Infective L3 Larvae Research Articles

Understanding Artemisia cina Ethyl Acetate Extract's Anthelmintic Effect on Haemonchus contortus Eggs and L3 Larvae: The Synergism of Peruvin Binary Mixtures.

Haemonchus contortus, a blood-feeding parasite in grazing sheep, causes economic losses. Drug resistance necessitates exploring plant-based anthelmintics like Artemisia cina (Asteraceae). The plant, particularly its ethyl acetate extract, shows anthelmintic activity against H. contortus. However, there is limited information on pharmacodynamic interactions in ethyl acetate compounds. The study aims to identify pharmacodynamic interactions in the ethyl acetate extract of A. cina with anthelmintic effects on H. contortus eggs and L3 larvae using binary mixtures. Bioactive compounds were isolated via chromatography and identified using spectroscopic techniques. Pharmacodynamic interactions were assessed through binary mixtures with a main compound. Four bioactive compounds were identified: 1-nonacosanol, hentriacontane, peruvin, and cinic acid. Binary mixtures, with peruvin as the main compound, were performed. Peruvin/1-nonacosanol-hentriacontane and peruvin/cinic acid mixtures demonstrated 1.42-fold and 4.87-fold increased lethal effects in H. contortus L3 infective larvae, respectively, at a 0.50LC25/0.50LC25 concentration. In this work, we determined the synergism between bioactive compounds isolated from the ethyl acetate extract of A. cina and identified unreported compounds for the specie.

Bio-guided isolation of a new sesquiterpene from Artemisia cina with anthelmintic activity against Haemonchus contortus L3 infective larvae.

Haemonchus contortus is a blood-feeding gastrointestinal parasite that impacts grazing sheep, causing economic losses in animal production. Due to its anthelmintic resistance, alternative antiparasitic treatments like plant-based anthelmintics are necessary to explore. Artemisia cina (Asteraceae) is a plant whose n-hexane extract and ethyl acetate extract exhibit anthelmintic activity against H. contortus, the n-hexane more active. To discover additional bioactive metabolites, a chemical analysis was performed on ethyl acetate extract, which presented an LC90 of 3.30 mg/mL and allowed the isolation of 11-[(1R,5S,7R,8R,10S,)-1,8-dihydroxy-5,10-dimethyl-4-oxodecahydroazulen-7-yl] acrylic acid. This new sesquiterpene was identified through one and two-dimensional NMR. The compound was named cinic acid and displayed an LC50 of 0.13 (0.11-0.14) mg/mL and LC90 of 0.40 (0.37-0.44) mg/mL, which, compared with ethyl acetate extract larvicidal activity, was 256-fold more active at LC50 and 15.71-fold at LC90. In this study, a new sesquiterpene with larvicidal activity against H. contortus L3 infective larvae was isolated from the ethyl acetate extract of Artemisia cina.

Potential of Nucleic Acid Receptor Ligands to Improve Vaccination Efficacy against the Filarial Nematode Litomosoides sigmodontis.

More than two-hundred-million people are infected with filariae worldwide. However, there is no vaccine available that confers long-lasting protection against filarial infections. Previous studies indicated that vaccination with irradiated infective L3 larvae reduces the worm load. This present study investigated whether the additional activation of cytosolic nucleic acid receptors as an adjuvant improves the efficacy of vaccination with irradiated L3 larvae of the rodent filaria Litomosoides sigmodontis with the aim of identifying novel vaccination strategies for filarial infections. Subcutaneous injection of irradiated L3 larvae in combination with poly(I:C) or 3pRNA resulted in neutrophil recruitment to the skin, accompanied by higher IP-10/CXCL10 and IFN-β RNA levels. To investigate the impact on parasite clearance, BALB/c mice received three subcutaneous injections in 2-week intervals with irradiated L3 larvae in combination with poly(I:C) or 3pRNA prior to the challenge infection. Vaccination with irradiated L3 larvae in combination with poly(I:C) or 3pRNA led to a markedly greater reduction in adult-worm counts by 73% and 57%, respectively, compared to the immunization with irradiated L3 larvae alone (45%). In conclusion, activation of nucleic acid-sensing immune receptors boosts the protective immune response against L. sigmodontis and nucleic acid-receptor agonists as vaccine adjuvants represent a promising novel strategy to improve the efficacy of vaccines against filariae and potentially other helminths.

