Infective Larvae Research Articles

Synergistic Interaction Effect of Artemisia cina n-hexane Extract and Tagetes lucida Ethyl Acetate Extract on Haemonchus Contortus

PurposeWe analysed the possible synergistic activity among active extracts from Artemisia cina and Tagetes lucida combinations on Haemonchus contortus, a nematode parasitising sheep.MethodsThe work was carried out in vitro on eggs and infective larvae (L3) of H. contortus. The results were analysed with SAS 9.1, applying the ANOVA and Tukey test, and the lethal concentration (LC) values LC50 and LC90 were determined with regression analysis, employing Proc Probit of SAS 9.1. Additionally, the lethal concentration (LC) was calculated with LC50 and LC90 to determine the synergistic effect.ResultsThe results demonstrated a high efficacy of the two plants studied on both nematode eggs and L3 larvae as well as of their combinations. The highest egg hatching inhibition was obtained with a 50/50 combination, and the best larvae mortality was obtained with 25% A. cina and 75% T. lucida at 10 mg/mL. Additionally, this combination showed a synergistic effect.ConclusionThe two plant species studied here can be applied as natural anthelmintic alternatives due to their high bioactive effect and synergistic response.

Green synthesis and anthelmintic activity of silver nanoparticles using Morus Alba Fruit extract against different stages of equine strongyles.

The current study was designed to green synthesize silver nanoparticles (GRAgNPs) using Morus alba fruit extract and evaluate their nematicidal effects against strongyle nematodes compared to commercial silver nanoparticles (CAgNPs) in vitro. The nanoparticles were characterized by Ultraviolet-visual absorption spectrography, transmission electron microscopy, and X-ray diffraction. Next, uptake of AgNPs by the first stage larvae (L1), egg hatch inhibition (EHI) and the motility of infectious larvae (L3s), and the ultrastructural analysis of the eggs and worms were conducted. Moreover, some of oxidative/nitrosative stress indicators, including total antioxidant status content (TAC), protein carbonylation (PCO), lipid peroxidation (MDA), and DNA damage were assessed in the homogenized samples of strongyle L3s. We found that the GRAgNPs had spherical shape, 20-30nm in diameter with rough surface. Following incubation with GRAgNPs at concentrations of 43.40, 21.70 and 10.85 ppm and CAgNPs at concentrations of 43.40 and 21.70, EHI was more than 90%. In addition, concentrations of 43.40 and 21.70 ppm of GRAgNPs led to inhibition of larval motility by more than 90%. The LC50 at 24h of treatment for GRAgNPs and CAgNPs was determined to be 8.62 and 10.34 ppm, respectively. GRAgNPs and CAgNPs, in a concentration-dependent manner, resulted in the induction of oxidative/nitrosative stress evidenced by decreased TAC levels, and increased levels of MDA and PCO, together with increased DNA damage. The uptake of AgNPs by the L1 larvae revealed that FITC labeled GRAgNPs fluoresced with high intensity largely in the intestinal area. Scanning Electron Microscopy analysis of eggs and larvae revealed that GRAgNPs penetrated the cuticle of larvae, changed the tegmentum, and ultimately killed the worm. In conclusion, GRAgNPs had more robust anthelminthic effects than the standard antiparasitic and CAgNPs. They could be considered as a promising antiparasitic agent.

Transcriptomic changes in the microsporidia proliferation and host responses in congenitally infected embryos and larvae

