Evaluation of resistance in a selected field strain of Haemonchus contortus to ivermectin and moxidectin using the Larval Migration on Agar Test

Haemonchus contortus is one of the most common and economically significant causes of disease in small ruminants worldwide, and the control programs of parasitic nematodes - including H. contortus - rely mostly on the use of anthelmintic drugs. The consequence of the use of this, as the sole sanitary strategy to avoid parasite infections, was the reduction of the efficacy of all chemotherapeutic products with a heavy selection for resistance. The widespread of anthelmintic resistance and the difficulty of its early diagnosis has been a major concern for the sustainable parasite management on farms. The objective of this research was to determine and compare the ivermectin (IVM) and moxidectin (MOX) effect in a selected field strain of H. contortus with a known resistance status, using the in vitro larval migration on agar test (LMAT). Third stage larvae of the selected isolate were obtained from faecal cultures of experimentally infected sheep and incubated in eleven increasing diluted concentrations of IVM and MOX (6, 12, 24, 48, 96, 192, 384, 768, 1536, 3072 and 6144µg/mL). The dose-response sigmoidal curves were obtained using the R² value of >0.90 and the lethal concentration (LC50) dose for the tested anthelmintic drugs using a four-parameter logistic model. The LC50 value for MOX was significantly lower than IVM (1.253µg/mL and 91.06µg/mL), identifying the H. contortus isolate as considerably less susceptible to IVM compared to MOX. Furthermore, the LMAT showed a high consistency (p<0.0001) and provided to be a useful diagnostic tool for monitoring the resistance status of IVM and MOX in H. contortus field isolate, as well as it may be used for official routine drug monitoring programs under the Ministry of Agriculture (MAPA) guidance.

Development and application of polymerase chain reaction test for detection of Conidiobolus lamprauges

Conidiobolomycosis is a granulomatous disease caused by the fungus Conidiobolus spp. in humans and animals. Traditional technique for diagnosis of the disease is isolation of the agent associated with the presence of typical clinical signs and pathological conditions. The aim of this study was to describe the development of a specific polymerase chain reaction (PCR) test for Conidiobolus lamprauges to detect the fungus in clinical samples. Samples from suspected animals were collected and submitted to isolation, histopathological analysis and amplification by PCR. DNA from tissues was subjected to PCR with fungi universal primers 18S rDNA gene, and specific primers were designed based on the same gene in C. lamprauges that generated products of about 540 bp and 222 bp respectively. The culture was positive in 26.6% of clinical samples. The PCR technique for C. lamprauges showed amplification of DNA from fresh tissues (80%) and paraffin sections (44.4%). In conclusion, the PCR technique described here demonstrated a high sensitivity and specificity for detection of fungal DNA in tissue samples, providing a tool for the rapid diagnosis of C. lamprauges.

Development of a Real-time PCR test for porcine group A rotavirus diagnosis

Group A Rotavirus (RVA) is one of the most common causes of diarrhea in humans and several animal species. A SYBR-Green Real-Time polymerase chain reaction (PCR) was developed to diagnose RVA from porcine fecal samples, targeting amplification of a 137-bp fragment of nonstructural protein 5 (NSP5) gene using mRNA of bovine NADH-desidrogenase-5 as exogenous internal control. Sixty-five samples were tested (25 tested positive for conventional PCR and genetic sequencing). The overall agreement (kappa) was 0.843, indicating 'very good' concordance between tests, presenting 100% of relative sensitivity (25+ Real Time PCR/25+ Conventional PCR) and 87.5% of relative sensitivity (35- Real Time PCR/40- Conventional PCR). The results also demonstrated high intra- and inter-assay reproducibility (coefficient of variation ≤1.42%); thus, this method proved to be a fast and sensitive approach for the diagnosis of RVA in pigs.

A survey of the generation of ocean waves in a test basin

At present stage the analytical design of wave tolerance for floating structures and vessels is still imperfect due to the mutually complex and nonlinear phenomena between structures and waves. Wave tolerance design is usually carried out through iterative evaluations of results from model tests in a wave basin, and this is done in order to reach a final structural design. The wave generation has then become an important technology in the field of the coastal and ocean engineering. This paper summarizes the facilities of a test basin and a wave maker in Japan and also surveys the methodology of the generation of ocean waves in a test basin.