Examination of Horizontal Transmission of Nippostrongylus brasiliensis in Mice to Assess Biosecurity Risks.

Mice are commonly infected with Nippostrongylus brasiliensis (Nb) to study their immune responses. However, biosecurity measures have not been established for housing Nb-infected mice and rats. Transmission reportedly does not occur when infected mice are cohoused with naive mice. To test this, we inoculated female NOD. Cg-Prkdcscid Il2rgtm1Wjl /Sz(NSG;n = 12) and C57BL/6J (B6;n = 12) mice with 750 Nb L₃ larvae. These mice were then cohoused with naïve NSG ( n = 24) and B6 ( n = 24) mice (1 infected and 2 naïve mice per cage (24 cages) for 28 d in static microisolation cages that were changed every 14 d. We also did several studies to determine the conditions that favor horizontal transmission. First, we assessed in vitro development to the L₃ stage of Nb egg-containing fecal pellets maintained under 4 environmental conditions (dry, moist, soiled bedding, and control). Second, we assessed infection of naïve NSG mice ( n = 9) housed in microisolation cages that contained soiled bedding spiked with infective L₃ larvae (10,000/cage). Third, we gavaged NSG mice ( n = 3) with Nb eggs to model the potential for infection after coprophagy. We found that naïve NSG (9 of 24) and B6 (10 of 24) mice cohoused with an infected cagemate passed Nb eggs in feces as early as 1 d after cohousing and intermittently thereafter for varying periods. This shedding was presumably the result of coprophagy because adult worms were not detected in the shedding mice at euthanasia. Although eggs developed in vitro into L₃ larvae under moist and control environmental conditions, none of the NSG mice housed in cages with L₃ -spiked bedding or gavaged with eggs became infected with Nb. These findings indicate that infectious horizontal transmission does not occur when mice are housed with Nb-shedding cage mates in static microisolation cages with a 14-d cage-changing interval. Results from this study can be used to inform biosecurity practices when working with Nb-infected mice.

An improved model for the population dynamics of cattle gastrointestinal nematodes on pasture: parameterisation and field validation for Ostertagia ostertagi and Cooperia oncophora in northern temperate zones

Gastrointestinal nematodes (GIN) are amongst the most important pathogens of grazing ruminants worldwide, resulting in negative impacts on cattle health and production. The dynamics of infection are driven in large part by the influence of climate and weather on free-living stages on pasture, and computer models have been developed to predict infective larval abundance and guide management strategies. Significant uncertainties around key model parameters limits effective application of these models to GIN in cattle, however, and these parameters are difficult to estimate in natural populations of mixed GIN species. In this paper, recent advances in molecular biology, specifically ITS-2 rDNA ‘nemabiome’ metabarcoding, are synthesised with a modern population dynamic model, GLOWORM-FL, to overcome this limitation. Experiments under controlled conditions were used to estimate rainfall constraints on migration of infective L3 larvae out of faeces, and their survival in faeces and soil across a temperature gradient, with nemabiome metabarcoding data permitting species-specific estimates for Ostertagia ostertagi and Cooperia oncophora in mixed natural populations. Results showed that L3 of both species survived well in faeces and soil between 0 and 30 °C, and required at least 5 mm of rainfall daily to migrate out of faeces, with the proportion migrating increasing with the amount of rainfall. These estimates were applied within the model using weather and grazing data and use to predict patterns of larval availability on pasture on three commercial beef farms in western Canada. The model performed well overall in predicting the observed seasonal patterns but some discrepancies were evident which should guide further iterative improvements in model development and field methods. The model was also applied to illustrate its use in exploring differences in predicted seasonal transmission patterns in different regions. Such predictive modelling can help inform evidence-based parasite control strategies which are increasingly needed due climate change and drug resistance. The work presented here also illustrates the added value of combining molecular biology and population dynamics to advance predictive understanding of parasite infections.

