Practical High-Throughput Method to Screen Compounds for Anthelmintic Activity against Caenorhabditis elegans.

In the present study, we established a practical and cost-effective high throughput screening assay, which relies on the measurement of the motility of Caenorhabditis elegans by infrared light-interference. Using this assay, we screened 14,400 small molecules from the "HitFinder" library (Maybridge), achieving a hit rate of 0.3%. We identified small molecules that reproducibly inhibited the motility of C. elegans (young adults) and assessed dose relationships for a subset of compounds. Future work will critically evaluate the potential of some of these hits as candidates for subsequent optimisation or repurposing as nematocides or nematostats. This high throughput screening assay has the advantage over many previous assays in that it is cost- and time-effective to carry out and achieves a markedly higher throughput (~10,000 compounds per week); therefore, it is suited to the screening of libraries of tens to hundreds of thousands of compounds for subsequent evaluation and development. The present phenotypic whole-worm assay should be readily adaptable to a range of socioeconomically important parasitic nematodes of humans and animals, depending on their dimensions and motility characteristics in vitro, for the discovery of new anthelmintic candidates. This focus is particularly important, given the widespread problems associated with drug resistance in many parasitic worms of livestock animals globally.

A fatal case after an intravenous injection of levamisole.

Levamisole is a drug originally prescribed as an antihelmintic. Because of the occurrence of severe cases of agranulocytosis and leukoencephalitis it was removed from the French market in 1998 for human use, while it remains available for veterinary use. Nowadays in France its only use in humans is regulated by authorization for temporary use for its immunomodulatory properties in the treatment of nephritic syndrome.A 52-year-old man was found dead at his farm. Injection points were observed on his arm and a syringe containing a dark orange-brown liquid was found near the body. At his home, the discovery of a letter highlighted suicidal intent. Analysis of the aforementioned liquid, peripheral blood and urine confirmed the unique presence of levamisole. The femoral blood concentration of levamisole was of 25 mg/L whereas the femoral blood concentrations reported in cases of fatalities after cocaine use do not exceed 0.0056 mg/L. In humans, levamisole can be detected in biological samples after cocaine use as this drug is also an adulterant and one of its metabolites (aminorex) seems to have amphetamine-like properties. In this case, the man consumed levamisole from time to time for its stimulant and strengthening effects.Cases of fatal poisoning using levamisole are very rare and poorly documented, which makes the interpretation of postmortem blood levamisole concentration difficult.

Nematocidal Effect of Oyster Culinary-Medicinal Mushroom Pleurotus ostreatus (Agaricomycetes) against Haemonchus contortus.

The nematocidal effect of Pleurotus ostreatus (white variety of oyster mushroom) aqueous extract (AE) was evaluated against Haemonchus contortus eggs and infective larvae (L3) in vitro and in artificially infected gerbils (Meriones unguiculatus). The chemical analyses indicated that constituents of AE are tridecanoic, tetradecanoic, linolelaidic, 9,15-octadecadienoic, and oxalic acids. P. ostreatus extract inhibited larval hatching by 100% at the concentration of 2.24 mg/mL and (50% effective concentration) EC50 of 0.73 mg/mL. In the larval development test, AE induced a larvicidal effect at the concentration of 50 mg/mL and EC50 of 17.24 mg/mL. The larval migration test revealed a reduction of 94.7% at a concentration of as low as 4 mg/mL and EC50 of 1.25 mg/mL. No significant effects of treatment with P. ostreatus AE were seen on H. contortus in the gerbil model. Thus, our results demonstrate an important nematocidal in vitro effect of P. ostreatus AE against the parasite H. contortus. However, further investigations are necessary to confirm the anthelmintic potential of P. ostreatus extract in small ruminants.

Discovery of potential ovicidal natural products using metabolomics.

