A multiplex PCR for the detection of Brucella spp. and Leptospira spp. DNA from aborted bovine fetuses

Bovine brucellosis and leptospirosis are important causes of bovine abortion around the world. Both diseases can be serologically diagnosed, but many factors may cause false positive and negative results. Direct methods based on bacteriological isolation are usually employed, but they are difficult, time consuming and dangerous. Monoplex polymerase chain reaction (PCR) have been successfully described for the detection of Brucella spp. and Leptospira spp. Aiming at improvement in the direct diagnosis, a multiplex PCR (mPCR) for the detection of these agents in aborted bovine fetuses is described. The detection threshold of the mPCR was evaluated in experimentally contaminated bovine clinical samples using a conventional proteinase K/SDS or a boiling-based extraction protocols. The mPCR was applied to two groups of clinical samples: 63 episodes of bovine abortion and eight hamsters experimentally infected with Leptospira interrogans serovar pomona. Adopting microbiological isolation as reference, the test showed a sensitivity of 100% in both groups of clinical samples. Seven samples collected from bovine fetuses were Brucella spp. culture negative but showed positive results in mPCR. Regarding Leptospira spp. detection, similar results were observed in three bovine clinical samples. All hamsters infected with Leptospira were positive in both microbiological culture and mPCR. The boiling extraction protocol showed better results in some clinical samples, probably by the removal of PCR inhibitors by heat treatment. The high sensitivity, simplicity and the possibility of detection of both bacteria in a single tube reaction support the use of the mPCR described in the routine diagnosis.     

Piscine reovirus (PRV) in wild Atlantic salmon,Salmo salar L., and sea‐trout,Salmo trutta L., in Norway

This is the first comprehensive study on the occurrence and distribution of piscine reovirus (PRV) in Atlantic salmon, Salmo salar L., caught in Norwegian rivers. PRV is a newly discovered reovirus associated with heart and skeletal muscle inflammation (HSMI), a serious and commercially important disease affecting farmed Atlantic salmon in Norway. A cross‐sectional survey based on real‐time RT‐PCR screening of head kidney samples from wild, cultivated and escaped farmed Atlantic salmon caught from 2007 to 2009 in Norwegian rivers has been conducted. In addition, anadromous trout (sea‐trout), Salmo trutta L., caught from 2007 to 2010, and anadromous Arctic char, Salvelinus alpinus (L.), caught from 2007 to 2009, were tested. PRV was detected in Atlantic salmon from all counties included in the study and in 31 of 36 examined rivers. PRV was also detected in sea‐trout but not in anadromous Arctic char. In this study, the mean proportion of PRV positives was 13.4% in wild Atlantic salmon, 24.0% in salmon released for stock enhancement purposes and 55.2% in escaped farmed salmon. Histopathological examination of hearts from 21 PRV‐positive wild and one cultivated salmon (Ct values ranging from 17.0 to 39.8) revealed no HSMI‐related lesions. Thus, it seems that PRV is widespread in Atlantic salmon returning to Norwegian rivers, and that the virus can be present in high titres without causing lesions traditionally associated with HSMI.     

Bioactivity of extracts and isolated compounds from Vitex polygama (Verbenaceae) and Siphoneugena densiflora (Myrtaceae) against Spodoptera frugiperda (Lepidoptera: Noctuidae)

The effects of crude extracts, fractions and isolated compounds from Vitex polygama Cham. and Siphoneugena densiflora Berg were evaluated on the development of Spodoptera frugiperda JE Smith, a destructive insect pest of corn and several other crops. The extracts and fractions were incorporated into an artificial diet at 1 mg g(-1) and offered to the insect during its larval stage. Length and viability of larval and pupal stages as well as pupal weight were assessed. Isolated compounds were tested through superficial contamination of the diet at 0.1 mg g(-1). Weight and viability of ten-day-old larvae were determined. Methanolic and hydroalcoholic S. densiflora extracts caused 100% larval mortality, while leaf and fruit hydroalcoholic extracts from V. polygama were the most active. Among the isolated compounds, flavonoids presented the best insecticidal results, and tannins the best larval growth inhibition.     