ILC2s Control Microfilaremia During Litomosoides sigmodontis Infection in Rag2-/- Mice.

Group 2 innate lymphoid cells (ILC2s) are inducers of type 2 immune responses, but their role during filarial infection remains unclear. In the present study, we used the Litomosoides sigmodontis rodent model of filariasis to analyze ILC2s during infection in susceptible BALB/c mice that develop a chronic infection with microfilaremia and semi-susceptible C57BL/6 mice that eliminate the filariae shortly after the molt into adult worms and thus do not develop microfilaremia. ILC2s (CD45+ Lineage- TCRβ- CD90.2+ Sca-1+ IL-33R+ GATA-3+) were analyzed in the pleural cavity, the site of L. sigmodontis infection, after the infective L3 larvae reached the pleural cavity (9 days post infection, dpi), after the molt into adult worms (30dpi) and during the peak of microfilaremia (70dpi). C57BL/6 mice had significantly increased ILC2 numbers compared to BALB/c mice at 30dpi, accompanied by substantially higher IL-5 and IL-13 levels, indicating a stronger type 2 immune response in C57BL/6 mice upon L. sigmodontis infection. At this time point the ILC2 numbers positively correlated with the worm burden in both mouse strains. ILC2s and GATA-3+ CD4+ T cells were the dominant source of IL-5 in L. sigmodontis-infected C57BL/6 mice with ILC2s showing a significantly higher IL-5 expression than CD4+ T cells. To investigate the importance of ILC2s during L. sigmodontis infection, ILC2s were depleted with anti-CD90.2 antibodies in T and B cell-deficient Rag2-/- C57BL/6 mice on 26-28dpi and the outcome of infection was compared to isotype controls. Rag2-/- mice were per se susceptible to L. sigmodontis infection with significantly higher worm burden than C57BL/6 mice and developed microfilaremia. Depletion of ILC2s did not result in an increased worm burden in Rag2-/- mice, but led to significantly higher microfilariae numbers compared to isotype controls. In conclusion, our data demonstrate that ILC2s are essentially involved in the control of microfilaremia in Rag2-/- C57BL/6 mice.

Microfilariae Trigger Eosinophil Extracellular DNA Traps in a Dectin-1-Dependent Manner

Eosinophils mediate protection against filarial nematodes. Our results demonstrate that eosinophil extracellular traps (EETosis) are induced by microfilariae and infective L3 larvae of Litomosoides sigmodontis. These extracellular DNA traps inhibit microfilariae motility in a DNA- and contact-dependent manner invitro. Accordingly, microfilariae-injection triggers DNA release in an eosinophil-dependent manner invivo and microfilariae covered with DNA traps are cleared more rapidly. Using dectin-1, we identify the required receptor for the microfilariae-induced EETosis, whereas signaling via other C-type lectin receptors, prior priming of eosinophils, and presence of antibodies are not required. The DNA released upon microfilariae-induced EETosis is mainly of mitochondrial origin, but acetylated and citrullinated histones are found within the traps. We further demonstrate that the presented DNA-dependent inhibition of microfilariae motility by eosinophils represents a conserved mechanism, as microfilariae from L.sigmodontis and the canine heartworm Dirofilaria immitis induce ETosis in murine and human eosinophils.

Mansonella ozzardi

The human filarial nematode Mansonella ozzardi occurs widely in the Neotropical region from southern Mexico to northwestern Argentina. It causes mansonellosis and is transmitted by blackflies of the genus Simulium and biting midges of the family Ceratopogonidae. The embryonic unsheathed microfilariae with sharp, unnucleated tails are detectable in the blood (and occasionally in the skin) 1 day after being released by the fertilized adult female worms. After being ingested by a vector, the microfilariae reach the insect’s thoracic musculature through the hemocoel and develop, after two moults, into infective L3 larvae that migrate to the head and mouth parts of the vector.