A Test Bed Applied Mobile Robot Navigation

It is presented a test bed applied to studies on dynamics, control, and navigation of mobile robots. A cargo ship scale model was chosen, which can be radio-controlled or operated autonomously through an embedded control system. A control program, which manages on board mission execution, is implemented on a microcontroller. Navigation is based on an electronic compass, which includes automatic compensation for pitch and roll motions. Heading control loop is based on this sensor, and on a rudder positioning system. A propulsion control system is also implemented. Typical manoeuvres as the turning test and "zig-zag", were implemented and tested. They are included on a manoeuvre library, and can be accessed independently or in combined modes. The embedded system is also in charge of signal acquisition and storing during the missions. It is possible to analyse experiments on identification of ship dynamics, control, and navigation, through the data transferred to a PC by serial communication. Navigation is going to be improved by including inertial sensors on board, and a DGPS. Preliminary tests are aimed to ship identification, and manoeuvrability, using free model tests. Future steps include extending this system for developing other mobile robots as, ROVs, AUVs, and aerial vehicles.

Sorption and desorption of Suflafenacil in two soils in the state of São Paulo with different physical and chemical attributes

A study was conducted to evaluate the sorption and desorption of 14C herbicide saflufenacil (pyrimidinedione) in two soils in the State of São Paulo, classified as Red Yellow Latosol with clayey texture (LVA-1) and medium texture (LVA-2), using the batch method through isotherms. The soils were air dried and sieved a 2 mm mesh. The radioactivity was determined by liquid scintillation spectrometry in acclimatized room (25 ± 2 °C). Sorption isotherms were conducted for 5 concentrations of saflufenacil (5.0; 2.5; 1.0; 0.5 and 0.05 μg mL-1) and the results were adjusted to the Freundlich equation, thus obtaining the parameters of sorption followed by two extractions with 0.01 M CaCl2 to determine desorption parameters similarly to sorption. The results showed that saflufenacil sorption was low for both soils studied, being greater for the LVA with higher organic matter content. The desorption coefficients were greater than their sorption coefficients, suggesting the occurrence of hysteresis. The sorption and desorption isotherms (classified as type C isotherms), hysteresis and the t-test between the angular coefficient of the respective isotherms showed that both the sorption and desorption occur with equal intensity.

Comparative uptake and metabolism of 2-[14C]-2,4-dichlorophenoxyacetic acid in callus cultures of monocot (Dioscorea spp.) and dicot (Nicotiana tabacum L.) plants

(Comparative uptake and metabolism of 2-[14C]-2,4-dichlorophenoxyacetic acid in callus cultures of monocot (Dioscorea spp.) and dicot (Nicotiana tabacum L.) plants). The uptake and metabolism of 2-[14C]-2,4-dichlorophenoxyacetic acid (2,4-D) were investigated in leaf calluses of Nicotiana tabacum, tuber calluses of Dioscorea opposita and calluses derived from zygotic embryos, leaves and petioles of Dioscorea composita. Striking similarities were evident in the patterns of 2,4-D metabolites and their chemical characteristics in the three callus types of D. composita compared, but significant differences were detected among the patterns of rnetabolites in the three species studied. Preliminary investigations on the stability of various metabolites (separated using TLC) by hydrolysis showed that sugar esters appeared to be the major metabolites in tobacco whilst in yams (D. opposita) glycosides were shown to be the main ones, which indicated a similarity between plants of Gramineae and Dioscoreaceae in terms of 2,4-D metabolism. Release of 2,4-D from tobacco callus cells upon their transfer to 2,4-D-free medium was detected and the implications of this are discussed in relation to the cultural conditions necessary to induce morphogenesis in vitro.

Test of hypotheses about herbivory and chemical defences of Qualea parviflora (Vochysiaceae) in Brazilian Cerrado

(Test of hypotheses about herbivory and chemical defences of Qualea parviflora (Vochysiaceae) in Brazilian Cerrado). Qualea parviflora Mart. (Vochysiaceae), a widely distributed tree found in different habitats in Brazilian Cerrado (savanna), provides resources for a great variety of insects. In this study, we tested two hypotheses about plant investment in anti-herbivore defences along a fertility gradient in Cerrado: the carbon/nitrogen balance (CNBH) and resource availability (RAH). We also investigated how the pattern of herbivory varies through the year and among three types of vegetation in Brazilian Cerrado - campo sujo, cerrado sensu strictu and cerradão. Sampling was conducted in three types of vegetation and in rainy (January and November) and dry months (April and July). Damage on 20 completely expanded leaves, leaf nutrients, sclerophlylly, total phenols and tannins were recorded for each plant (n = 30). When leaves were young, less sclerophyllous, and with higher concentration of nutrients and tannins, damage by herbivores was about 7% in cerrado sensu stricto and 3% in campo sujo. Mature leaves did not show any significant difference on herbivory among habitats, that varied from 6 to 9%. Nutrient availability to plants is an important factor determining production of secondary metabolites in Q. parviflora, corroborating the CNBH. The absence of correlation between damaged leaf area and tannin concentration did not corroborate the RAH, suggesting that tannin production is not strongly influenced by herbivores on Q. parviflora.

