Larval Development Assay Research Articles

Use of Viscous medium to study anthelmintic drug action in Caenorhabditis elegans

Caenorhabditis elegans is an appealing tool for experimental evolution and for working with antiparasitic drugs, from understanding the molecular mechanisms of drug action and resistance to uncover new drug targets. We present a new methodology for studying the impact of antiparasitic drugs in C. elegans. Viscous medium was initially designed for C. elegans maintenance during long-term evolution experiments. Viscous medium provides a less structured environment than the standard nematode growth media agar, yet the bacteria food source remains suspended. Further, the Viscous medium offers the worm population enough support to move freely, mate, and reproduce at a rate comparable to standard agar cultures. Here, the Viscous medium was adapted for use in antiparasitic research. We observed a similar sensitivity of C. elegans to anthelmintic drugs as in standard liquid media and statistical difference to the standard agar media through a larval development assay. Using Viscous medium in C. elegans studies will considerably improve antiparasitic resistance research, and this medium could be used in studies aimed at understanding long-term multigenerational drug activity.

Soil legacies in maize-edible legume intercropping alter maize growth and reduce Spodoptera frugiperda larval feeding

Plant-soil feedback can influence aboveground interactions between plants and herbivores by affecting plant chemistry. Such interactions can be utilized in pest management. However, cropping systems such as maize-legume intercropping (MLI) can influence these interactions which is not well understood. In this study, we explored effects of MLI systems on soil physico-chemical properties, maize growth, larval feeding and development of fall armyworm (Spodoptera frugiperda). We used sterile soil and soil conditioned by different MLI and maize-monoculture cropping systems to explore these interactions. Soil samples that included soil conditioned by different MLI and maize-monoculture cropping systems were collected from smallholder farmer fields in eastern Kenya, where different MLI and maize-monoculture cropping systems were being practiced. These soil samples were compared with sterile soils for physico-chemical properties using black oxidation and Walkley methods. Three-weeks-old maize plants grown in the different soil treatments in the greenhouse were used for larval feeding and development assays. Neonate S. frugiperda larvae were allowed to feed on maize leaf discs for 24 hours and another set of plants were inoculated with the neonates for 15 days and the larval survival and development monitored. Soil obtained from different maize-edible legume intercropping systems had a higher pH, electrical conductivity, nitrogen, organic carbon, potassium, phosphorus, calcium, magnesium, exchangeable acidity, copper, clay and silt compared to maize-monoculture and sterile soil. Maize plants grown in MLI soil had better growth parameters compared to those in maize-monoculture and sterile soils. A high correlation was found between pH and plant biomass, while no significant correlation with other physico-chemical properties was noted. There were significant differences in larval feeding by S. frugiperda neonates when exposed to constitutive and induced maize leaf discs with more leaf tissue fed on maize grown in maize-monoculture and sterile soil. When allowed to feed for 15 days, S. frugiperda larval weight and length were significantly lower on maize plants grown in soils conditioned by MLI than those grown in soil conditioned by maize-monoculture and sterile soils. Findings from this study show how conditioning soil by MLI systems improve soil health, maize growth and reduces S. spodoptera larval feeding and development.

Antioxidant, angiotensin converting enzyme inhibitory and anthelmintic activities of bark essential oil of Cinnamomum bejolghota (Buch.-Ham.) Sweet from North East India

Cinnamomum bejolghota (Buch.-Ham.) Sweetisa traditional anthelmintic and cardiotonic medicinal plant. The essential oil extracted from the bark of C. bejolghota was characterized by gas chromatography-mass spectrometry and 28 constituents were identified. Monoterpenoids were found to be dominating chemical constituents with α-terpineol (18.57 %) as the major component of the essential oil. The antioxidant activity of the essential oil was evaluated by DPPH radical, β-carotene bleaching and reducing power assay methods. The essential oil showed in-vitro dose-response hindering activity against angiotensin-converting enzyme with an IC50 value of 33.43 ± 0.46 µg/mL.The essential oil also exhibited potent anthelmintic property in-vitro against Caenorhabditis elegans with an ED50value of 80.53 ± 3.57 µg/mLin adult mortality assay. The inhibitory activity of essential oil against larval development assay showed an ED50value of 72.40 ± 2.68 µg/mL against C. elegans larva. Further, the egg hatching assay using C. eleganseggs demonstrated an ED50 value of 68.27 ± 3.51 µg/mL with this essential oil. Thus, the ethnomedicinal claim has been validated for the bark essential oil, which provides an excellent scope for the development of the C. bejolghota bark essential oil as novel herbal antihypertensive and anthelmintic agents.