Plant extracts are a potential source of new compounds for nematode control and may be an excellent alternative for the control gastrointestinal nematodes that are resistant to conventional anthelmintics. However, research involving natural products is a complex process. The main challenge is the identification of bioactive compounds. Online analytical techniques with universal detectors, such as high-performance liquid chromatography-mass spectrometry (HPLC-MS), together with metabolomics could enable the fast, accurate evaluation of a massive amount of data, constituting a viable option for the identification of active compounds in plant extracts. This study focused on the evaluation of the ovicidal activity of ethanol extracts from 17 plants collected from the Pantanal wetland in the state of Mato Grosso do Sul, Brazil, against eggs of Haemonchus placei using the egg hatchability test. The ethanol extracts were obtained using accelerated solvent extraction. The data on ovicidal activity, mass spectrometry and metabolomics were evaluated using HPLC-DAD-MS, partial least squares regression analysis (PLS-DA) and a correlation map (univariate correlation analyses) to detect compounds that have a positive correlation with biological activity. Among the ten metabolites with the best correlation coefficients, six were phenylpropanoids, two were triterpene saponins, one was a brevipolide, and one was a flavonoid. Combinations of metabolites with high ovicidal action were also identified, such as phenylpropanoids combined with the triterpene saponins and the flavonoid, flavonoids combined with iridoid and phenylpropanoids, and saponins combined with phenylpropanoid. The positive correlation between classes of compounds in plants belonging to different genera and biological activity (as previously identified in the literature) reinforces the robustness of the statistical data and demonstrates the efficacy of this method for the selection of bioactive compounds without the need for isolation and reevaluation. The proposed method also enables the determination of synergism among the classes, which would be impracticable using traditional methods. The present investigation demonstrates that the metabolomic technique was efficient at detecting secondary metabolites with ovicidal activity against H. placei. Thus, the use of metabolomics can be a tool to accelerate and simplify bioprospecting research with plant extracts in veterinary parasitology.

Nematicidal activity of 'major royal jelly protein'-containing glycoproteins from Acacia honey.

Parasitic nematodes infect more than two billion people worldwide particularly in developing countries. We previously reported nematicidal activity of natural honey using model nematode Caenorhabditis elegans. In this study, characterization of nematicidal effects of natural honey and its glycoproteins has been carried out. Chromatographically separated honey glycoproteins showed potent anti-C. elegans activity (LD50 = 100 ng proteins/µL). Honey glycoproteins with molecular masses of ∼260 kD and ∼160 kD comprised of 'major royal jelly protein-1'-containing complexes. In these complexes, MRJP1 was present in different glycosylation forms. Quantitative PCR based gene expression assays described molecular functions of C. elegans affected by honey and honey glycoproteins. Expression of 14 gene transcripts associated with key cellular and molecular functions including energy metabolism, cytoskeleton, cell division, transcription and translation was analyzed. Acacia honey exerted a concentration-dependent alteration of gene transcripts involved in the citric acid cycle (mdh-1 and idhg-1) and cytoskeleton (act-1, act-2, and arp6). Likewise, MRJP1-containing glycoproteins caused down-regulation of arp-6 and idhg-1; and up-regulation of act-1 and mdh-1 gene transcripts. Consistent down-regulation of isocitrate dehydrogenase encoding idhg-1 gene which is among the rate-controlling enzymes of the citric acid cycle was considered as main biochemical factor involved in the nematicidal activity of honey and MRJP-containing glycoproteins. Acacia honey suppressed the expression of gene transcripts encoding actin-2, while honey glycoproteins did not. Hence, honey partly exerted anti-C. elegans activity by decreasing the transcription of actin-2 gene transcripts, demonstrated by a defect in the movement and egg laying. Moreover, arp-6 gene transcripts encoding actin-related protein 6 was significantly and constantly down-regulated by honey and honey proteins.

Effects of biochar amendment on sorption, dissipation, and uptake of fenamiphos and cadusafos nematicides in sandy soil.

BACKGROUND: The application of biochar to soil is supposed to alter its adsorption/desorption potential toward pesticides, thereby affecting their bioavailability and efficacy. This is particularly relevant in the case of nematicides because these pesticides are directly applied to soil. RESULTS: Biochar was produced from date palm (PB) and eucalyptus (EB) waste at 450 °C and added at a rate of 1% to a sandy soil. The half-life (t½ ) of fenamiphos was increased from 2.7 to 18.3 and 18.6 days in PB- and EB-amended soils, respectively. By contrast, the half-life of cadusafos was unaffected. Freundlich Kf values increased from 1.22 and 0.39 (µg1-Nf g-1 mLNf ) to 4.49 and 6.84 in 1% PB-amended soil, and to 3.49 and 4.62 in 1% EB-amended soil for cadusafos and fenamiphos, respectively. Plant uptake of both nematicides in tomato seedlings was reduced by approximately 97% (cadusafos) and 85% (fenamiphos). Although nematicide efficacy against Meloidogyne incognita was not altered at the recommended dosage, it was negatively affected at a half-dose rate. Under these conditions, it decreased from 43.1% in unamended sandy soil to only 18.3% in 1% PB-amended soil. CONCLUSIONS: Biochar addition increased the sorption capacity of soil. This resulted in a decrease of nematicide bioavailability, together with a reduction of both the dissipation rate and uptake by tomato plants. © 2018 Society of Chemical Industry.