S100A8/S100A9 deficiency increases neutrophil activation and protective immune responses against invading infective L3 larvae of the filarial nematode Litomosoides sigmodontis.

Neutrophils are essentially involved in protective immune responses against invading infective larvae of filarial nematodes. The present study investigated the impact of S100A8/S100A9 on protective immune responses against the rodent filarial nematode Litomosoides sigmodontis. S100A9 forms with S100A8 the heterodimer calprotectin, which is expressed by circulating neutrophils and monocytes and mitigates or amplifies tissue damage as well as inflammation depending on the immune environment. Mice deficient for S100A8/A9 had a significantly reduced worm burden in comparison to wildtype (WT) animals 12 days after infection (dpi) with infective L3 larvae, either by the vector or subcutaneous inoculation, the latter suggesting that circumventing natural immune responses within the epidermis and dermis do not alter the phenotype. Nevertheless, upon intradermal injection of L3 larvae, increased total numbers of neutrophils, eosinophils and macrophages were observed within the skin of S100A8/A9-/- mice. Furthermore, upon infection the bronchoalveolar and thoracic cavity lavage of S100A8/A9-/- mice showed increased concentrations of CXCL-1, CXCL-2, CXCL-5, as well as elastase in comparison to the WT controls. Neutrophils from S100A8/A9-/- mice exhibited an increased in vitro activation and reduced L3 larval motility more effectively in vitro compared to WT neutrophils. The depletion of neutrophils from S100A8/A9-/- mice prior to L. sigmodontis infection until 5dpi abrogated the protective effect and led to an increased worm burden, indicating that neutrophils mediate enhanced protective immune responses against invading L3 larvae in S100A8/A9-/- mice. Interestingly, complete circumvention of protective immune responses in the skin and the lymphatics by intravenous injection of L3 larvae reversed the phenotype and resulted in an increased worm burden in S100A8/A9-/- mice. In summary, our results reveal that lack of S100A8/S100A9 triggers L3-induced inflammatory responses, increasing chemokine levels, granulocyte recruitment as well as neutrophil activation and therefore impairs larval migration and susceptibility for filarial infection.

Profiling microRNAs through development of the parasitic nematode Haemonchus identifies nematode-specific miRNAs that suppress larval development

Parasitic nematodes transition between dramatically different free-living and parasitic stages, with correctly timed development and migration crucial to successful completion of their lifecycle. However little is known of the mechanisms controlling these transitions. microRNAs (miRNAs) negatively regulate gene expression post-transcriptionally and regulate development of diverse organisms. Here we used microarrays to determine the expression profile of miRNAs through development and in gut tissue of the pathogenic nematode Haemonchus contortus. Two miRNAs, mir-228 and mir-235, were enriched in infective L3 larvae, an arrested stage analogous to Caenorhabditis elegans dauer larvae. We hypothesized that these miRNAs may suppress development and maintain arrest. Consistent with this, inhibitors of these miRNAs promoted H. contortus development from L3 to L4 stage, while genetic deletion of C. elegans homologous miRNAs reduced dauer arrest. Epistasis studies with C. elegans daf-2 mutants showed that mir-228 and mir-235 synergise with FOXO transcription factor DAF-16 in the insulin signaling pathway. Target prediction suggests that these miRNAs suppress metabolic and transcription factor activity required for development. Our results provide novel insight into the expression and functions of specific miRNAs in regulating nematode development and identify miRNAs and their target genes as potential therapeutic targets to limit parasite survival within the host.