Effect of aqueous extract of Azadirachta indica leaves on gastrointestinal nematodes in ruminants: an in vitro study

High rates of gastrointestinal parasite infestation lead to the frequent use of anthelmintics for their control, resulting in resistance in the main species of gastrointestinal nematodes. Natural plant extracts, particularly those from Azadirachta indica A. Juss leaves, have been described as having antiparasitic, antimicrobial, insecticidal, antioxidant, and anticorrosive activities, which are related to the diversity of their chemical composition. This research aimed to evaluate the effect of the aqueous extract of A. indica leaves on the control of gastrointestinal nematodes in ruminants. To achieve this, an in vitro test was performed using the larval development assay, with fecal samples obtained from Pelibuey sheep, Criolla goats in the development category, and Criolla calf cattle for the culture of gastrointestinal nematode larvae. Three concentrations (minimum, medium, and maximum) were evaluated for each animal species against Haemonchus placei and Cooperia sp larvae for cattle and Haemonchus contortus, Trichostrongylus sp, and Trichuris for sheep and goats. The survival percentage indicator was evaluated at 0, 24, 48, and 72 h. The research concluded that the minimum concentration was valid for sheep and goats but not for cattle, and the medium and maximum concentrations were effective against all three animal species, resulting in 100% larval mortality. Keywords: Azadirachta, drug effects, Ruminants, Trichostrongyloidea

Biotic potential and pathogenicity of a Haemonchus contortus susceptible laboratory isolate compared to a resistant field isolate in Brazil

The high prevalence of anthelmintic resistance of Haemonchus contortus is threatening sheep farming. In this context, resistant populations’ replacement using susceptible isolates appears to be a strategy to overcome this situation. The aim of the present study was to evaluate the ability of a susceptible H. contortus laboratory isolate in comparison with a multiresistant field isolate to infect lambs and to study its behaviour in field conditions. Sixteen worm-free lambs were experimentally infected with 4000 infective larvae (L3) of a H. contortus anthelmintic-resistant field isolate (Group R, n = 8) or a laboratory isolate susceptible to anthelmintics (Group S, n = 8). Groups were placed in separate clean pastures, where they were kept for 266 days. Faecal egg counts (FEC) showed maximum values between 21- and 42-days post-infection (PI) and decreased over time, with no statistical differences between groups (P > 0.05). The pathogenicity of the isolates was similar, as lambs from both groups showed a decrease in packed cell volume and total plasma protein values in the beginning of the study, coinciding with the highest FECs. Production of L3 were similar for both groups, when evaluating faecal cultures kept in vitro or L3 recovered from pasture samples and from faecal pats collected from the paddocks, which suggests that both isolates have the same potential of development in vitro and in the environment. The degree of resistance/susceptibility to thiabendazole (TIA), levamisole (LEV), monepantel (MON) and ivermectin (IVE) was evaluated using the Larval Development Assay (RESISTA-Test©) 160 days PI. The resistant isolate showed a high resistance factor (RF) to TIA (141), LEV (822), MON (34) and IVE (196), while the RF of the susceptible isolate remained below three, which indicates high susceptibility to all drugs tested. Our study demonstrates that the susceptible isolate was able to infect lambs and did not show higher pathogenicity nor any differences on its biotic potential when compared to the resistant field isolate. These results indicate that the susceptible isolate has the potential to be used to replace a resistant population of H. contortus, which should be validated in long-term studies under commercial sheep farming conditions.

Broad-Spectrum Inhibitors for Conserved Unique Phosphoethanolamine Methyltransferases in Parasitic Nematodes Possess Anthelmintic Efficacy.

