Evaluating anthelmintic activity through Caenorhabditis elegans egg hatching assay.

The Caenorhabditis elegans egg hatching methodology is a valuable tool for assessing the anthelmintic activity of drugs and compounds and evaluating anthelmintic drug efficacy. Isolated eggs from gravid adults are exposed to different concentrations of selected drugs and the percentage of egg hatching is determined with respect to the control condition. The assay allows the construction of concentration-response curves and determination of EC50 or EC90 values for egg hatching inhibition. Also, it allows measurements of inhibition as a function of time of exposure. This approach addresses the urgent need for new anthelmintics, as resistance to current treatments poses a significant challenge in parasitic nematode infection. This resistance not only affects humans but also animals and plants, causing significant economic losses in livestock farming and agriculture. By using the free-living nematode C. elegans as a parasitic model organism, researchers can efficiently screen for potential treatments and assess drug combinations for synergistic effects. Importantly, this assay offers a cost-effective and accessible alternative to traditional methods, eliminating the need for specialized infrastructure, hosts, and trained animal maintenance personnel. Additionally, the methodology closely mimics natural conditions, providing insights into egg development and potential therapeutic targets. This method allows for evaluating the direct negative impact of drugs on egg hatching, which correlates with long-term anthelmintic effects, offering advantages in preventing or reducing the transmission and spread of worm infections by eggs. Overall, this approach represents a significant advancement for anthelmintic discovery, offering both practical applications and avenues for further scientific research. •The C. elegans egg hatching assay is a robust and effective method for assessing the anthelmintic potential of various drugs and compounds, allowing the generation of concentration-response curves.•By leveraging the free-living nematode C. elegans as a parasitic model organism, this method facilitates efficient screening of potential treatments and evaluation of drug combinations.•The method addresses the urgent need for new anthelmintics, offering a cost-effective and accessible alternative to traditional approaches.

METARHIZIUM ANISOPLIAE SENSU LATO NATIVE TO LIVESTOCK SOILS CAUSES HIGH MORTALITY ON RHIPICEPHALUS MICROPLUS LARVAE, ADULTS AND AFFECTS THEIR REPRODUCTION.

The acaricidal effect of 14 strains of Metarhizium anisopliae sensu lato isolated from soil of livestock farms in the Mexican tropics was evaluated against larvae and engorged females, and during the laying and hatching of eggs of Rhipicephalus microplus (Ixodida: Ixodidae). For each fungal strain, the larvae mortality percentage was evaluated through a larval immersion test, while the reproductive efficiency indices in engorged females were measured using adult immersion tests at a dose of 1 × 108 conidia/ml. All strains of M. anisopliae (s.l.) proved to be highly effective against R. microplus larvae (66-100%) and engorged females (100%). The strains also showed a good effect in inhibiting egg laying (16.45-56.38%) and a moderate effect in decreasing egg hatching (5.24-32.68%). Two strains demonstrated to be effective against all development phases of R. microplus in an integrated manner.

eIF3 subunit M regulates blood meal digestion in Rhodnius prolixus affecting ecdysis, reproduction, and survival.

In triatomines, blood-feeding triggers many physiological processes including post embryonic development and reproduction. Different feeding habits, such as hematophagy, can shape gene functions to meet the challenges of each type of diet. The gut of blood-sucking insects faces particular challenges after feeding due to the quantity and the quality of the food ingested. A comparison of transcriptomic and proteomic data indicates that post transcriptional regulation of gene expression is crucial in the triatomine gut. It was proposed that eukaryotic translation initiation factor 3 subunit m (eIF3m) and eIF3e define 2 different eIF3 complexes with a distinct affinity for the different mRNAs, thus selecting the set of mRNAs to be translated and constituting a post transcriptional mode of regulation of gene expression. Because the eIF3m is mainly expressed in the gut, we evaluated its relevance in Rhodnius prolixus physiology through RNA interference-mediated gene silencing. The knockdown of eIF3m reduced the digestion rate, affecting the processes triggered by a blood meal. Its silencing inhibited molting and caused premature death in nymphs while impaired ovary development, oviposition and increased resistance to starvation in adult females. The survival of males after feeding (resistance to starvation) was not affected by eIF3m knockdown. The information regarding the eIF3m function in insects is scarce and the phenotypes observed in R. prolixus upon eIF3m silencing are different and more severe than those previously described in Drosophila melanogaster, indicating a pleiotropic role of this gene in triatomines.