Genotoxic activity of the insecticide Nuvacron (Monocrotophos) detected by the micronucleus test in bone marrow erythrocytes of mice and in CHO cells

The organophosphorus insecticide Nuvacron (Monocrotophos) is a very toxic agent widely utilized in Brazilian agriculture. To evaluate the clastogenic potential of this insecticide, in vivo and in vitro micronucleus (MN) assay experiments were carried out on Swiss mice and on Chinese hamster ovary (CHO) cells, respectively. Nuvacron administered at doses of 2.5 and 5.0 mg/kg induced a statistically significant increase in the frequencies of MN detected in polychromatic bone marrow erythrocytes from animals (six/group) treated ip 24 h before. Exponentially growing CHAO cells were treated continuously (16h) with Nuvacron diluted in water to final concentrations of 1, 10, 100, 200, and 400 mug/ml. Three experiments were carried out using the cytokinesis-block method and a total of 6000 binucleated cells were scored to determine MN frequencies. A statistically significant increase in the frequencies of MN was observed for the cells treated with 1 and 10 mug/ ml Nuvacron. A marked decrease in cell proliferation rates was observed for CHO cultures treated with higher concentrations. These data demonstrate that Nuvacron has a genotoxic effect on both in vivo and in vitro mammalian test systems.

Effect of urea on biomimetic aggregates

The effect of urea on biomimetic aggregates (aqueous and reversed micelles, vesicles and monolayers) was investigated to obtain insights into the effect of the denaturant on structured macromolecules. Direct evidence obtained from light scattering (static and dynamic), monolayer maximum isothermal compression and ionic conductivity measurements, together with indirect evidence from fluorescence photodissociation, fluorescence suppression, and thermal reactions, strongly indicates the direct interaction mechanism of urea with the aggregates. Preferential solvation of the surfactant headgroups by urea results in an increase in the monomer dissociation degree (when applied), which leads to an increase in the area per headgroup and also in the loss of counterion affinities

Standardization of a computerized method for calculating autonomic function test responses in healthy subjects and patients with diabetes mellitus

The objectives of the present study were 1) to compare results obtained by the traditional manual method of measuring heart rate (HR) and heart rate response (HRR) to the Valsalva maneuver, standing and deep breathing, with those obtained using a computerized data analysis system attached to a standard electrocardiograph machine; 2) to standardize the responses of healthy subjects to cardiovascular tests, and 3) to evaluate the response to these tests in a group of patients with diabetes mellitus (DM). In all subjects (97 healthy and 143 with DM) we evaluated HRR to deep breathing, HRR to standing, HRR to the Valsalva maneuver, and blood pressure response (BPR) to standing up and to a sustained handgrip. Since there was a strong positive correlation between the results obtained with the computerized method and the traditional method, we conclude that the new method can replace the traditional manual method for evaluating cardiovascular responses with the advantages of speed and objectivity. HRR and BPR of men and women did not differ. A correlation between age and HRR was observed for standing (r = -0.48, P<0.001) and deep breathing (r = -0.41, P<0.002). Abnormal BPR to standing was usually observed only in diabetic patients with definite and severe degrees of autonomic neuropathy.

Interaction between NO and oxytocin: Influence on LHRH release

Nitric oxide synthase (NOS)-containing neurons have been localized in various parts of the CNS. These neurons occur in the hypothalamus, mostly in the paraventricular and supraoptic nuclei and their axons project to the neural lobe of the pituitary gland. We have found that nitric oxide (NO) controls luteinizing hormone-releasing hormone (LHRH) release from the hypothalamus acting as a signal transducer in norepinephrine (NE)-induced LHRH release. LHRH not only releases LH from the pituitary but also induces sexual behavior. On the other hand, it is known that oxytocin also stimulates mating behavior and there is some evidence that oxytocin can increase NE release. Therefore, it occurred to us that oxytocin may also stimulate LHRH release via NE and NO. To test this hypothesis, we incubated medial basal hypothalamic (MBH) explants from adult male rats in vitro. Following a preincubation period of 30 min, MBH fragments were incubated in Krebs-Ringer bicarbonate buffer in the presence of various concentrations of oxytocin. Oxytocin released LHRH at concentrations ranging from 0.1 nM to 1 µM with a maximal stimulatory effect (P<0.001) at 0.1 µM, but with no stimulatory effect at 10 µM. That these effects were mediated by NO was shown by the fact that incubation of the tissues with NG-monomethyl-L-arginine (NMMA), a competitive inhibitor of NOS, blocked the stimulatory effects. Furthermore, the release of LHRH by oxytocin was also blocked by prazocin, an a1-adrenergic receptor antagonist, indicating that NE mediated this effect. Oxytocin at the same concentrations also increased the activity of NOS (P<0.01) as measured by the conversion of [14C]arginine to citrulline, which is produced in equimolar amounts with NO by the action of NOS. The release of LHRH induced by oxytocin was also accompanied by a significant (P<0.02) increase in the release of prostaglandin E2 (PGE2), a mediator of LHRH release that is released by NO. On the other hand, incubation of neural lobes with various concentrations of sodium nitroprusside (NP) (300 or 600 µM), a releaser of NO, revealed that NO acts to suppress (P<0.01) the release of oxytocin. Therefore, our results indicate that oxytocin releases LHRH by stimulating NOS via NE, resulting in an increased release of NO, which increases PGE2 release that in turn induces LHRH release. Furthermore, the released NO can act back on oxytocinergic terminals to suppress the release of oxytocin in an ultrashort-loop negative feedback

