lambing rates were similar (p = .13) between SF (20.9%) and VT (15.9%). Embryos recovered by non‐surgical route after cervical dilation treatment and later cryopreserved by either slow freezing or vitrification produced reasonable pregnancy rates after FTET.     

Comparison of ketamine and S(+)-ketamine, with romifidine and diazepam, for total intravenous anesthesia in horses

OBJECTIVE: To compare the quality of induction and recovery, degree of muscle relaxation, clinically apparent potency and cardiopulmonary effects of racemic ketamine or S(+)-ketamine when used for total intravenous anesthesia in horses. STUDY DESIGN: Prospective randomized clinical trial ANIMALS: Sixteen healthy stallions (323 +/- 99 kg), with a mean age of 6.2 years, undergoing castration. METHODS: Horses were pre-medicated with romifidine IV, 15 minutes before induction of anesthesia. Each animal was then randomly allocated to receive either diazepam and ketamine (DK) or diazepam and S(+)-ketamine (DKS) at similar doses to induce anesthesia. For maintenance of anesthesia, 1/4 of the initial bolus of ketamine alone or S(+)-ketamine alone was administered, as required. Heart rate (HR), respiratory rate (RR) and systolic blood pressure were measured before and at 10-minute intervals during recumbency. Time from induction to lateral recumbency, time from induction to first additional dose, time from last additional dose to return to sternal posture and time from last additional dose to standing were recorded, and a subjective evaluation of quality of induction, endotracheal intubation, muscle relaxation and quality of recovery was recorded. RESULTS: The quality of the induction and duration of anesthesia were similar in both groups. HR, RR and systolic blood pressure were not significantly different between groups. Although some animals which received DKS showed some minor excitatory effects (25% of them) during the induction of anesthesia, these animals received 32% fewer doses for the maintenance of anesthesia and the recovery scores were better. CONCLUSIONS AND CLINICAL RELEVANCE: S(+)-ketamine showed some advantages over racemic ketamine, such as less anesthetic agent being required and better overall recovery from anesthesia. Further studies are needed to obtain the optimum induction dose for the S(+)-ketamine.     

Bovine colostrum enriched with lyophilized bovine colostrum stimulates intestinal epithelium renewal of Holstein calves in the first days of life

Consumption of a second meal of colostrum with high quality could contribute to the intestinal epithelium development, especially if there is poor supply of colostrum just after birth. The effect of a second colostrum meal was evaluated on histomorphometry of the intestinal mucosa of newborn Holstein calves fed with high‐ and low‐quality first colostrum. Seventy‐two calves were fed with a first colostrum meal with high (HFM, close to 100 mg/ml) or low (LFM, close to 30 mg/ml) IgG concentration. At 12 hr of life, three treatments of second colostrum feeding were applied to the calves either fed high or low first colostrum: calves fed with low (LOW—close to 30 mg/ml) or high (HIGH—close to 100 mg/ml) IgG concentration; and colostrum enriched with lyophilized bovine colostrum with high IgG concentration (ENRICHED—higher than 120 mg/ml), resulting in six groups. Intestinal samples were collected after 24 and 72 hr of life. In the distal jejunum and ileum, LOW showed higher villus height than ENRICHED (p < .05). In the distal jejunum, greater villus perimeter was observed in the LOW compared to ENRICHED at 24 hr (p < .05). In ileum, LFM showed higher villus perimeter compared to HFM (p < .05). LOW showed the highest villus height‐to‐crypt depth ratio in the medium and distal jejunum and ileum, p < .05. ENRICHED and HFM showed decreased muscle layer thickness in the proximal and distal jejunum respectively (p < .05). The results reveal that the high concentration of total solids, crude protein, IgG and IGF‐I of colostrum with high quality worsened the absorptive area, but may have stimulated the activity of cell division in intestinal crypts. Considering the present results, bovine colostrum enriched with lyophilized bovine colostrum stimulates intestinal epithelium renewal of Holstein calves in the first days of life.