Report: An ex vivo up-take of levamisole molecules by cestode (Monezia expensa) of goat (Capra hirsa) and its detection through RP-HPLC.

Detection of various molecules of drugs remained a prime issue especially in tissues of animals, humans and in their target parasites. The cestode/tapeworms pose a dilemma because of their weird body composition and uptake pattern of nutrients and medicines especially through absorption by tegument. We selected levamisole; thought to be potent antiparasitic/ani-cestodal drug. The uptake of levamisole (LEV) through cestodeal tissues is studied through HPCL in this paper. High performance liquid chromatography technique has been utilized to know the uptake of levamisole in tissues of cestodes of Goat (Monezia expensa) in small ruminants. The drug was exposed to M. expensa by in vitro till its death or a parasite ceases its movement. The tissue/ part of proglattids of the M. expensa were homogenized with some modifications and levamisole extraction was performed with liquid phase extraction method. The evaporation of solvent was done and the residual cestodal tissues were cleaned by solid phase. After the solid phase extraction method, the recovery of drug, detection and quantification of levamisole from cestodal tissues was determined through Reverse Phase Column High Performance Liquid Chromatography (RP-HPLC). Levamisole (LEV) molecules assay was obtained on a C18 reverse-phase (20um, 6mm x 150mm) column at flow rate of 1ml/min using acetonitrile and ammonium acetate as mobile phase and UV detection was done at 254nm. The development of method of Levamisole (LEV) detection from cestodal tissues by HPLC in vitro samples has been demonstrated first time in Pakistan, which can provide the solution of parasitic control and provide in sight in to the uptake of anti cestodal drugs either against human or livestock parasites.

Kainic acid in the seaweed Palmaria palmata (dulse).

Twenty samples of the seaweed Palmaria palmata (dulse) purchased mainly from commercial Internet shops on the European market were analysed by a liquid chromatograph coupled with a tandem mass spectrometer method for the content of kainic acid, a naturally occurring neurotoxic compound in P. palmata. Kainic acid levels in the samples ranged widely from trace levels to approximately 560 µg g-1 dry weight.

Off-tarp emissions, distribution, and efficacy of carbonated fumigants in a low permeability film tarped field.

Carbonated fumigants have been shown to distribute quickly and uniformly in sandy soils and improve pest control efficacy for annual crops. Low permeability films, such as VaporSafe® (TIF), could further improve fumigant dispersion by effectively retaining the fumigant in soil; however, there is a concern that the TIF can lead to higher off-tarp edge emissions. An orchard field trial was conducted to determine the off-tarp emissions, distribution, efficacy, and fate of carbonated Telone® C35 [63.4% 1,3-dichloropropene (1,3-D), 34.7% chloropicrin (CP)] that was shank-injected at 46cm soil depth. Treatments included carbonated fumigants at full- or 2/3 rates and a full rate of regular (nitrogen-pressurized) fumigants covered with standard polyethylene (PE) film, TIF, or no surface seal. Fumigant emissions at the regular tarp edge (25cm from the shank line) peaked at 3.98µgm-2s-1 for 1,3-D and 0.05µgm-2s-1 for CP. The addition of a TIF tarp extension (to 85cm from the shank line) reduce peak off-tarp emissions to <1 and <0.03µgm-2s-1 for 1,3-D and CP, respectively. Fumigant concentration under TIF was usually at least twice that under PE regardless of carbonation. Carbonation at 345KPa with 1.5% of dissolved CO2 did not significantly improve fumigant dispersion in soil profile compared to the conventional nitrogen pressurization. In a citrus nematode bioassay, only the 2/3 rate of carbonated fumigation treatment sealed with PE failed to control nematodes at 25cm away from shank line. This research indicates that a 60-cm TIF extension can effectively reduce off-tarp edge emissions in strip fumigation treatments. While the adaptability of carbonation of fumigants is still questionable, further research efforts are needed in finding effective solutions to control plant parasitic nematodes, which remain a challenge in orchard fumigation.