Assessment of genetic resistance and abomasal tissues expression of Yichang white goat experimentally challenge with Haemonchus contortus infection

Our previous association study revealed the mutation in candidate immune genes (NOD1 & NLRP9) was significantly associated with FEC of Haemonchus contortus infection in Yichang white goats, but the relative expression of mRNA of those genes associated with resistance to H. contortus was not investigated. Aim of the current experiment was to evaluate the susceptible and resistant individuals to nematode infection within the population of Yichang white goat (YWG) and assess the differential level of mRNA expression of those candidate genes in the abomasal tissues of susceptible and resistant goats. Fecal egg count (FEC) was determined using a modified McMaster technique, and the hematological parameter was measured by Mindray auto hematology analyzer. Phenotype data were collected and analyzed using a generalized linear model with SAS statistical program. Field investigate revealed that the prevalence (76%) with maxium parasite load (734.34±84.21epg) of H. contortus occurred in August within the experimental flock FEC in resistant group (103.38±1.20epg) and susceptible group (1180.25±43.53epg) group demonstrated the presence of two distinct goat populations within this breed. Four resistant and four susceptible goats were selected from each group. The parasite infection was established by artificially challenge with 5000 infective L3 larvae of H. contortus. Abomasal tissues were collected from all experimental goats after 42 days of post-infection. FEC, Body weight, packed cell volume, and hemoglobin value were significantly different (P < 0.01) between resistant and susceptible group of goats. Quantitative real-time PCR in abomasal tissues revealed that the expression level of mRNA for NOD1 (P < 0.00001), IFNG (P < 0.0001), NLRP9, TLR8, IL32, (P < 0.001) and IGF1 (P < 0.01) was higher in resistant goat compared to susceptible, except SFTPA1. These findings revealed the presence of genetic resistant individuals to H. contortus within the goat breed and expression of NOD1 and NLRP9 genes proved the positive finding of our previous study. Presence of genetic resistant individuals in Yichang white goat YWG breed could be a good candidate for selective breeding and highly expressed genes related to resistant could be used as biomarkers to develop H. contortus resistant goat population.
 Progressive Agriculture 30 (2): 194-208, 2019

Viability of Anisakis spp. Larvae After Direct Exposure to Different Processing Media and Non-Thermal Processing in Anchovy Fillets

Anisakiasis is fish-borne zoonoses caused by nematodes of the genus Anisakis, contracted by the ingestion of live L3 infective larvae through consumption of raw, undercooked or thermally unprocessed seafood products, such as carpaccio, and white marinated and dry-salted anchovies. In order to maintain the organoleptic properties of the product, the freezing of fish prior to processing is often ignored, especially in households, and traditional processing methods are not sufficient to kill Anisakis larvae. In this study, we investigated the survival and resistance of Anisakis spp. larvae in different processing solutions including varying salt and sugar content, lemon juice, acetic acid, alcohol, wine, and apple vinegar. We also performed a viability test of larvae during processing in anchovy fillets. When exposed directly to different NaCl concentrations, larvae were killed after approximately three days in the strongest (35%) and 10 days in the weakest solution (5%). In lemon juice and lemon juice with added acetic acid, the survival of larvae was around 5 days. In intact alcohol vinegar, larvae were killed under less than 48 h, while in the solution with water their resistance was prolonged to almost 40 days. In fillets, larvae showed increased resistance during carpaccio and white wine vinegar marinades and only dry salting was effective in destroying Anisakis spp. larvae.

Expression of worm resistance in sheep selected for low worm-egg counts fed at maintenance or above-maintenance level

The present study reports on changes in faecal worm-egg counts (WEC), larval composition, and the number of worms at different developmental stages in young sheep sourced from a flock selected for reduced faecal worm-egg counts over 15 years. The sheep were individually penned and fed a maintenance (1.0 M) or a 1.5 times maintenance (1.5 M) diet over two periods, namely, worm-free and infection phases. They were dosed weekly with 10000 Trichostrongylus colubriformis and 10000 Teladorsagia circumcincta L3 infective larvae for 11 weeks. Sheep on the 1.5 M diet had lower WEC and higher bodyweights than did sheep on the 1.0 M diet. A significant decline in the percentage T. colubriformis occurred during the experiment, but no concomitant change in T. circumcincta was noticed. Resistant sheep had significantly (P < 0.001) fewer worms at necropsy, and also shed significantly (P < 0.001) fewer worm eggs during the experiment. Restricted feeding reduced bodyweight significantly (P < 0.001) and had a small but significant (P < 0.04) effect on the faecal worm-egg output and on the number of T. colubriformis worms (P < 0.01) in both the control and resistant sheep. The study showed that sheep selected for low WEC resulted in significantly (P < 0.001) lower WEC than in an unselected control line fed at a maintenance (1.0 M) and at an above-maintenance (1.5 M) level. Restricted feeding reduced bodyweight and had a small negative, and inconsistent, effect on the faecal worm-egg output in both the control and resistant lines.