Potential Utilization of Green Tide-Forming Macroalgae from Patos Lagoon,Rio Grande-RS,Brazil

ABSTRACT

This study was conducted to evaluate the biochemical and nutritional characteristics of naturally occurring estuarine macroalgae in the Patos Lagoon (Rio Grande, Brazil). The study of these macroalgae, dominated by Ulva and Cladophora species, contributes to current research on the potential utilization of green tide-forming algae. Although high levels of carotenoids (378 ± 0.02 µg g^?1) and antioxidant activity (75.0% for DPPH) were found in the naturally occurring macroalgae, these macroalgae cannot be used in the food industry due to the presence of toxic levels of arsenic (exceeding the thresholds of both French and Brazilian legislation for food). Regarding the potential of the studied macroalgae as a biofuel source, the low lipid and very high carbohydrate contents suggest a possible use in the fermentation of ethanol, butanol, or methane.     

Simple Technologies for Converting Rest Raw Materials of Atlantic Salmon (Salmo salar) into High-Quality,Valuable, and Tasty Feed Ingredients

Rest raw materials as viscera, heads, and frames from farmed Atlantic salmon (Salmo salar) were hydrolyzed with the use of endogenous enzymes and the commercial enzymes Protamex and a mixture of Papain and Bromelain. Composition of the prehydrolysis mixture clearly influenced the quality of final hydrolysate and process kinetics. An increased proportion of viscera increased the amount of endogenous enzymes, which influenced hydrolysis kinetics, the extent of hydrolysis, and increased bitterness. Commercial enzymes, in addition to endogenous enzymes, are not always more yield efficient or economically beneficial but can be used to improve the taste and to ease the separation of oil from the hydrolysate fraction. Hydrolysis of denatured proteins in rest raw materials was hardly detectable. Optimum temperature should therefore be selected to avoid protein denaturation and simultaneously maintain high enzymatic activity. Hydrolysates preferably contain a low concentration of oil, and this work shows that high-quality oil can be separated before hydrolysis by mild thermal treatment. The previous separation of oil did not influence hydrolysate yield and decreased the concentration of lipids in the final hydrolysate. The initial separation of oil also increased productivity of the hydrolysis reactor, due to the reduction of hydrolysis volume.     

Quality and Shelf Life of Liver of Farmed Cod (Gadus morhua)

ABSTRACT

Livers of Atlantic cod (Gadus morhua) are traditionally used in cod liver oil production or consumed cooked or canned. The farming of cod is a relatively new industry in Norway. The aim of this study was to determine quality and shelf life of fresh liver from farmed cod during chilled storage on ice by hydrolysis and oxidation state and sensory quality and the influence on canned liver. In two experiments, livers from farmed cod were stored chilled and sampled from Days 0 to 13, respectively. Quality, measured as hydrolytic and oxidation degradation, was reduced after 7 days of storage, while sensory quality was reduced after 4 days. Free fatty acids increased from Day 7 in both experiments, while peroxide value and anisidine value showed no change when the livers were single wrapped. Rancid odor was the first sign of oxidation and was registered after three to four days of storage. Canning within 2 days of storage prevented leakage of oil from the canned livers. Sensory analyses of oxidation are recommended as a sensitive and rapid method to detect oxidation of chilled cod liver.     

Evaluation of immunological, parasitological and molecular methods for the diagnosis of Schistosoma mansoni infection before and after chemotherapy treatment with praziquantel in experimentally infected Nectomys squamipes