In humans, nematode infections are prevalent in developing countries, causing long-term ill health, particularly in children. Worldwide, nematode infections are prevalent in livestock and pets, affecting productivity and health. Anthelmintic drugs are the primary means of controlling nematodes, but there is now high prevalence of anthelmintic resistance, requiring urgent identification of new molecular targets for anthelmintics with novel mechanisms of action. Here, we identified orthologous genes for phosphoethanolamine methyltransferases (PMTs) in nematodes within the families Trichostrongylidae, Dictyocaulidae, Chabertiidae, Ancylostomatoidea, and Ascarididae. We characterized these putative PMTs and found that they possess bona fide PMT catalytic activities. By complementing a mutant yeast strain lacking the ability to synthesize phosphatidylcholine, the PMTs were validated to catalyze the biosynthesis of phosphatidylcholine. Using an in vitro phosphoethanolamine methyltransferase assay with PMTs as enzymes, we identified compounds with cross-inhibitory effects against the PMTs. Corroboratively, treatment of PMT-complemented yeast with the PMT inhibitors blocked growth of the yeast, underscoring the essential role of the PMTs in phosphatidylcholine synthesis. Fifteen of the inhibitors with the highest activity against complemented yeast were tested against Haemonchus contortus using larval development and motility assays. Among them, four were found to possess potent anthelmintic activity against both multiple drug-resistant and susceptible isolates of H. contortus, with IC50 values (95% confidence interval) of 4.30 μM (2.15-8.28), 4.46 μM (3.22-6.16), 28.7 μM (17.3-49.5), and 0.65 μM (0.21-1.88). Taken together, we have validated a molecular target conserved in a broad range of nematodes and identified its inhibitors that possess potent in vitro anthelmintic activity.

The Brown Alga Bifurcaria bifurcata Presents an Anthelmintic Activity on All Developmental Stages of the Parasitic Nematode Heligmosomoides polygyrus bakeri.

The current control of gastrointestinal (GI) parasitic nematodes mainly relies on the widespread use of anthelmintics, which has inevitably led to resistance. Therefore, there is an urgent need to find new sources of antiparasitic compounds. Macroalgae represent a rich source of active molecules and are widely described as having medicinal properties. In the present study, we investigated the potential anthelmintic activity of aqueous extracts from three species of algae (Bifurcaria bifurcata, Grateloupia turuturu and Osmundea pinnatifida) on the murine parasite Heligmosomoides polygyrus bakeri. Using a set of complementary in vitro tests, including larval development assays, egg hatching tests and nematicidal activity assays on larvae and adults, we report the nematicidal activity of aqueous extracts of B. bifurcata. In addition, aqueous extract fractionation using liquid/liquid partitioning with a solvent of increasing polarity was performed in order to identify the groups of active molecules underlying the anthelmintic activity. Non-polar extracts (heptane, ethyl acetate) demonstrated high anthelmintic potential, highlighting the role of non-polar metabolites such as terpenes. Here, we highlight the strong anthelmintic potential of the brown alga B. bifurcata on a mouse model of GI parasites, thus confirming the strong interest in algae as natural alternatives for the control of parasitic nematodes.

MiR-34-5p, encoded by Spodoptera frugiperda, participates in anti-baculovirus by regulating innate immunity in the insect host

Spodoptera frugiperda is one of the significant migratory pests. Baculovirus, an insect-specific microbial insecticide, has been used to control pests. microRNA-34 (miR-34) regulates insect development and innate immunity to pathogens. This study explored the critical functions of miR-34-5p, encoded by S. frugiperda, in the anti-AcMNPV by targeting the JAK/STAT immune pathway. Integrating immune signal pathways analysis, developmental expression patterns, larval development, and genomic replication assay, we focused on the stat, a miR-34-5p target gene, and comprehensively revealed a novel mechanism of physiological and anti-virus function in S. frugiperda. miR-34-5p inhibited AcMNPV proliferation by targeting stat and provided an unfavorable environment for the virus by affecting the host genome replication and promoting the cell to undergo apoptosis. Moreover, stat silence suppressed viral and host genome replication, impaired viral proliferation by suppressing the transcript level of viral early gene ie1, ie2, and promoted apoptosis by affecting p35 expression. This study highlighted that the miR-34-5p target gene stat played physiological functions in the insect antiviral immune and development, which provided a basis for creating target nucleic acid pesticides against S. frugiperda and prepared AcMNPV virus strains inactivated by miR-34-5p to escape host immunity.