Toxicity of the bionematicide 1,4-naphthoquinone on non-target soil organisms.

The main goal of the present study was to evaluate the ecotoxicological effects of 1,4-naphthoquinone (1,4-NTQ), a natural-origin compound presenting nematicidal activity, that can be obtained from walnut husk, in plants and soil invertebrates, including non-target soil nematode communities. This research was part of an ongoing project that aims to develop environmentally-friendly nematicides obtained from agricultural residues. The battery of ISO tests included emergence and growth of corn (Zea mays) and rape (Brassica napus); avoidance with the earthworm Eisenia andrei and the collembolan Folsomia candida; and reproduction with the previous species plus the enchytraeid Enchytraeus crypticus. A novel soil nematode community assay was also performed. ISO tests and nematode assays were conducted using a natural uncontaminated soil that was spiked with a range of 1,4-NTQ concentrations. Toxicity of 1,4-NTQ was found for all test-species and the most sensitive were F. candida and E. andrei. After 7 days of exposure to 1,4-NTQ, nematode abundance decreased along the concentration gradient, and a partial recovery was observed after 14 days (1,4-NTQ <48 mg kg-1 soil). The number of nematode families consistently decreased in both periods. Overall, results indicate that a 1,4-NTQ concentration of <20 mg kg-1 could be environmentally safe but preliminary data suggest that it might be ineffective for the target-nematodes, root-knot nematodes, Meloidogyne spp., and root-lesion nematodes, Pratylenchus spp. In addition, if higher dosages of 1,4-NTQ bionematicide are necessary, the potential recovery of non-target organisms under real field scenarios also needs to be assessed.

Plasma pharmacokinetics and muscle residue dynamics of mebendazole in Carassius auratus.

Mebendazole is approved for use in aquatic animals and is widely used in Chinese aquaculture. We developed a pharmacokinetic and residue analysis for mebendazole levels in the goldfish (Carassius auratus). Plasma and muscle samples of C. auratus were taken after oral administration of 10 mg/kg mebendazole. The maximal drug plasma concentration of 0.55 mg/L was achieved at 48 hr and then declined with the elimination half-life (T1/2ß ) of 7.99 hr. Administration of 10 mg/kg by oral gavage for 5 successive days resulted in a peak mebendazole concentration of 0.70 mg/kg in muscle at 96 hr after the last dose. The drug was then eliminated at a relatively slow rate from muscle with T1/2ß of 68.41 hr. There was no detectable mebendazole in any muscle samples at 24 days postadministration. The AUClast in plasma and muscle was 19.42 and 105.33 mg hr/L, respectively. These data provide information for dosage recommendations and withdrawal time determinations for mebendazole use in aquariums.

Bioactive secondary metabolites with multiple activities from a fungal endophyte.

In order to replace particularly biohazardous nematocides, there is a strong drive to finding natural product-based alternatives with the aim of containing nematode pests in agriculture. The metabolites produced by the fungal endophyte Fusarium oxysporum 162 when cultivated on rice media were isolated and their structures elucidated. Eleven compounds were obtained, of which six were isolated from a Fusarium spp. for the first time. The three most potent nematode-antagonistic compounds, 4-hydroxybenzoic acid, indole-3-acetic acid (IAA) and gibepyrone D had LC50 values of 104, 117 and 134 µg ml-1 , respectively, after 72 h. IAA is a well-known phytohormone that plays a role in triggering plant resistance, thus suggesting a dual activity, either directly, by killing or compromising nematodes, or indirectly, by inducing defence mechanisms against pathogens (nematodes) in plants. Such compounds may serve as important leads in the development of novel, environmental friendly, nematocides.