Fasciola hepatica and rumen flukes - in vitro evaluation of main commercial anthelmintics

Background: Trematode infections are of great importance as they affect the health of many species of mammals as cattle, sheep and goat. Fasciola hepatica represents the main trematode zoonosis and risks to human and cattle and paramphistomosis is one emerging parasitic diseases of ruminants widely distributed in the world. The economic expenses are incurred by the use of ineffective anthelmintics for trematode control. Besides to faecal egg count reduction test (FECRT) to determine the anthelmintic efficacy, can be used in vitro assays, by this the aim of the study was to determine the lethal doses (LD) with hatching egg test (EHT) of the main commercial anthelmintics used for the control of trematodes in cattle. Materials, Methods & Results: Liver and rumen were examined from cattle slaughtered in Tabasco, Chiapas and Campeche states from Mexico. F. hepatica eggs were recovered from gallbladder and rumen fluke eggs collected from adult parasites in saline solution. Subsequently, the hatching egg assays were performed placing 100 trematode eggs in distilled water in each one of 96 wells of polystyrene plates. After making the appropriate dilutions, several concentrations of commercial anthelmintics were evaluated, ranging from 0.04 to 80.63 mM for triclabendazole + 0.046 to 96.87 mM febendazole (TC+FBZ), from 0.04 to 91 mM for rafoxanide (RAFOX), from 0.02 to 43.74 mM for closantel (CLOS), from 0.036 to 76.18 mM for clorsulon + 0.002 to 3.31 mM ivermectin (CLORS+ IVM) and from 0.163 to 334.47 mM for nitroxynil (NITROX). A control group (water) was included in each plate. Lethal doses were obtained using the Probit procedure and analysis of the means with a one-way statistical design. Most drugs used against rumen fluke eggs presented a high LD50 and therefore were ineffective to cause egg mortality, such was the case of RAFOX that presented LD50 from 4,580 to 10,790 µg/mL (7 to 17 mM). CLOS presented the lowest LD50 (80 µg/mL or 0.12 mM) on rumen fluke eggs. TC+FBZ was found to be effective drug against the development of F. hepatica eggs in many samples. In the same way NITROX showed a low LD50 (37 to 63 µg/mL or 0.13 to 0.22 mM), but RAFOX presented a highest LD50 (1,450 µg/mL or 2.32 mM). Discussion: The present study focused on screen the ovicidal activity and determining in vitro lethal doses 50 of main commercial anthelmintics used to control F. hepatica and rumen fluke as rapid tests in a tropical region from Mexico. The FECRT is the main method to detect effectiveness of anthelmintic and other method is the coproantigen reduction test (CRT) by ELISA. Both tests require many infected animals depending the number of treatments and by this the egg hatch assay (EHA) represent a complementary diagnosis of effectiveness of anthelmintic products to compare between regions and even between farms, because few animals are required from the farm to collect trematode eggs, and it is possible to know the effectiveness against various anthelmintics at the same time. Efficacy studies on trematodes using egg hatching tests are scarce, although they have the advantage that they can be applied to both F. hepatica and rumen fluke. TC+FBZ was one of the most effective products in inhibiting the development of F. hepatica eggs. However, RAFOX showed low effectiveness against trematode eggs, with very high lethal doses. These results agree with a study that show low efficacy against the development of Paramphistomum cervi eggs and with the FECRT test reductions of 75% and 80.58% were obtained, in times from 7 to 84 days after treatment with RAFOX. NITROX and CLORS were drugs that had good efficacy on the development of F. hepatica eggs. A differential response between liver and rumen fluke was observed. The anthelmintics used against rumen fluke eggs show low ovicidal activity and in Fasciola hepatica TC+FBZ show the best activity

In Vitro Anthelmintic Activity of a Hydroalcoholic Extract from Guazuma ulmifolia Leaves against Haemonchus contortus.