Anthelmintic activity of Moringa oleifera and Azadirachta indica against gastrointestinal nematodes of wild sheep

Gastrointestinal nematodes (GINs) are serious issue for health of wild sheep kept in captivity. Chemically synthesized anthelmintics are regularly used to control these parasites. In recent years anthelmintic resistance and remnant of drugs in animal products leads to use of medicinal plants as alternative to anthelmintics. In current study, the efficacy of aqueous, methanolic and ethanolic dried leaf extracts of medicinal plants Moringa oleifera and Azadirachta indica were tested for in vitro ovicidal and larvicidal activities against Haemonchus, Trichuris, Coccidia and Trichostrongylus; naturally acquired nematodes isolated from wild sheep (Ovis orientalis orientalis). Six concentrations of these plants extract (1.56, 3.13, 6.25, 12.5, 25 and 50 mg/ml) were evaluated using egg hatching assay (EHA) and larval development assay (LDA) in three replicates. To compare treatment effects, untreated and treated (0.1% ivermectin) controls were used. The aqueous, methanolic and ethanolic leaf extracts showed anthelmintic activities against isolated genera of nematodes but the inhibition was maximum (99%) in ethanol extract of M. oleifera followed by methanol extract (97%) at maximum concentration tested at (50mg/ml). The overall findings of this study shows that Moringa oleifera and Azadirachta indica leaf extracts possess significant anthelmintic efficacy against GINs of sheep and these could be a natural alternative to synthetic anthelmintics to treat the worm infections in animals.

Comparative analysis on transcriptomics of ivermectin resistant and susceptible strains of Haemonchus contortus.

BackgroundIvermectin (IVM) is one of the most important and widely used anthelmintics in veterinary medicine. However, its efficacy is increasingly compromised by widespread resistance, and the exact mechanism of IVM resistance remains unclear for most parasitic nematodes, including Haemonchus contortus, a blood-sucking parasitic nematode of small ruminants.MethodsIn this study, an H. contortus IVM-resistant strain from Zhaosu, Xinjiang, China, was isolated and assessed by the control test, faecal egg count reduction test (FECRT) and the larval development assay (LDA). Subsequently, comparative analyses on the transcriptomics of IVM-susceptible and IVM-resistant adult worms of this parasite were carried out using RNA sequencing (RNA-seq) and bioinformatics.ResultsIn total, 543 (416 known, 127 novel) and 359 (309 known, 50 novel) differentially expressed genes (DEGs) were identified in male and female adult worms of the resistant strain compared with those of the susceptible strain, respectively. In addition to several previously known candidate genes which were supposed to be associated with IVM resistance and whose functions were involved in receptor activity, transport, and detoxification, we found some new potential target genes, including those related to lipid metabolism, structural constituent of cuticle, and important pathways such as antigen processing and presentation, lysosome, autophagy, apoptosis, and NOD1-like receptor signalling pathways. Finally, the results of quantitative real-time polymerase chain reaction confirmed that the transcriptional profiles of selected DEGs (male: 8 genes, female: 10 genes) were consistent with those obtained by the RNA-seq.ConclusionsOur results indicate that IVM has multiple effects, including both neuromuscular and non-neuromuscular targets, and provide valuable information for further studies on the IVM resistance mechanism in H. contortus.Graphical Supplementary InformationThe online version contains supplementary material available at 10.1186/s13071-022-05274-y.

Effects of sodium perchlorate and 6-propylthiouracil on metamorphosis and thyroid gland histopathology in the European common frog (Rana temporaria).

Several chemicals have been identified as thyroid hormone axis disrupting chemicals (THADCs) able to interfere with the thyroid hormone system during fetal life and early life stages, thereby impairing neurodevelopment in mammals and inducing development and growth disorders in fish and amphibians. However, identification of THADCs is particularly challenging, and thyroid modalities are currently only assessed in vivo by mammalian and amphibian tests. The aquatic African clawed frog (Xenopus laevis/tropicalis) is the model species of the amphibian test guidelines developed by the OECD and the United States Environmental Protection Agency, but as most European amphibians are semi-aquatic, concern has been raised whether the sensitivity of native European species is comparable to Xenopus. A shortened version of the OEDC test guideline 241 (Larval Amphibian Growth and Development Assay, LAGDA) was used to investigate the effects of two model THADCs on the metamorphosis and thyroid histopathology in the European common frog (Rana temporaria). R. temporaria eggs were collected on the field and exposed till metamorphic climax to sodium perchlorate (11.9-426.5 μg/L perchlorate concentrations) and 6-propylthiouracil (PTU: 1.23-47.7mg/L). PTU severely delayed metamorphosis and affected several thyroid gland histopathological endpoints at slightly lower concentrations compared to Xenopus. As opposed to what was described in similar Xenopus studies, we observed no effect of perchlorate on the investigated endpoints. Interspecies differences may be linked to mechanisms of action.