Atividade antagonista do óleo essencial de Lippia alba (Mill. ) N. E. Brown (Verbenaceae) sobre Meloidogyne incognita (Kofoid & White) Chitwood / Antagonist activity of the essential oil Lippia alba (Mill. ) N. E. Brown (Verbenaceae) on Meloidogyne incognita (Kofoid & White) Chitwood

RESUMO O controle de fitonematóides é uma tarefa difícil. A alta infestação de nematoides no solo obriga os produtores a usarem doses mais elevadas de nematicidas, ou ainda, a aumentarem a freqüência das aplicações, geralmente no solo, ocasionando maior potencial de dano ao homem e ao ambiente. Além disso, nematicidas convencionais vêm sofrendo grandes restrições de uso em muitos países. Desse modo, a busca de novas medidas de controle de fitonematóides é uma prioridade da agricultura sustentável. Desta forma, objetivou-se estudar o efeito de óleos essenciais de Lippia Alba, na mortalidade de juvenis pré-parasitas do segundo estádio de Meloidogyne incognita. O ensaio foi montado em delineamento inteiramente casualizado, com 4 repetições, em esquema fatorial 3×4 (3 quimiotipos x 4 concentrações) empregando-se três diferentes quimiotipos de L. Alba (I, II, III) nas concentrações de 0, 100, 500 e 1000 ppm, coletados em diferentes horas do dia (7, 9, 13, 16 e 19h). Os dados coletados (% de mortalidade) foram submetidos à análise de variância e os valores médios comparados por meio do teste de Tukey. Foi possível concluir que Lippia alba contém, em seu óleo essencial, compostos com efeitos significativos na mortalidade de juvenis de segundo estádio (J2) de M. incognita. Dessa forma, é importante destacar que o acentuado efeito nematicida do óleo essencial de L. alba sugere a possibilidade de seu uso no controle de M. incognita.

ABSTRACT The control of nematodes is difficult. The high infestation of nematodes in the soil requires the producers use higher doses of nematicides, increasing the frequency of applications, usually in soil, causing negative effects to humans and the environment. Furthermore, conventional nematicides have suffered great use restrictions in many countries. Thus, the search for new measures to control nematodes is a priority of sustainable agriculture. Thus, the objective of this study was available the effect of essential oils from Lippia Alba against Meloidogyne incognita. The experiment was conducted in a completely randomized design with four replications in a factorial 3x4 (3 chemotypes and 4 concentrations) using three different chemotypes of L. Alba (I, II, III) at concentrations of 0, 100, 500 and 1000 ppm, collected in different times of day (7 AM, 9 AM, 1 PM, 4 PM e 7 PM). The data collected (% mortality) were subjected to analysis of variance and mean values were compared using the Tukey test. It was able to conclude that L. alba contains in its essential oil, compounds with significant effects on mortality of M. incognita. Thus, it is important to note that the greatest nematicide effect of essential oil of L. alba suggests the possibility of its use to control M. incognita.

Eficácia de Chenopodium ambrosioides (erva-de-santa-maria) no controle de endoparasitos de Coturnix japônica (codorna japonesa) / Effectiveness of Chenopodium ambrosioides (santa maria herb) for controlling Coturnix japonica (japanese quail) endoparasites

O objetivo foi testar in vitro e in vivo a eficácia da planta medicinal Chenopodium ambrosioidesLinnaeus, 1786 (erva-de-santa-maria), nas formas fitoterápica e homeopática, como meios alternativos para o controle de endoparasitos de Coturnix japonica Temminck & Schlegel, 1849 (codorna japonesa), um sério problema que afeta a criação e desempenho de aves domésticas, ocasionando morte quando muito intenso, retardo de crescimento, redução de índice de conversão alimentar e aumento na suscetibilidade às doenças infecciosas. As metodologias utilizadas foram preconizadas por Coles et al. (1992), creditada pela World Association for the Advancement of Veterinary Parasitology (WAAVP). A pesquisa evidenciou a presença dos gêneros Ascaridiae Eimeria. O ensaio in vitro demonstrou alta taxa de redução na inibição de eclosão de ovos de Ascaridiasp. (100,00%) e significativa taxa de redução na destruição de oocistos de Eimeriasp. (47,06%). O ensaio in vivodemonstrou alta taxa de redução na contagem de ovos de Ascaridiasp. nas fezes (100,00%) e expressiva taxa de redução na contagem de oocistos de Eimeriasp. nas fezes (60,33%). Chenopodium ambrosioides mostrou em certos momentos superioridade frente ao produto tradicional (Thiabendazole/Mebendazole) e índices superiores aos preconizados pelo Ministério da Agricultura do Brasil e Organização Mundial da Saúde como indicativos de eficácia.

