Despite the enormous disease burden, treatments are limited for soil-transmitted helminthiasis. The handful of anthelmintic drugs suffer from variable and often insufficient efficacy against the pathogens. Resistance development, caused by high drug pressure, is an ongoing concern. New treatments are needed, and combination therapies may represent a promising therapeutic strategy. We explored emodepside as a partner in combination therapy and characterized drug-interaction effects with key anthelmintics in vitro against different soil-transmitted helminths (STHs). Drug-interaction studies were carried out in human recombinant cytochromes P450 assays. Combination studies revealed species-, stage-, and drug-dependent interaction profiles. For benzimidazole-emodepside combinations, potentiation was only observed against Ancylostoma ceylanicum larvae, while against adult stages, up to threefold potentiation or synergy (ΣFIC < 0.9) was observed for Heligmosomoides polygyrus and Trichuris muris, especially for albendazole and albendazole sulfoxide. Combinations of emodepside with macrocyclic lactones showed mostly strong synergy against H. polygyrus larvae (ΣFIC 0.06-2.52), while other species trended toward antagonism (ΣFIC >1.5). Against adult stages, potentiation or synergism was observed in H. polygyrus and T. muris, while against Strongyloides ratti, only the moxidectin-emodepside combination was synergistic (ΣFIC 0.69-0.99). For nAChR agonists, levamisole showed the strongest effect against Necator americanus larvae, while oxantel pamoate exhibited potentiation or synergistic effects across multiple species and life stages. CYP450 inhibition assays reveal no relevant drug-drug interactions. This study provides a thorough evaluation of emodepside as a combination partner against soil-transmitted helminths in vitro, paving the way for in vivo studies and ultimately a novel combination therapy with emodepside against STH infections.

Foot-and-mouth disease (FMD) control still depends largely on inactivated vaccines; however, improving vaccine efficacy without reformulating existing vaccines remains an important practical challenge.

The aim of the present study was to evaluate the protection against tissue cyst formation from T. gondii in pigs immunized with a DNA vector plasmid encoding partial rop18 and sag1 genes. Ten pigs were divided into three groups (G): G1, and G2 received 150 μg of pcDNA3.1+rop18, and 150 μg of pcDNA3.1+sag1 by intradermal (ID) and intramuscular (IM) routes, respectively. G3 received 300 μg of pcDNA 3.1 alone by IM. All groups received 2% (v/v) of levamisole as an adjuvant. Four immunizations were carried out on days 0, 14, 35, and 56; the challenge was performed with ∼ 4 × 10[3] oocysts of the ME49 strain by oral route at day 70, and animals were culled on day 100. Humoral immune response of IgM and IgG antibodies was assessed using indirect ELISA, and tissue cysts were evaluable by mouse bioassay. The results showed that the animals immunized with the DNA vaccine (sag1+rop18) developed partial protection against the formation of tissue cysts, however, more studies with higher number of pigs should be conducted.

Rainbow trout (Oncorhynchus mykiss) eggs are a rich source of polyunsaturated fatty acids (PUFAs), with levels exceeding those found in fish meat, and are also a notable source of bioactive omega-3 fatty acids, primarily eicosapentaenoic acid (EPA)and docosahexaenoic acid (DHA). However, despite their nutritional advantages, their in vivo immunomodulatory properties remain poorly understood. This study explored the immunomodulatory potential of the lipid extract from O. mykiss eggs combined with PEG6000 (OM-PEG) in a cyclophosphamide (CY)-induced immunosuppressive BALB/c mouse model. Mice were randomly assigned to nine groups (n = 3/group): normal control, CY, CY + ginseng (100 mg/kg), CY + levamisole (40 mg/kg), CY + PEG6000 (10 mg/kg), and four CY + OM-PEG groups (50-200 mg/kg). In CY-treated mice, prophylactic oral administration of OM-PEG (50-200 mg/kg body weight) protected against changes in spleen size and index. It also significantly stimulated splenic immune cells, including T cells, B cells, and natural killer (NK) cells (p < 0.05). Furthermore, cell proliferation, nitric oxide (NO) production, and phagocytic activity were enhanced in peritoneal macrophages (p < 0.05). The mRNA expression of immunostimulatory cytokines and mediators, including interleukin-1β (IL-1β), IL-2, IL-4, IL-6, inducible nitric oxide synthase (iNOS), TNF-α, IFN-γ, and COX-2, was significantly upregulated in both splenic lymphocytes and peritoneal macrophages of CY-treated mice (p < 0.05). Consequently, these results suggest that OM-PEG6000, prepared by incorporating lipids isolated from O. mykiss eggs into PEG6000, contributes to immune system activation and has potential as a functional food ingredient due to its immune-enhancing efficacy.