Mutualism between Gut-Borne Yeasts and Their Host, Thaumatotibia leucotreta, and Potential Usefulness in Pest Management

Simple SummaryThe false codling moth is an indigenous pest of the citrus industry in southern Africa. It is a major threat as it can cause substantial fruit damage through larval feeding. The microorganisms associated with insects are often studied for their potential to kill their host instead of how they could aid them. However, in recent years, this aspect of their interaction has received much attention. The codling moth has been shown to have a close relationship with the naturally occurring yeast found within their digestive tract. These beneficial yeasts help the adult female moths locate a suitable host for egg laying. During their larval phase, these yeasts help them develop faster and increase their chance of survival. These interactions could be manipulated for the purposes of biological control, as one might be able to combine these yeasts with existing biological control agents to improve their effectiveness. These yeasts may also provide additional methods for monitoring field populations of insect pests. In this study, we identified yeasts that occur naturally in the guts of false codling moth larvae and investigated whether any of them are beneficial to their host.Thaumatotibia leucotreta is endemic to southern Africa and is highly significant for various fruit industries, including the South African citrus industry, due to its classification as a phytosanitary pest. Mutualistic associations between C. pomonella, closely related to T. leucotreta, and yeasts have previously been described and reported to reduce larval mortality and enhance larval development. Here, we determined which yeast species occur naturally in the gut of T. leucotreta larvae and investigated whether any of the isolated yeast species affect their behaviour and development. Navel oranges infested with T. leucotreta larvae were collected from geographically distinct provinces in South Africa, and the larvae were processed for analysis of naturally occurring associated yeasts. Six yeast species were isolated and identified from the guts of these T. leucotreta larvae via PCR amplification and sequencing of the ITS region of rDNA and D1/D2 domain of large ribosomal subunit. Larval development and attraction assays were conducted, and T. leucotreta larvae that fed on Navel oranges inoculated with yeast had accelerated developmental periods and reduced mortality rates. Neonate T. leucotreta were also attracted to YPD broth cultures inoculated with yeast for feeding. Oviposition preference assays were conducted with adult T. leucotreta females. Navel oranges inoculated with yeast were shown to influence the oviposition preference of adult females. Yeasts harbour the potential for use in biocontrol, especially when combined with other well-established control methods. This study provides a platform for future research into incorporating yeast with current biological control agents as a novel option for controlling T. leucotreta in the field.

Evaluation of in vitro methods of anthelmintic efficacy testing against Ascaridia galli.

To investigate methods for in vitro assessment of anthelmintic efficacy against the chicken nematode Ascaridia galli this study firstly evaluated sample preparation methods including recovery of eggs from excreta using different flotation fluids and induced larval hatching by the deshelling-centrifugation method and the glass-bead method with or without bile. It then evaluated two in vitro assays, the in-ovo larval development assay (LDA) and larval migration inhibition assay (LMIA), for anthelmintic efficacy testing against A. galli using fresh eggs and artificially hatched larvae, respectively. Four anthelmintics, thiabendazole (TBZ), fenbendazole (FBZ), levamisole (LEV) and piperazine (PIP) were employed using an A. galli isolate of known susceptibility. The results suggested that the LDA and LMIA could successfully be used to generate concentration response curves for the tested drugs. The LDA provided EC50 values for inhibition of egg embryonation of 0.084 and 0.071 μg/ml for TBZ and FBZ, respectively. In the LMIA, the values of effective concentration (EC50) of TBZ, FBZ, LEV and PIP were 105.9, 6.32, 349.9 and 6.78 × 107 nM, respectively. For such in vitro studies, a saturated sugar solution showed high egg recovery efficiency (67.8%) and yielded eggs of the highest morphological quality (98.1%) and subsequent developmental ability (93.3%). The larval hatching assays evaluated did not differ in hatching efficiency but the deshelling-centrifugation method yielded larvae that had slightly better survival rates. For final standardization of these tests and establishment of EC50 reference values, tests using isolates of A. galli of defined resistance status need to be performed.