Resistance of Djallonké sheep to <i>Haemonchus contortus</i> under artificial challenge

Gastrointestinal parasitism is a major constraint for small ruminant production. Coupled to the development of drug resistance in parasite populations, it is of high interest to identify animals with tolerance to nematode infections. In this study we evaluated Djallonke and Sahelian sheep resistance to Haemonchus contortus infection in Burkina Faso by challenging a total of 35 lambs (21 Djallonke and 14 Sahelian). Five weeks following deworming, a single dose of 5,000 L3 infective larvae of H. contortus were inoculated to each lamb. Body weight (BW), fecal egg counts (FEC), packed cell volume (PCV) and FAffa Malan CHArt (FAMACHA) eye scores were recorded at days 0; 28; 35 and 42 following artificial infection. The body weight between days 28 and 42, the FAMACHA scores, PCV, FEC and lnFEC28-lnFEC35 were significantly correlated. Mean BW of Djallonke did not change significantly throughout the experiment while a significant loss of weight (p=0.04) was observed in Sahelian sheep. There were no breed effects on PCV, FEC and lnFEC (p>0.05), while effects of sex and measurement time on PCV and FAMACHA scores were significant (p<0.05), i.e., in Djallonke the FEC declined from 3945.14 to 3235.71 from days 28 to 35, and remained constant thereafter. The results of this study indicate that Djallonke breed is able to maintain its body weight despite the parasite burden. Together with the high variability found in FEC and PCV, Djallonke breed might be considered in selection program for internal parasite resistance. Keywords : Haemonchus contortus , Sheep, FaffaMalanChart, Parasitism, Burkina Faso.

The occurrence of Contracaecum sp. larvae (Nematoda: Anisakidae) in four teleostean species from Lake Nasser, Egypt: morphological and molecular studies

BackgroundNematodes of the family Anisakidae are parasites of many fishes and aquatic invertebrates which act as intermediate or paratenic hosts, while mammals and fish-eating birds are definitive hosts. Infective L3 larvae may be incidentally taken by human through eating raw or undercooked fish meat, causing anisakidosis. The main purpose of this study is to provide a basis for the future investigations to discover the genetic diversity of this widely distributed parasite nematodes of fishes and fish eating animals and their effect on fisheries and public health in Egypt and worldwide.ResultsOne thousand, one hundred and fifteen specimens belong to nine fish species were collected from Lake Nasser, Egypt, and examined for infection with Anisakid larvae. Four fish species (Oreochromis niloticus, Tilapia galilaea, Lates niloticus, and Hydrocynus forskahlii) were found infected with third stage larvae of Contracaecum spp. No other Anisakid nematodes were detected. Larvae were found in the body cavity adhering to mesenteries by a thin membrane, except in Oreochromis niloticus and Tilapia galilaea were found free in branchial chambers. The highest prevalence was recorded in L. niloticus (100%) and H. forskahlii (82%). The mean intensity of infections were 0.17–4.12 and 5.1–10.3 in L. niloticus and H. forskahlii respectively. For further identification, the internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA from isolated larvae (n = 54) were amplified by PCR, followed by single-strand conformation polymorphism (SSCP) analysis which revealed five possible profiles.ConclusionLight and scanning electron microscope studies revealed that all anisakid larvae in the present study showed the most typical features of the genus Contracaecum. The sequencing (n = 28) and sequence and phylogenetic analyses showed that the present nematode larvae are likely belonging to C. multipapillatum.

Angiostrongylus cantonensis: An optimized cultivation of this parasitic nematode under laboratory conditions.