The purpose of the present study was to assess the ovicidal and larvicidal activity of a hydroalcoholic extract (HAE) and their fractions (aqueous, Aq-F and organic, EtOAc-F) from Guazuma ulmifolia leaves using Haemonchus contortus as a biological model. The egg hatching inhibition (EHI) and larval mortality against infective larvae (L3) tests were used to determine the anthelmintic effect of the treatments. The extract and fractions were tested at different concentrations against eggs and L3. Additionally, distilled water and methanol were used as negative controls and ivermectin as a positive control. The extract and fractions were subjected to HPLC analysis to identify the major compounds. The HAE displayed the highest ovicidal activity (100% EHI at 10 mg/mL). Fractionation of the HA extract allowed increasing the nematicidal effect in the EtOAc-F (100% EHI at 0.62 mg/mL and 85.35% mortality at 25 mg/mL). The phytochemical analysis of the extract and fractions revealed the presence of kaempferol, ethyl ferulate, ethyl coumarate, flavonol, luteolin, ferulic acid, luteolin rhamnoside, apigenin rutinoside, coumaric acid derivative, luteolin glucoside, and quercetin glucoside. These results suggest that G. ulmifolia leaves could be potential candidates for the control of H. contortus or other gastrointestinal parasitic nematodes.

The role of neuropeptides in regulating ecdysis and reproduction in the hemimetabolous insect Rhodnius prolixus.

In ecdysozoan animals, moulting entails the production of a new exoskeleton and shedding of the old one during ecdysis. It is induced by a pulse of ecdysone that regulates the expression of different hormonal receptors and activates a peptide-mediated signalling cascade. In Holometabola, the peptidergic cascade regulating ecdysis has been well described. However, very little functional information regarding the neuroendocrine regulation of ecdysis is available for Hemimetabola, which display an incomplete metamorphosis. We use Rhodnius prolixus as a convenient experimental model to test two hypotheses: (1) the role of neuropeptides that regulate ecdysis in Holometabola is conserved in hemimetabolous insects; and (2) the neuropeptides regulating ecdysis play a role in the regulation of female reproduction during the adult stage. The RNA interference-mediated reduction of ecdysis triggering hormone (ETH) mRNA levels in fourth-instar nymphs resulted in lethality at the expected time of ecdysis. Unlike in holometabolous insects, knockdown of eth and orcokinin isoform A (oka) did not affect oviposition in adult females, pointing to a different endocrine regulation of ovary maturation. However, eth knockdown prevented egg hatching. The blockage of egg hatching appears to be a consequence of embryonic ecdysis failure. Most of the first-instar nymphs hatched from the eggs laid by females injected with dsRNA for eclosion hormone (dsEH), crustacean cardioactive peptide (dsCCAP) and dsOKA died at the expected time of ecdysis, indicating the crucial involvement of these genes in post-embryonic development. No phenotypes were observed upon corazonin (cz) knockdown in nymphs or adult females. The results are relevant for evolutionary entomology and could reveal targets for neuropeptide-based pest control tools.

In vitro assessment of the efficacy of protein exudates from seeds against Haemonchus contortus.

Nematodes develop resistance to the most common commercially available drugs. The aim of this study was to identify and evaluate the action of protein exudates from Mimosa caesalpiniifolia, Leucaena leucocephala, Acacia mangium, and Stylosanthes capitata seeds on the gastrointestinal nematode Haemonchus contortus. The exuded proteins were precipitated, dialyzed, lyophilized, and assessed for their effect on egg hatching and artificial larval exsheathment inhibition. Proteome analysis of the protein extracts was also performed. Although no egg-hatching inhibition was observed, all exudates showed efficacy in inhibiting the larval exsheathment of H. contortus larvae with an EC50 varying from 0.61 to 0.26 mg P mL-1. Proteomic analysis revealed the presence of proteases, protease inhibitors, chitinases, and lectins among other proteins in the exudates. Most of the exuded proteins belong to the oxidative stress/plant defense and energy/carbohydrate metabolism functional clusters. This study concluded that the bioactive proteins from different classes exuded by seeds of M. caesalpiniifolia, L. leucocephala, A. mangium, and S. capitata show stage-specific inhibition against H. contortus.

In vitro ovicidal effect of a Senecio brasiliensis extract and its fractions on Haemonchus contortus.

BACKGROUND: Haemonchosis affects sheep husbandry and its treatment is often compromised due to the development of anthelminthic resistance. Plant-derived bioactive compounds have been studied as alternative to control Haemonchus contortus. The objective of this study was to evaluate the effect of Senecio brasiliensis extracts on H. contortus egg hatching and infective larvae migration. RESULTS: The aqueous extract from dried and fresh plant and alkaloid-enriched fraction of the previously dried leaves of S. brasiliensis inhibited H. contortus egg hatching. The main plant compound in alkaloid fraction was integerrimine, a pyrrolizidine alkaloid (PA). However, the aqueous extract from dried plant displayed higher efficacy when compared to their alkaloid enriched or non-polar fractions, meaning that, although PAs contributed to the ovicidal effect, other compounds in the plant can also contribute to their effect. Furthermore, the aqueous extract from dried plant also had higher efficacy than aqueous extract from fresh plant in larvae migration inhibition. Finally, extract from dried plant presented low in vitro cytotoxic effect. CONCLUSION: Taken together our results suggest a good anthelmintic effect of S. brasiliensis, especially when aqueous extract is prepared from dried plant. Further in vivo studies should be performed focused on forms of administration of this extract in rearing sheep.