Multiple drug resistance in hookworms infecting greyhound dogs in the USA

Ancylostoma caninum is the most prevalent nematode parasite of dogs. We confirmed multiple-drug resistance (MDR) in several A. caninum isolates to all anthelmintic drug classes approved for the treatment of hookworms in dogs in the USA. Cases of MDR hookworms appear to be highly overrepresented in greyhounds. The aims of this study were to evaluate the drug-resistant phenotypes and genotypes of the A. caninum infecting greyhounds. Fecal samples from greyhounds of the USA were acquired from two greyhound adoption kennels, one active greyhound racing kennel, and three veterinary practices. Fecal egg counts (FECs) were performed on fecal samples from 219 greyhounds, and despite treatment with anthelmintics, the mean FEC was 822.4 eggs per gram (EPG). Resistance to benzimidazoles and macrocyclic lactones were measured using the egg hatch assay (EHA) and the larval development assay (LDA), respectively. We performed 23 EHA and 22 LDA on either individual or pooled feces, representing 54 animals. Mean and median IC50 and IC95 values for the EHA were 5.3 μM, 3.6 μM, and 24.5 μM, 23.4 μM, respectively. For the LDA, the median IC50 value was >1000 nM. These values ranged 62–81 times higher than our susceptible laboratory isolate. Only post-treatment samples were available. For samples collected <10 days post-treatment with albendazole, moxidectin, or a combination of febantel-pyrantel-moxidectin, the mean FEC were 349, 333, and 835 EPG, respectively. We obtained DNA from hookworm eggs isolated from 70 fecal samples, comprised of 60 individual dogs and 10 pools. Deep sequencing of the isotype 1 β-tubulin gene only revealed the presence of the F167Y (TTC>TAC) resistance polymorphism in 99% of these samples. These clinical, in vitro, and genetic data provide strong evidence that greyhound dogs in the USA are infected with MDR A. caninum at very high levels in prevalence and infection intensity.

Ecotoxicological screening of UV-filters using a battery of marine bioassays

The present study aimed to assess the toxicity of seven UV-filters: zinc oxide nanoparticles (nZnO, particle size <100 nm), titanium dioxide nanoparticles (nTiO2, primary particle size 21 nm), 2-ethylhexyl-4-methoxycinnamate (EHMC), 4-methylbenzylidene camphor (4-MBC), avobenzone (AVO), octocrylene (OCTO) and benzophenone-3 (BP-3) on three species: Aliivibrio fischeri (inhibition of bioluminescence), Phaeodactylum tricornutum (growth inhibition) and Ficopomatus enigmaticus (larval development success). Results showed nTiO2 to be the most toxic for P. tricornutum (EC50 0.043 mg L−1), while no effect was observed in A. fischeri and F. enigmaticus. EHMC was the most toxic to A. fischeri (EC50 0.868 mg L−1 (15 min) and 1.06 mg L−1 (30 min)) and the second most toxic to P. tricornutum. For F. enigmaticus, the lowest percentages of correct development resulted from 4-MBC exposure, with EC50 of 0.836 mg L−1. Overall, AVO induced low toxicity to every assessed species and OCTO was the least toxic for F. enigmaticus larvae. Considering the results obtained for F. enigmaticus, further larval development assays were performed with nZnO and EHMC under different light (light vs darkness) and temperature (20 and 25 °C) conditions, showing higher percentages of correct development at 25 °C, independently on light/darkness conditions. Under different temperature and photoperiod conditions, nZnO was more toxic than EHMC. Overall, nZnO and EHMC were among the most toxic UV filters tested and, when testing the effects of these UV-filters with temperature the results highlight that the impacts are liable to be lessened at higher temperatures (25 °C compared with 20 °C), in the case of this estuarine polychaete species. Nevertheless, further experiments are necessary to describe the effects of these two UV-filters at different organization levels, to study the toxicity of eventual degradation by-products and to provide more information on the combination of different stressors.

First Report of Multiple Drug-resistant Gastrointestinal Nematodes of Sheep in Arbat District, Sulaymaniyah, Iraq Detected By in Vivo and in Vitro Methods.