The aim was in vitro and in vivo to test the effectiveness of Chenopodium ambrosioides Linnaeus, 1786 (santa maria herb) medicinal plant, in regard to phytotherapeutic and homeopathic forms as alternative methods to control Coturnix japonica Temminck & Schlegel, 1849 (japanese quail) endoparasites. The parasitosis is a serious problem affecting domestic poultry raising and performance causing death, delay in grow, food conversion rate reduction and increase of susceptibility to infectious diseases. Methodologies were advocated by Coles et al. (1992), corroborated by World Association for the Advancement of Veterinary Parasitology (WAAVP). Presence of the genera Ascaridia and Eimeria was displayed by this survey. In vitro essay demonstrated high reduction rate on eggs eclosion inhibition of Ascaridia sp. (100.00%) and significant reduction rate on oocyst destruction of Eimeria sp. (47.06%). In vivo essay demonstrated high fecal egg counting reduction rate of Ascaridia sp. (100.00%) and expressive fecal oocyst counting reduction rate of Eimeriasp. (60.33%). C. ambrosioides showed upper rates front traditional product (Thiabendalol/Mebendazol) as well as to those ones advocated by the Brazilian Ministry of Agriculturel and the World Health Organization as effectiveness indicative.

Selective extraction and concentration of mebendazole in seawater samples using molecularly imprinted polymer as sorbent.

A high selective pre-treatment method for the extraction and analysis of mebendazole in environmental water samples was developed based on molecularly imprinted solid-phase extraction (MISPE). The mebendazole imprinted polymers were synthesized in acetonitrile using methacrylic acid and ethylene glycol dimethacrylate as functional monomer and cross-linker respectively. The imprinted materials showed high adsorption ability for mebendazole and were applied as special solid-phase extraction sorbents for selective separation of mebendazole. An off-line MISPE procedure was developed for the purification and enrichment of mebendazole from natural seawater samples prior to high-performance liquid chromatography analysis. The recoveries of spiked seawater on the MISPE cartridges were from 83.0% to 90.6%, and the values of the relative standard deviation were in the range of 2.78-4.13% (n=3). The satisfied results showed that this pre-treatment methodology for extracting mebendazole in seawater was simple and effective.

A comprehensive approach to the determination of two benzimidazoles in environmental samples.

Among the various pharmaceuticals regarded as emerging pollutants, benzimidazoles--represented by flubendazole and fenbendazole--are of particular concern because of their large-scale use in veterinary medicine and their health effects on aquatic organisms. For this reason, it is essential to have reliable analytical methods which can be used to simultaneously monitor their appearance in environmental matrices such as water, sediment and tissue samples. To date, however, such methods relating to these three matrices have not been available. In this paper we present a comprehensive approach to the determination of both drugs in the mentioned above matrices using liquid chromatography-ion trap mass spectrometry (LC-MS/MS). Special attention was paid to the sample preparation step. The optimal extraction methods were further validated by experiments with spiked water, sediment and fish tissue samples. Matrix effects were established. The following absolute recoveries of flubendazole and fenbendazole were achieved: 96.2% and 95.4% from waters, 103.4% and 98.3% from sediments, and 98.3% and 97.6% from fish tissue samples, respectively. Validation of the LC-MS/MS methods enable flubendazole and fenbendazole to be determined with method detection limits: 1.6 ng L(-1) and 1.7 ng L(-1) in water samples; 0.3 ng g(-1) for both compounds in sediment samples, and 3.3 ng g(-1) and 3.5 ng g(-1) in tissue samples, respectively. The proposed methods were successfully used for analysing selected pharmaceuticals in real samples collected in northern Poland. There is first data on the concentration in the environment of the target compounds in Poland.

Closantel plasma and milk disposition in dairy goats: assessment of drug residues in cheese and ricotta.