In this study, the anthelmintic potential of the ethanolic and aqueous extracts of Moringa oleifera leaves and Embelia ribes seeds was evaluated against Haemonchus contortus by both in vitro (egg hatch assay, EHA; larval development assay, LDA; and adult mortality assay, AMA) and in vivo assays (faecal egg count reduction test, FECRT). Ethanolic extract of M. oleifera (MOEE) exhibited maximum inhibitory effects on egg hatching (87.77%) and larval development (86.97%) along with complete adult mortality (100% after 12 hours of incubation) @ 16.0 mg/mL and 32.0 mg/mL, respectively. However, ethanolic extract of E. ribes (EREE) @ 16.0 mg/mL concentration exhibited poor ovicidal (22.69%) but moderate larvicidal (77.90%) and adulticidal (80% mortality after 12 hours of incubation) effects. In EHA, MOEE showed a moderate IC50 of 5.721 mg/mL, while EREE failed to achieve 50% inhibition at tested concentrations (1-32 mg/mL). For the LDA, both MOEE and EREE exhibited moderate IC50 values of 4.131 mg/mL and 2.882 mg/mL, respectively. The aqueous extract of M. oleifera (MOAE) exhibited highest inhibitory effect on egg hatching (89.12%), larval development (89.94%), and also caused significant mortality (100%) of adult H. contortus in comparison to the standard drugs and other plant extracts evaluated in this study. In contrast, ERAE showed no significant effect on egg hatching or adult worm viability but notably inhibited larval development (86.97%). Both of the plant extracts contained phytochemicals like phenolic compounds, tannins, flavonoids, and alkaloids with anthelmintic properties. Further, in vivo trial using MOAE @ 150 mg/kg BW exhibited 72.42% FEC reduction on day 10 post-treatment as compared to fenbendazole (70.92%) and levamisole (89.01%) treated groups. The MOAE appeared effective in combating the resistant H. contortus worms. This study formed a base for further in vivo evaluation of anthelmintic efficacy of the mixture of Moringa and Embelia extracts and to standardise the optimum concentration and dose for its field applicability against GI parasitism in animals.

Echinacea purpurea polysaccharide (EPP), a key bioactive constituent of the natural immunostimulant E. purpurea, shows potential as a poultry feed additive. This study evaluated EPP's impact on growth performance, serum biochemistry indices, organ index, antioxidant capacity, immunity, intestinal health, and microbiota in Mahuang chickens. A total of 180 one-day-old Mahuang chicks (initial average body weight 39.10 ± 0.51 g) were randomly divided into six groups (six replicates, five chicks per replicate): a basal diet with 0 (CON), 0.1 g/kg levamisole hydrochloride (LMS), 100 mg/kg EPP (EPP100), 200 mg/kg EPP (EPP200), 400 mg/kg EPP (EPP400), and 800 mg/kg EPP (EPP800) for 42 days. The results showed that EPP did not affect serum biochemical indices and organ index, but significantly improved growth performance by increasing body weight gain (d 21) (p < 0.05). Additionally, EPP improved intestinal (duodenum, jejunum, and ileum) morphology by increasing villus height and villus height-to-crypt depth ratio, and decreasing crypt depth (d 21 and d 42) (p < 0.05). Com-pared to the CON and LMS groups, EPP significantly increased superoxide dismutase activity (p = 0.011) and total antioxidant capacity activity (p = 0.001) (d 21), elevated the levels of immunoglobulin (Ig) M (p < 0.001) and secretory IgA (p = 0.038) (d 21), and upregulated intestinal barrier-related genes (d 21 and d 42) (p < 0.05). 16S rRNA gene sequencing revealed that EPP treatment increased the Bacteroidetes/Firmicutes ratio. EPP also elevated the relative abundances of Bacteroides, Lactobacillus, Ruminococcus, Butyricicoccus, Oscillospira, and Akkermansia, while decreasing the relative abundance of Streptococcus (p < 0.05). Overall, the EPP groups outperformed the CON and LMS groups, with the high-dose groups (EPP400, EPP800) showing superior effects to the low-dose groups (EPP100, EPP200). In summary, this study confirmed that supplementing with EPP could boost growth performance, antioxidant capacity, immunity, intestinal barrier function, and cecal microbial community in Mahuang chickens without adverse effects, and the 400-800 mg/kg EPP appears to be optimal. This highlights the potential of EPP as a safe and effective alternative to synthetic immunostimulants in poultry production.