IntroductionThe control of gastrointestinal nematodes (GINs) in small ruminants is principally dependent on anthelmintic therapy, which encounters the rising problem of anthelmintic resistance (AR) development. Veterinarians reported anthelmintic failure in several sheep farms in Arbat District, Sulaymaniyah, northern Iraq, which called for a systematic study about the efficacy of three commonly used drugs: albendazole, ivermectin, and levamisole.Material and MethodsA faecal egg count reduction test (FECRT) was conducted to elucidate the anthelmintics’ efficacies, with coprocultures to determine the GINs parasitising sheep in the study area. Larval development assays (LDAs) were conducted to determine the drugs’ median inhibitory concentrations (IC50).ResultsThe FECRT revealed that AR was widespread to all three drugs, and ivermectin was the least effective, reducing the faecal egg counts (FECs) by 50.5% to 57.1%. The coprocultures revealed that the GIN genera of sheep in Arbat District were Trichostrongylus, Nematodirus, and Trichuris, and the resistance was mainly due to Trichostrongylus species. The mean IC50 of albendazole, ivermectin, and levamisole were 0.073 ng/mL, 7.97 ng/mL, and 1.43 ng/mL, respectively.ConclusionThis study is the first Iraqi report of AR confirmed by both in vivo FECRT and in vitro LDA methods.

Anthelmintic, antimycobacterial, antifungal, larvicidal and cytotoxic activities of acetone leaf extracts, fractions and isolated compounds from Ptaeroxylon obliquum (Rutaceae)

Ethnopharmacological relevancePtaeroxylon obliquum (Thunb.) Radlk, sneezewood, is a commonly used medicinal plant in South Africa for the treatment of parasitic infections in animals, tuberculosis (TB) and related symptoms, and other microbial infections. Aim of the studyIn this study anthelmintic, antifungal, antimycobacterial, larvicidal and cytotoxic activities of the acetone leaf extract of P. obliquum, solvent-solvent derived fractions of the extract and isolated compounds (obliquumol and a mixture of lupeol and β-amyrin) were determined. Materials and methodsThe in vitro anthelmintic activity was tested against Haemonchus contortus ova and larvae using the egg hatch inhibition and larval development assays. The antifungal activity was investigated using a serial microplate dilution method against Aspergillus fumigatus, Cryptococcus neoformans and Candida albicans. Antimycobacterial activity was evaluated by a tetrazolium violet-based broth microdilution method against pathogenic Mycobacterium tuberculosis and Mycobacterium bovis. The larvicidal activity was evaluated against Aedes aegypti. Cytotoxicity was determined using human liver (C3A) and Vero African green monkey kidney cell lines. ResultsObliquumol (ptaeroxylinol acetate) was more effective than the crude acetone extract against H. contortus ova and larvae with an LC50 of 95 μg/mL against the larvae. Obliquumol had very good antifungal activity with minimal inhibitory concentrations (MIC) values from 2 to 16 μg/mL. Lupeol and β-amyrin mixture also had good activity against C. neoformans and C. albicans with an MIC of 16 μg/mL against both fungi. The crude extract and fractions had average to weak antimycobacterial activity against the two pathogenic mycobacteria with MICs of 313–625 μg/mL for the extract and fractions tested. Obliquumol had good activity with an MIC of 63 μg/mL against pathogenic M. tuberculosis ATCC 27115. The acetone crude extract was toxic against Vero cells with an CC50 of 14.2 μg/mL. Obliquumol and the mixture of lupeol and β-amyrin were not toxic against both Vero and C3A cells at the highest concentration tested of 200 μg/mL and had good selectivity index values against the opportunistic fungal pathogens. No tested samples had any larvicidal activity against A. aegypti at the concentrations tested. ConclusionThe activity noted against H. contortus may support the ethnoveterinary use of this plant against animal parasites in South Africa although aqueous extracts were not active. The activity of obliquumol against opportunistic fungal pathogens was also promising. The cytotoxicity of the acetone leaf extract, however, raises concerns about the traditional use of P. obliquum to treat a wide range of diseases although if an aqueous extract is used the toxicity concern may not be relevant.

The Extended Amphibian Metamorphosis Assay: A Thyroid-Specific and Less Animal-Intensive Alternative to the Larval Amphibian Growth and Development Assay.