Angiostrongylus cantonensis (A. cantonensis), a parasitic nematode, is the important neurotropic pathogen which causes human angiostrongyliasis. It has a complex life-cycle and severe parasite-host interaction in contrast to free-living nematode. Establishment of a well-suited life-cycle and in vitro cultivation of A. cantonensis in the laboratory will be one of the key techniques to elucidate the mechanism of parasite-host interaction. However, the low survival and growth rate of worms is still to be the problem. We optimized the known life-cycle of A. cantonensis in the laboratory, showing that small in size, easy to breed, and high compatibility of Biomphalaria straminea precede the common snails as an intermediate host of A. cantonensis. Furthermore, the egg hatching rate in Ham's F-12 medium reached approximately 80% using the eggs of mature female adult worms. We also demonstrated that the survival of larvae could be sustained for more than 30days by in vitro cultivation of L1 larvae in DMEM with mixed antibiotics (100units/mL of penicillin G potassium, 50μg/mL of streptomycin sulfate, and 0.5μg/mL of amphotericin B) and L3, L4, and L5 larvae in Waymouth's medium with 20% fetal calf serum and mixed antibiotics. Infective L1 and L3 larvae kept high infective rate to the snail and rat after cultivation in these media, respectively. It will provide the basis for studying on genetic manipulations for functional genes, new drug screening, and the mechanism of parasite-host interaction of parasitic nematodes.

Susceptibility of ten Haemonchus contortus isolates from different geographical origins towards acetone:water extracts of polyphenol-rich plants. Part 2: Infective L3 larvae

This study explored the variation in susceptibility to acetone:water plant extracts between infective larvae (L3) of ten Haemonchus contortus isolates from different geographical origin. The L3 of 10 different isolates were exposed either to the acetone:water extract of a temperate plant (Onobrychis viciifolia) or a tropical plant (Acacia pennatula) and were evaluated with the larval exsheathment inhibition assay (LEIA). The L3 of each isolate were incubated with different concentrations of each extract (0, 25, 50, 100, 200, 400, 600, 800, 1000 and 1200μg/mL of phosphate buffered saline (PBS)). After incubation, the exsheathment process of L3 was induced using a solution with sodium hypochlorite (2%) and sodium chloride (16.5%). The proportion of exsheathed L3 was determined for each concentration at 0, 20, 40 and 60min. Effective concentrations 50% (EC50) and the corresponding 95% confidence intervals (95% CI) were calculated for every isolate with both extracts. Moreover, a resistance ratio (RR) was calculated for each extract to compare isolates, using the most susceptible isolate as the respective reference for each extract. To determine the role of polyphenols on the reported effect, a second set of incubations was made for each isolate and each extract, using the extracts at a concentration of 1200μg/mL PBS with or without polyvinylpolypyrrolidone (PVPP), a polyphenol blocking agent, and controls without extract. The ten different H. contortus isolates showed variation in susceptibility for each of the 2 extracts tested (P<0.05). The EC50 values for A. pennatula extract ranged from 36 to 501μg/mL (RR: 2.11–13.68). Meanwhile, the EC50 values for O. viciifolia extract ranged from 128 to 1003μg/mL (RR: 1.25–7.82). The use of PVPP revealed that polyphenols were responsible for the anthelmintic activity recorded for both extracts. However, tested H. contortus isolates suggested that susceptibility to one polyphenol-rich extract did not determine the susceptibility to the other polyphenol rich extract. The latter result indicated that the different H. contortus isolates varied in their susceptibility to the polyphenols present in each extract evaluated.