In low endemicity areas of schistosomiasis, the recommended diagnostic method of coprological examination results in an underestimation of infection cases. Alternative diagnostic methods have been developed, such as immunodiagnostic and molecular techniques. In this study we evaluated three methods used in the diagnosis of Schistosoma mansoni infection: parasitological (Kato-Katz), immunological (ELISA) and molecular (real time PCR), and also investigated the sensitivity of each technique in the cure determination after treatment with praziquantel using the water rat Nectomys squamipes, a natural reservoir for S. mansoni, as an experimental model. Two infection laboratory experiments were carried out. The first experiment aimed to observe the evolution of the immunological response in the first moments after infection and in the first months after treatment. The second experiment aimed to compare the efficacy of the three diagnostic techniques after infection and after treatment over a more extended time period. In the first experiment, 44% of the infected animals showed IgG reactivity after two weeks of infection, and 94% were positive based on serology 30 days after infection. The serological IgG titers increased just after infection but decreased gradually after treatment. In the second experiment, 89% of the animals showed positive IgG titers 22 days after infection. Only 68% of the animals showed positive results on the coproscopic diagnostic analysis and 79% did so by qPCR, 50 days after infection. Treated animals showed negative results on coproscopy one month after treatment but remained positive by serology even 12 months after treatment, although showing a decline in immunologic reaction after treatment. By qPCR analysis, all animals showed negative results three months after treatment, except for one animal. The parasitosis can be detected by coproscopy only six weeks after infection, and by serology 14 days after infection. The qPCR was a better diagnostic method for confirming the infection cure of S. mansoni. In early infection, this method was less efficient than serology but was slightly more efficient than the Kato-Katz method. We suggest that the methods should be used in low endemic areas as follows: serology should be used in the initial diagnosis in a population with potential positive cases; subsequently, coproscopy should be used in IgG positive cases to confirm the current infection; and qPCR should be used to evaluate the infection cure after treatment and is also a very valuable tool when there are cases showing positive IgG and negative coproscopy.     

Effects of latex from "Amapazeiro"Parahancornia amapa (Apocynaceae) on blowfly Chrysomya megacephala (Diptera: Calliphoridae) post-embryonic development

Nowadays, insect control is usually carried out using chemical insecticides, but insect resistance and other negative side effects have prompted the search for alternatives. Biopesticides provide a positive alternative to synthetic pesticides because they have low impact on the environmental, low toxicity to humans and low costs among other advantages. This research was carried out to evaluate the activity of Parahancornia amapa (Huber) Ducke (Apocynaceae) lyophilized latex on the post embryonic development of Chrysomya megacephala (F.) (Diptera: Calliphoridae). Larvae treated with 1.0% latex showed a shorter post embryonic development period (larval, pupal and newly hatched larvae to adult); whereas larvae treated with 3.0% latex provoked a prolongation of these periods. Viability (53%) was also very low at the newly hatched larvae to adult period for larvae treated with 3.0% latex, indicating that latex from P. amapa at high concentrations could change C. megacephala post embryonic development.     

Effect of high‐density lipoprotein on oocyte maturation and bovine embryo development in vitro

High‐density lipoprotein (HDL) is the main lipoprotein in the follicular fluid, and it has anti‐inflammatory, antioxidant and cryoprotectant properties. The anti‐inflammatory potential and antioxidant potential are derived from its lipid composition, especially the apolipoprotein AI (ApoAI) and paraoxonase 1 (PON1). The aim of this study was to evaluate the effect of HDL during in vitro maturation (IVM) on oocyte maturation and early bovine embryo development. For this, cumulus–oocyte complexes (COCs) were obtained from bovine ovaries collected at a local slaughterhouse. COCs (n = 2,250) were allocated into three groups (n = 50 COCs/group) according to the addition of HDL protein (HDL‐P) during IVM for 22 hr: 0 (control), 50 and 150 mg/dl. After IVM, COCs were inseminated (in vitro fertilization) and cultivated for 7 days. Total cholesterol concentration, total protein, triglycerides and ApoAI concentrations on IVM medium increased proportionally to HDL‐P addition. However, PON1 activity was not detected in any treatment. The addition of HDL‐P did not affect nuclear maturation rate, endogenous reactive oxygen species and glutathione levels in COCs (p > 0.05). The highest HDL‐P concentration (150 mg/dl) decreased cleavage and blastocyst rate (p < 0.05). Moreover, the HDL‐P 150 mg/dl group had lower cellular count/blastocyst than the 50 mg/dl group (p < 0.05). However, the addition of HDL‐P did not affect relative gene expression of evaluated genes. In conclusion, the complex HDL/ApoAI obtained from human plasma, in the absence of PON1 activity during in vitro oocyte maturation, decreased initial embryo development.