Closantel (CLS) is currently used in programs for the strategic control of gastrointestinal nematodes. CLS is extralabel used in different dairy goat production systems. From available data in dairy cows, it can be concluded that residues of CLS persist in milk. The current work evaluated the concentration profiles of CLS in plasma and milk from lactating orally treated dairy goats to assess the residues pattern in dairy products such as cheese and ricotta. Six (6) female Saanen dairy goats were treated orally with CLS administered at 10 mg/kg. Blood and milk samples were collected between 0 and 36 days post-treatment. The whole milk production was collected at 1, 4, 7, and 10 days post-treatment to produce soft cheese and ricotta. CLS concentrations in plasma, milk, cheese, whey, and ricotta were determined by HPLC. The concentrations of CLS measured in plasma were higher than those measured in milk at all sampling times. However, the calculated withdrawal time for CLS in milk was between 39 and 43 days postadministration to dairy goats. CLS residual concentrations in cheese (between 0.93 and 1.8 µg/g) were higher than those measured in the milk used for its production. CLS concentrations in ricotta were sixfold higher than those in the milk and 20-fold higher than those in the whey used for its production. The persistent and high residual concentrations of CLS in the milk and in the cheese and ricotta should be seriously considered before issuing any recommendation on the extralabel use of CLS in dairy goat farms.

Resonance Rayleigh scattering, frequency doubling scattering and absorption spectrum of the interaction for mebendazole with 12-tungstophosphoric acid and its analytical applications.

The interaction of mebendazole (MBZ) with 12-tungstophosphoric acid (TP) has been investigated by using resonance Rayleigh scattering (RRS) and frequency doubling scattering (FDS) combining with absorption spectrum. In pH 1.0 HCl medium, MBZ reacted with TP to form 3:1 ion-association complex. As a result, not only the spectrum of absorption was changed, but also the intensities of RRS and FDS were enhanced greatly. The maximum RRS, FDS and absorption wavelengths are located at 372, 392 and 260 nm, respectively. The increments of scattering intensity (ΔI) and absorption (ΔA) are directly proportional to the concentrations of MBZ in certain ranges. The detection limits (3σ) of RRS, FDS and absorption are 0.56, 0.86 and 130.16 ng/mL, respectively. The sensitivity of RRS method is higher than FDS and absorption methods. The optimum conditions of RRS method and the influence factors were discussed in the paper, in addition, the structure of ion-association complex and the reaction mechanism were investigated. Based on the ion-association reaction and its spectral response, the rapid, simple and sensitive RRS method for the determination of MBZ has been developed.

Investigation of the persistence of rafoxanide residues in bovine milk and fate during processing.

Rafoxanide is an effective treatment for the control of fluke infections in animals, but it is currently not permitted for treating animals whose milk is intended for human consumption. In this study, the persistence of rafoxanide residues in milk, and their migration to dairy products, was investigated following the treatment of six lactating dairy cows with Curafluke 10% oral drench. The highest concentration of rafoxanide residues detected in the individual cows milk ranged from 249 to 627 µg kg(-1) and occurred at 2-3 days post-treatment. At 2 and 23 days post-treatment (representing high and low residue concentrations) the milk was pooled into two independent aliquots, each containing the full day's milk produced by three cows. Milk products were made from pasteurised and unpasteurised milk. Pasteurisation appeared to have little impact on the stability of the residues. Rafoxanide concentrated sixfold in the cheese (week 0) compared to the starting milk (2070 vs. 349 µg kg(-1)) but was four times lower in whey (75 µg kg(-1)). Rafoxanide residues were up to 14 times higher in butter (week 0) than in the starting milk (5468 vs. 376 µg kg(-1)). Residues were found to further concentrate in butter and cheese at longer storage and ripening times, respectively. Skim-milk powder was manufactured from skim milk, and residues were 10-fold higher than in the starting skim milk (5468 vs. 376 µg kg(-1)) despite the 185°C temperature required for the process. Rafoxanide residues were stable in this skim-milk powder when stored at ambient temperature for at least 1 year. Results showed that detectable rafoxanide residues were excreted in milk for 47 days, and concentrated in the fat-based products. The analytical ranges of the UHPLC-MS/MS method used were 1.0-200 µg kg(-1) (milk and whey) and 10-2000 µg kg(-1) (other dairy products).

[Biologically active substances of preparation avercom].

The preparation avercom created on the basis of ethanol extracts from the biomass of Streptomyces avermitilis UCM Ac-2179, contains an antiparasitic antibiotic avermectin, as well as a complex of biologically active substances: amino acids, lipids, including nonsaturated fatty acids, and phytohormones, particularly: auxins, cytokinins, hybberellins. The above mentioned complex is characterized by nematocidical, phytostimulating and elicitor effect upon plants which has been confirmed with the results of production experiments on the cucumber variety Angelina.