The effect of bacterial lipopolysaccharide on gastric emptying in rats suffering from moderate renal insufficiency

The objective of the present study was to evaluate the response of rats suffering from moderate renal insufficiency to bacterial lipopolysaccharide (LPS, or endotoxin). The study involved 48 eight-week-old male SPF Wistar rats (175-220 g) divided into two groups of 24 animals each. One group underwent 5/6 nephrectomy while the other was sham-operated. Two weeks after surgery, the animals were further divided into two subgroups of 12 animals each and were fasted for 20 h but with access to water ad libitum. One nephrectomized and one sham-treated subgroup received E. coli LPS (25 µg/kg, iv) while the other received a sterile, pyrogen-free saline solution. Gastric retention (GR) was determined 10 min after the orogastric infusion of a standard saline test meal labeled with phenol red (6 mg/dl). The gastric emptying of the saline test meal was studied after 2 h. Renal function was evaluated by measuring the plasma levels of urea and creatinine. The levels of urea and creatinine in 5/6 nephrectomized animals were two-fold higher than those observed in the sham-operated rats. Although renal insufficiency did not change gastric emptying (median %GR = 26.6 for the nephrectomized subgroup and 29.3 for the sham subgroup), LPS significantly retarded the gastric emptying of the sham and nephretomized groups (median %GR = 42.0 and 61.0, respectively), and was significantly greater (P<0.01) in the nephrectomized rats. We conclude that gastric emptying in animals suffering from moderate renal insufficiency is more sensitive to the action of LPS than in sham animals

Memory-impairing effects of local anesthetics in an elevated plus-maze test in mice

Post-training intracerebroventricular administration of procaine (20 µg/µl) and dimethocaine (10 or 20 µg/µl), local anesthetics of the ester class, prolonged the latency (s) in the retention test of male and female 3-month-old Swiss albino mice (25-35 g body weight; N = 140) in the elevated plus-maze (mean ± SEM for 10 male mice: control = 41.2 ± 8.1; procaine = 78.5 ± 10.3; 10 µg/µl dimethocaine = 58.7 ± 12.3; 20 µg/µl dimethocaine = 109.6 ± 5.73; for 10 female mice: control = 34.8 ± 5.8; procaine = 55.3 ± 13.4; 10 µg/µl dimethocaine = 59.9 ± 12.3 and 20 µg/µl dimethocaine = 61.3 ± 11.1). However, lidocaine (10 or 20 µg/µl), an amide class type of local anesthetic, failed to influence this parameter. Local anesthetics at the dose range used did not affect the motor coordination of mice exposed to the rota-rod test. These results suggest that procaine and dimethocaine impair some memory process(es) in the plus-maze test. These findings are interpreted in terms of non-anesthetic mechanisms of action of these drugs on memory impairment and also confirm the validity of the elevated plus-maze for the evaluation of drugs affecting learning and memory in mice

Improvement of the indirect hemagglutination test for the detection of antibodies to Streptococcus pyogenes

An indirect hemagglutination test for a seroepidemiological survey of Streptococcus pyogenes infection was standardized. This is an improved modification of the indirect hemagglutination test which utilizes an unstable reagent prepared with fresh blood cells. Two types of bacterial antigens represented by extracellular products and purified streptolysin O were assayed, but only the former antigen gave good results. Pretreatment of the bacterial antigen with 0.15 M NaOH and neutralization to pH 5.5, as well as postfixation of sensitized red cells with 0.1% glutaraldehyde at 56oC for 30 min were found to be essential to give long stability to the reagent in liquid suspension, at least 9 months at 4oC. A total of 564 serum samples with high, moderate and low anti-streptolysin O antibodies as determined by the neutralization assay were studied by the indirect hemagglutination test using the new reagent. The sensitivity, specificity, efficiency, positive predictive value and negative predictive value of the test in relation to the neutralization assay were 0.950, 0.975, 0.963, 0.973, and 0.955, respectively. The kappa agreement index between the two techniques was high (0.926) and ranked as "almost perfect". Antibody levels detected by both techniques also presented a high positive correlation (rs = 0.726). Five reagent batches successively produced proved to be reproducible. Thus, the improved indirect hemagglutination test seems to be useful for public health laboratories.