The amphibian metamorphosis assay (AMA; US Environmental Protection Agency [USEPA] test guideline 890.1100 and Organisation for Economic Co‐Operation and Development test guideline 231) has been used for more than a decade to assess the potential thyroid‐mediated endocrine activity of chemicals. In 2013, in the context of the Endocrine Disruptor Screening Program of the USEPA, a Scientific Advisory Panel reviewed the results from 18 studies and recommended changes to the AMA test guideline, including a modification to a fixed‐stage design rather than a fixed‐time (i.e., 21‐d) design. We describe an extended test design for the AMA (or EAMA) that includes thyroid histopathology and time to metamorphosis (Nieuwkoop–Faber [NF] stage 62), to address both the issues with the fixed‐time design and the specific question of thyroid‐mediated adversity in a shorter assay than the larval amphibian growth and development assay (LAGDA; Organisation for Economic Co‐Operation and Development test guideline 241), using fewer animals and resources. A demonstration study was conducted with the EAMA (up to NF stage 58) using sodium perchlorate. Data analyses and interpretation of the fixed‐stage design of the EAMA are more straightforward than the fixed‐time design because the fixed‐stage design avoids confounded morphometric measurements and thyroid histopathology caused by varying developmental stages at test termination. It also results in greater statistical power to detect metamorphic delays than the fixed‐time design. By preferentially extending the AMA to NF stage 62, suitable data can be produced to evaluate thyroid‐mediated adversity and preclude the need to perform a LAGDA for thyroid mode of action analysis. The LAGDA remains of further interest should investigations of longer term effects related to sexual development modulated though the hypothalamus–pituitary–gonadal axis be necessary. However, reproduction assessment or life cycle testing is currently not addressed in the LAGDA study design. This is better addressed by higher tier studies in fish, which should then include specific thyroid‐related endpoints. Environ Toxicol Chem 2021;40:2135–2144. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

A deep-sea bacterium related to coastal marine pathogens.

SummaryEvolution of virulence traits from adaptation to environmental niches other than the host is probably a common feature of marine microbial pathogens, whose knowledge might be crucial to understand their emergence and pathogenetic potential. Here, we report genome sequence analysis of a novel marine bacterial species, Vibrio bathopelagicus sp. nov., isolated from warm bathypelagic waters (3309 m depth) of the Mediterranean Sea. Interestingly, V. bathopelagicus sp. nov. is closely related to coastal Vibrio strains pathogenic to marine bivalves. V. bathopelagicus sp. nov. genome encodes genes involved in environmental adaptation to the deep‐sea but also in virulence, such as the R5.7 element, MARTX toxin cluster, Type VI secretion system and zinc‐metalloprotease, previously associated with Vibrio infections in farmed oysters. The results of functional in vitro assays on immunocytes (haemocytes) of the Mediterranean mussel Mytilus galloprovincialis and the Pacific oyster Crassostrea gigas, and of the early larval development assay in Mytilus support strong toxicity of V. bathopelagicus sp. nov. towards bivalves. V. bathopelagicus sp. nov., isolated from a remote Mediterranean bathypelagic site, is an example of a planktonic marine bacterium with genotypic and phenotypic traits associated with animal pathogenicity, which might have played an evolutionary role in the origin of coastal marine pathogens.

Comparative Evaluation of Two In Vitro Tests for Detection of Ivermectin Resistance in Haemonchus contortus of Small Ruminants in Uttar Pradesh, India.

Resistance against ivermectin in India has been detected solely implementing in vivo tests and there is no published report of resistance employing in vitro methods. The present study was carried out to study ivermectin resistance in H. contortus of small ruminants by two in vitro tests and to determine their efficacy in effectively detecting ivermectin resistance in the field isolates. The present study was carried out in the small ruminant farms of the western region of Uttar Pradesh, India. Faecal samples with high egg per gram of faeces for H. contortus were selected. Two in vitro tests namely Micro agar larval development test and larval development assay were performed for assessing ivermectin resistance in field isolates of H. contortus by subjecting the eggs to different concentrations of ivermectin aglycone. The results of the two in vitro tests implicated towards the susceptibility of H. contortus isolates in the area under study. The standard deviations of the two tests was compared and was found to be statistically non-significant. It can be said that both in vitro tests are reliable for detecting resistance against different anthelmintics including ivermectin.