The validation of a semi-quantitative method for the rapid screening of whole blood and urine specimens using the Neogen® Gabapentin kit is presented. Decision points were validated at 1000 ng/mL for whole blood and urine. The validation design was based on ANSI/ASB 036 and internal laboratory requirements. Experiments included the evaluation of sensitivity, precision, specificity, carryover, hook effect, drift, ruggedness/robustness, interference, and an authentic case sample comparison. The experimental limit of detection (LOD) for gabapentin was determined to be at least 500 ng/mL in whole blood and urine. Excellent precision was demonstrated when the assay was evaluated using the mean of three replicates from five separate runs (n = 15) at the decision point and at concentration levels -50% and +100% of the decision point. The assay was reliably able to detect gabapentin without interference from matrix components or other substances routinely detected in authentic case samples with the exception of levamisole in urine if present at unusually high concentrations. Urine samples with 50 µg/mL or more of levamisole yielded a false positive screening result. Other case sample results were comparable to those obtained with liquid chromatography tandem high-resolution mass spectrometry or liquid chromatography tandem mass spectrometry for blood and urine, respectively. The Neogen® Gabapentin kit exhibited minimal cross-reactivity for pregabalin and demonstrated excellent precision and appropriate sensitivity in both whole blood and urine, making it an efficient and reliable method to screen for gabapentin.

Swarm behaviors are prevalent in nature and have inspired many engineering designs. However, achieving artificial swarms capable of reversibly migrating through fluids against gravity and transcending physical boundaries, such as solid-liquid and liquid-air interfaces, remains challenging. Here, we propose a strategy for generating enhanced rotating magnetic fields by synchronizing a global rotating field with a co-rotating local gradient. This enables three-dimensional (3D) movement of magnetic microparticles independent of physical boundaries and leads to reconfigurable collective behaviors, including reversible transitions between 2D and 3D tornado-like patterns, as well as between single and dual microswarms collectives. The generated tornado-like microswarm induces convection flows that enable entrapment and transport of substances within a confined spatial domain. This capability was used to promote chemical reaction within a confined region. As a further demonstration, the cardiotoxic compound-levamisole was vertically transported across intermediate barriers to induce localized arrhythmic beating of cardiomyocytes. These multifunctional magnetic microswarms hold promise for biological mechanism discovery and drug screening.

Anthelmintic drugs are used to control soil-transmitted helminths that infect a quarter of the world's human population. There is increasing concern about the development of resistance to anthelmintic drugs because of the limited number of compounds available and there is an unmet need for new resistance-busting drugs. Here we describe the presence of a previously unrecognized endogenous acetylcholine (ACh) analogue, β-alanine betaine, which may serve as an endogenous ligand for an alternate subfamily of nicotinic receptors (DEG-3/DES-2) that could be developed as novel drug targets because their analogues are not present in their human or animal hosts. We collected pseudocoelomic fluid from female Ascaris suum (a model for the human parasite Ascaris lumbricoides) and subjected it to HILIC-MS and HILIC-MS/MS to reveal signals consistent with ACh, choline, and β-alanine betaine but not betaine, propionylcholine, butyrylcholine, or butyrobetaine. The presence of β-alanine betaine was confirmed by comparison with an analytical standard. Injection of betaine into female A. suum produced no effect. However, injection of β-alanine betaine produced characteristic pretzel coiling and injection of levamisole produced a rod-like spastic paralysis. The differences between β-alanine betaine and levamisole suggested that they activate different nAChRs subfamilies. RT-PCR showed that message for members of the DEG-3/DES-2 subfamily of nAChR channels, which are betaine targets, were present in the intestine and body wall of A. suum. β-alanine betaine increased intracellular Ca2+ of the intestinal enterocytes and depolarized the membrane potential of body wall muscle cells. In N2 Caenorhabditis elegans, application of β-alanine betaine produced gradual inhibition of motility, which was reduced in acr-20, acr-23, deg-3/des-2 and lgc-41 null-mutants. These observations suggest that, in addition to ACh, β-alanine betaine - an analog of betaine produced under anaerobic conditions - may function as an endogenous ligand in anaerobic nematodes such as A. suum. An expanded repertoire of nicotinic acetylcholine receptor subfamilies in nematodes relative to mammals may reflect a corresponding need for diversification of cholinergic endogenous ligands in these organisms. This repertoire could allow their simpler neuronal system to perform more complex controls and be exploited for development of different and novel subfamily selective cholinergic anthelmintics.

Levamisole, a synthetic imidazothiazole derivative, remains a cornerstone in the management of frequently relapsing nephrotic syndrome and steroid-dependent nephrotic syndrome in resource-limited settings. While its efficacy as a steroid-sparing agent is well-established, its safety profile is complicated by rare but severe autoimmune phenomena. We present two distinct paediatric cases of levamisole-induced vasculitis from India.The first case involves a female child in middle childhood who developed multisystemic vasculitis (pulmonary, renal and cutaneous) following an accidental dose escalation. The second involves a female child, also in middle childhood, who developed a similar phenotype after prolonged, stable therapy. Both cases were marked by the presence of anti-neutrophil cytoplasmic antibody and resolved promptly on drug withdrawal. These cases underscore that levamisole-induced autoimmunity is not confined to specific ethnicities or illicit drug contaminants but is a direct iatrogenic risk that clinicians must monitor closely in the paediatric population.