Activities and Ergonomics of Workers in Broiler Hatcheries

The objective this study was to assess ergonomic factors, posture and biomechanics of workers of a broiler egg hatchery. The analysis of ergonomic factors was based on physical work load, thermal environment, and exposure to light and noise. The posture of workers was analyzed using photographic records which were evaluated by the software program OWAS (Ovako Working Posture Analysing System). A biomechanics analysis was also performed based on the photographs taken of the employee at various angles, which were used as inputs to the Michigan two-dimensional biomechanical model software program. The results show that certain activities can be considered unhealthy due to the exposure of employees to physical and thermal overload. The continuous noise levels and lighting were outside the range considered adequate by the regulations of the Brazilian Ministry of Labor. The manner in which certain activities are carried out when associated with weight and poor posture can result in body lesions in broiler hatchery employees. It is therefore necessary to apply specific ergonomic programs, including scheduled breaks, training, and other measures in order to reduce or to eliminate the risks involved in these activities.

Activities and Ergonomics of Workers in Broiler Hatcheries

The objective this study was to assess ergonomic factors, posture and biomechanics of workers of a broiler egg hatchery. The analysis of ergonomic factors was based on physical work load, thermal environment, and exposure to light and noise. The posture of workers was analyzed using photographic records which were evaluated by the software program OWAS (Ovako Working Posture Analysing System). A biomechanics analysis was also performed based on the photographs taken of the employee at various angles, which were used as inputs to the Michigan two-dimensional biomechanical model software program. The results show that certain activities can be considered unhealthy due to the exposure of employees to physical and thermal overload. The continuous noise levels and lighting were outside the range considered adequate by the regulations of the Brazilian Ministry of Labor. The manner in which certain activities are carried out when associated with weight and poor posture can result in body lesions in broiler hatchery employees. It is therefore necessary to apply specific ergonomic programs, including scheduled breaks, training, and other measures in order to reduce or to eliminate the risks involved in these activities.(AU)

Chemical composition and efficacy in the egg-hatching inhibition of essential oil of Piper aduncum against Haemonchus contortus from sheep

Piper aduncum L., Piperaceae, has been used to treat mainly inflammatory diseases and has shown several biological activities such as insecticidal and larvicidal. The aim of this study was to analyze the chemical composition of essential oil of P. aduncum and its efficacy to egg-hatching inhibition of Haemonchus contortus from sheep. The essential oil was obtained from leaves and analysed by gas chromatography coupled to flame ionization detector and gas chromatography coupled to mass spectrometry. It was possible to characterize 22 different substances, among them monoterpenes (80.6%) and sesquiterpenes (13.9%). The major compound was identified as 1,8-cineole (55.8%). Eggs of the nematode were exposed to four concentrations of the essential oil. Levamisole phosphate was used as positive control. The essential oil showed to be effective in inhibiting H. contortus hatchability and the LC90 was calculated as 8.9 mg.ml-1. These results can point out the P. aduncum essential oil and its chemical components as potential alternative to control of H. contortus .

In vitro effects of Musa x paradisiaca extracts on four developmental stages of Haemonchus contortus.

This study was carried out to evaluate the in vitro effect of Musa x paradisiaca stem and leaf against the parasitic nematode of small ruminants Haemonchus contortus. Three extracts (aqueous, methanolic and/or dichloromethane) of Musa x paradisiaca stem and leaf were tested in vitro on four developmental stages of H. contortus using egg hatch assay (EHA), larval development assay (LDA), L3 migration inhibition assay (LMI) and adult worm motility assay (AWM). The highly significant (P<0.0001) ability to stop larval development (inhibition >67% for each extract) and the negative effect of the dichloromethane extract of leaf on adult worm motility (43% of inhibition of motility after 24h of incubation) compared to the negative controls, suggest anthelmintic properties of Musa x paradisiaca stem and leaf against H. contortus. The active principles responsible for the activity could be secondary metabolites such as terpenoid and flavonoid compounds present in the leaf and stem of the plant.