Development of Dirofilaria immitis and Dirofilaria repens in Aedes japonicus and Aedes geniculatus

BackgroundThe mosquito-borne filarial nematodes Dirofilaria immitis and Dirofilaria repens primarily affect dogs but also cats, causing heartworm disease or subcutaneous dirofilariosis, respectively, and both may also cause zoonotic diseases in humans. Several mosquito species have been reported as competent vectors for these nematodes, but no data are available for the invasive mosquito species Aedes japonicus (Theobald, 1901). The objective of this study was to describe the development of both D. immitis and D. repens under standardised experimental laboratory conditions in mosquitoes.MethodsFor this purpose, both a laboratory strain and field-collected individuals of the invasive mosquito species Ae. japonicus and, for comparative purposes, a laboratory strain of Aedes geniculatus, a rare indigenous species sharing habitats with Ae. japonicus, and of the tropical species Aedes aegypti were used. Anticoagulated microfilariaemic blood was fed at a density of 3000 mf/ml to mosquitoes with a hemotek system. Blood-fed mosquitoes were incubated at 27 °C and 85% relative humidity, and specimens were dissected under the microscope at pre-set time points to observe developmental stages of both Dirofilaria species. Additionally, real-time PCRs were carried out in some microscopically negative samples to determine the infection rates.ResultsIn field-collected Ae. japonicus infectious L3 larvae of both D. immitis and D. repens developed, rendering this mosquito species an efficient vector for both filarial species. Additionally, Ae. geniculatus was shown to be an equally efficient vector for both filarial species. Aedes japonicus mosquitoes from a laboratory colony were refractory to D. immitis but susceptible to D. repens, whereas Ae. aegypti was refractory to both filarial species.ConclusionsTo our knowledge, Aedes japonicus was for the first time shown to be an efficient vector for both D. immitis and D. repens, indicating that this invasive and locally highly abundant species may contribute to a transmission of filarial worms. The data emphasize the necessity to perform vector competence studies with local mosquito populations as basis for risk assessments. We further demonstrated that detection of filarial DNA in a mosquito species alone does not allow to draw reliable conclusions with regard to its vector competence.

Litomosoides sigmodontis: a jird urine metabolome study.

The neglected tropical disease onchocerciasis affects more than 35 million people worldwide with over 95% in Africa. Disease infection initiates from the filarial parasitic nematode Onchocerca volvulus, which is transmitted by the blackfly vector Simulium sp. carrying infectious L3 larvae. New treatments and diagnostics are required to eradicate this parasitic disease. Herein, we describe that a previously discovered biomarker for onchocerciasis, N-acetyltyramine-O-glucuronide (NATOG) is also present in urine samples of jirds infected with the onchocerciasis model nematode Litomosoides sigmodontis. Increased NATOG values paralleled a progressing infection and demonstrated that quantification of NATOG in this rodent model can be utilized to track its infectivity. Moreover, our findings suggest how NATOG monitoring may be used for evaluating potential drug candidates.

Equine Cyathostominae Can Develop to Infective Third Stage Larvae on Straw Bedding

Reasons for performing studyTo determine whether horses could become infected with cyathostomins when bedded in deep litter straw.ObjectivesThe specific objective of the study was to determine whether cyathostomin eggs could hatch and develop to infective larval stages on straw bedding.Study designExperimental study.MethodsFour horticultural incubators were set up to simulate 3 straw bedding scenarios, and one grass turf control. Faeces were placed on 12 plots, and larval recoveries performed on samples of straw/grass over a 17‐day period. The plots within incubators A, B, and C contained dry straw, watered straw, and deep litter straw, respectively. Plot 1 of each incubator contained a faecal pat of a horse that tested negative for strongyle eggs – these were to serve as negative control plots. Plots 2 and 3 of each incubator contained a faecal pat from horses that had average faecal worm egg counts (FWEC) of 269 epg and 921 epg, respectively. A thermostat within each incubator was set to maintain an environmental temperature of approximately 20°C.ResultsNo L3 larvae were recovered from the control plots of each incubator, and none were recovered from any of the plots within Incubator A. L3 larvae were first detected on plots 2 and 3 of Incubator B on Day 8, and on plot 3 of incubators C and D on Day 10.ConclusionsIt is evident that equine Cyathostominae can develop to infective L3 larvae on straw bedding, but only when the straw is moist. Therefore, it may be speculated that a horse bedded in deep litter straw may become infected by ingesting the infective L3 larvae contaminating the straw.Ethical animal research: Not applicable. Source of funding: E.C. McGirr was supported by a World Horse Welfare Undergraduate Bursary 2014. Competing interests: None declared.