Partial worm replacement (PR), consisting of the elimination of an anthelmintic-resistant parasite population from the host but not from the environment, was evaluated as a tool to improve anthelmintic treatment efficacy in White Dorper (DO), Santa Inês (SI), and Texel (TX) sheep breeds. Artificially infected ewes were kept either in a nematode-free paddock (total replacement - TR) or in a paddock naturally contaminated with resistant helminth larvae (PR) and compared with untreated ewes harboring resistant helminths kept in a naturally contaminated paddock (control - C) over two breeding seasons. In the third breeding season, because of reestablishment of resistance, ewes from the PR and TR groups were reinfected with the susceptible isolate, and their lambs were evaluated every 21 days for fecal egg count (FEC), packed cell volume (PCV), live body weight, FAMACHA© score, and body condition score (BCS). Benzimidazole (F200Y) and levamisole (S168T) resistance-associated polymorphisms were genotyped in Haemonchus contortus larvae. Lambs in the C group had higher FEC and lower PCV than lambs in the other groups, in addition to lower final weight than in the TR group (p < 0.05). DO lambs showed higher FEC, lower PCV, and higher final weight, whereas SI lambs showed higher PCV (p < 0.05). FAMACHA© 3 predominated in the C group and in DO lambs (p < 0.05), which also required significantly more anthelmintic treatments. Reinfection reduced the allele frequencies associated with benzimidazole and levamisole resistance, mainly in the PR group, positively affecting the evaluated parameters. Although reversion of anthelmintic efficacy was transient, strategic reinfection at the end of the dry season may offer an alternative for producers unable to implement TR. When combined with the use of resistant breeds, this approach may help reduce production losses in countries where effective drug classes are unavailable.

The status of anthelmintic resistance against benzimidazole (BZ), levamisole (LV) and ivermectin (IVM) was assessed in Haemonchus contortus at ten sheep flocks in Gangetic plains of India by using larval development assay (LDA) and PCR based molecular assays like allele specific PCR (AS-PCR) targeting SNP at F200Y of β- tubulin isotype-1 gene of H. contortus (BZ resistance) and PCR targeting insertion or deletion of 63 bp in acr-8b gene of H. contortus (LV resistance). Resistance to benzimidazole was detected in 10/10 flocks, levamisole resistance in 3/10 flocks, and ivermectin resistance in 2/10 flocks. Multiple resistance to albendazole, levamisole and ivermectin was observed in both Ayodhya flocks, whereas resistance to albendazole and levamisole was observed in one Etah flock. The findings highlight the emergence of multiple anthelmintic resistance in H. contortus in Uttar Pradesh, India. Proper monitoring of anthelmintic resistance and sound managemental practices are urgently required to mitigate further spread of anthelmintic resistance across India.

Mixed cryoglobulinemia is a small-vessel vasculitis mediated by cold-precipitable immune complexes, and chronic hepatitis C virus (HCV) infection is its most common cause. Cutaneous disease, classically palpable purpura of the lower limbs, is the most frequent manifestation but is readily mistaken for cellulitis or other dermatoses, delaying diagnosis. We describe a 61-year-old woman with chronic HCV, a long smoking history, and active cocaine use who experienced more than a year of recurrent, painful, pruritic erythematous - purpuric eruptions of the lower extremities that were repeatedly treated empirically as cellulitis without benefit. She also reported new easy bruising and skin fragility. Inflammatory markers were elevated, an extended antinuclear antibody panel was negative, and serum cryoglobulins were positive; immunofixation of the cryoprecipitate demonstrated type III (polyclonal) mixed cryoglobulinemia. Urinalysis showed hematuria with low-grade proteinuria, indicating renal involvement, and complement C4 was at the low end of normal. The findings established HCV-associated type III mixed cryoglobulinemic vasculitis. Cocaine history required consideration of levamisole-adulterated cocaine vasculopathy, an important mimic. She was started on corticosteroids with rheumatology co-management and HCV-directed direct-acting antiviral therapy as the cornerstone of treatment. Following completion of antiviral therapy and achievement of sustained virologic response, cutaneous vasculitis resolved, urinalysis normalized with no evidence of active glomerulonephritis, and the corticosteroid dose was successfully tapered. The patient remains on prednisone 5 mg daily with ongoing taper. This case underscores cryoglobulinemic vasculitis as a cause of recurrent, cellulitis-like, lower limb eruptions, and the primacy of antiviral cure in achieving vasculitis remission.