Resistance to freezing temperatures in Aedes (Ochlerotatus) albifasciatus (Macquart) eggs (Diptera: Culicidae) from two different climatic regions of Argentina.

Aedes (Ochlerotatus) albifasciatus (Macquart) has the capacity to proliferate in different kinds of climates within its distribution range in South America. With the aim of studying local thermal adaptations of eggs, we exposed egg stocks from two climatically different localities: temperate humid pampa (Buenos Aires) and cold arid Patagonian (Sarmiento), to freezing conditions and then evaluated the effect on some features at this level. First, we thermally described the substrate where this species lays its eggs in the arid region. A typical thermal condition during winter was 10 h at -12° C. Second, we evaluated the effect of freezing on primary hatching (vs total hatching) and embryo survival. We also compared the proportion of embryonated eggs from both populations. The proportions of embryonated eggs were not different between localities, with averages of 78% and 83% in Sarmiento and Buenos Aires, respectively. Survival was equally successful after freezing in the two localities with an average range between 94-99%. Whether or not the eggs from Buenos Aires and Sarmiento were under freezing conditions, hatching was more than 98% after the first flooding. The results suggest that eggs of Ae. albifasciatus from Sarmiento and Buenos Aires have the same ability to survive at extreme temperatures (<0° C), showing a regional thermal adaptation rather than a local one.

The effect of abiotic factors on the hatching of Moina micrura Kurz, 1874 (Crustacea: Cladocera) ephippial eggs

The roles of some abiotic factors in controlling the hatching rates of Moina micrura ephippial eggs were investigated. Determination of optimum hatching conditions would be important in developing the use of this species as a food for fish larvae in aquaculture. Ephippia were exposed to different treatments in the laboratory, and monitored for hatching over a period of seven days. Optimum hatching conditions were: pH 5-9, temperature 25ºC, photoperiod eight or more hours light per day and light intensity equal to or greater than 850 lux. Differences in water ionic concentrations (from deionized water to 880 mg.L FONT FACE=Symbol>- /FONT>1 of selected salts) had no effect.

Foram investigados os efeitos de alguns fatores abióticos na taxa de eclosão dos ovos efipiais de Moina micrura. O conhecimento das melhores condições para a eclosão desses ovos pode ser importante na utilização dessa espécie para a aqüicultura, como alimento para larvas de peixes. Os efípios foram expostos a diferentes experimentos em laboratório por um período de sete dias. As melhores condições de eclosão foram: pH de 5 a 9, temperatura de 25ºC, fotoperíodo de oito ou mais horas de luz e intensidade luminosa igual ou maior que 850 lux. Diferentes concentrações de íons na água (de água deionizada e até 880 mg.L FONT FACE=Symbol>- /FONT>1 de sais selecionados) não influenciaram a eclosão.

The effect of abiotic factors on the hatching of Moina micrura Kurz, 1874 (Crustacea: Cladocera) ephippial eggs

The roles of some abiotic factors in controlling the hatching rates of Moina micrura ephippial eggs were investigated. Determination of optimum hatching conditions would be important in developing the use of this species as a food for fish larvae in aquaculture. Ephippia were exposed to different treatments in the laboratory, and monitored for hatching over a period of seven days. Optimum hatching conditions were: pH 5-9, temperature 25ºC, photoperiod eight or more hours light per day and light intensity equal to or greater than 850 lux. Differences in water ionic concentrations (from deionized water to 880 mg.L FONT FACE=Symbol>- /FONT>1 of selected salts) had no effect.

Foram investigados os efeitos de alguns fatores abióticos na taxa de eclosão dos ovos efipiais de Moina micrura. O conhecimento das melhores condições para a eclosão desses ovos pode ser importante na utilização dessa espécie para a aqüicultura, como alimento para larvas de peixes. Os efípios foram expostos a diferentes experimentos em laboratório por um período de sete dias. As melhores condições de eclosão foram: pH de 5 a 9, temperatura de 25ºC, fotoperíodo de oito ou mais horas de luz e intensidade luminosa igual ou maior que 850 lux. Diferentes concentrações de íons na água (de água deionizada e até 880 mg.L FONT FACE=Symbol>- /FONT>1 de sais selecionados) não influenciaram a eclosão.