Congenital infection caused by vertical transmission of microsporidia N. bombycis can result in severe economic losses in the silkworm-rearing industry. Whole-transcriptome analyses have revealed non-coding RNAs and their regulatory networks in N. bombycis infected embryos and larvae. However, transcriptomic changes in the microsporidia proliferation and host responses in congenitally infected embryos and larvae remains unclear. Here, we simultaneously compared the transcriptomes of N. bombycis and its host B. mori embryos of 5-day and larvae of 1-, 5- and 10-day during congenital infection. For the transcriptome of N. bombycis, a comparison of parasite expression patterns between congenital-infected embryos and larva showed most genes related to parasite central carbon metabolism were down-regulated in larvae during infection, whereas the majority of genes involved in parasite proliferation and growth were up-regulated. Interestingly, a large number of distinct or shared differentially expressed genes (DEGs) were revealed by the Venn diagram and heat map, many of them were connected to infection related factors such as Ricin B lectin, spore wall protein, polar tube protein, and polysaccharide deacetylase. For the transcriptome of B. mori infected with N. bombycis, beyond numerous DEGs related to DNA replication and repair, mRNA surveillance pathway, RNA transport, protein biosynthesis, and proteolysis, with the progression of infection, a large number of DEGs related to immune and infection pathways, including phagocytosis, apoptosis, TNF, Toll-like receptor, NF-kappa B, Fc epsilon RI, and some diseases, were successively identified. In contrast, most genes associated with the insulin signaling pathway, 2-oxacarboxylic acid metabolism, amino acid biosynthesis, and lipid metabolisms were up-regulated in larvae compared to those in embryos. Furthermore, dozens of distinct and three shared DEGs that were involved in the epigenetic regulations, such as polycomb, histone-lysine-specific demethylases, and histone-lysine-N-methyltransferases, were identified via the Venn diagram and heat maps. Notably, many DEGs of host and parasite associated with lipid-related metabolisms were verified by RT-qPCR. Taken together, simultaneous transcriptomic analyses of both host and parasite genes lead to a better understanding of changes in the microsporidia proliferation and host responses in embryos and larvae in N. bombycis congenital infection.

Low toxicity contributes to Sporothrix globosa invade the skin of patients in low-epidemic areas of China.

This study aims to assess the clinical characteristics of sporotrichosis in low-endemic areas of China, including the prevalence geography, genotypic traits of patients, clinical manifestations, and strain virulence and drug sensitivities. The objective is to improve the currently used clinical management strategies for sporotrichosis. Retrospective data were collected from patients diagnosed with sporotrichosis through fungal culture identification. The isolates from purified cultures underwent identification using CAL (Calmodulin) gene sequencing. Virulence of each strain was assessed using a Galleria mellonella (G. mellonella) larvae infection model. Invitro susceptibility testing against commonly used clinical antifungal agents for sporotrichosis was conducted following CLSI criteria. In our low-endemic region for sporotrichosis, the majority of cases (23) were observed in middle-aged and elderly women with a history of trauma, with a higher incidence during winter and spring. All clinical isolates were identified as Sporothrix globosa (S. globosa). The G. mellonella larvae infection model indicated independent and dose-dependent virulence among strains, with varying toxicity levels demonstrated by the degree of melanization of the G. mellonella. Surprisingly, lymphocutaneous types caused by S. globosa exhibited lower invitro virulence but were more common in affected skin. In addition, all S.globosa strains displayed high resistances to fluconazole, while remaining highly susceptible to terbinafine, itraconazole and amphotericin B. Given the predominance of elderly women engaged in agricultural labour in our region, which is a low-epidemic areas, they should be considered as crucial targets for sporotrichosis monitoring. S. globosa appears to be the sole causative agent locally. However, varying degrees of melanization in larvae were observed among these isolates, indicating a divergence in their virulence. Itraconazole, terbinafine and amphotericin B remain viable first-line antifungal options for treating S.globosa infection.

Pathogenicity, infection process, physiological and biochemical effects of Metarhizium rileyi against Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) larvae

BackgroundMetarhizium rileyi is an important entomopathogenic fungus which has a wide range of host pests. In this work, the virulence of M. rileyi EFNL-08 against 3rd instar larvae of the fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), was quantified. Additionally, the infection process and aspects of the host immunological response were described in the laboratory.ResultsBioassay results proved that M. rileyi was highly virulent to 3rd instar S. frugiperda larvae with 98.33% mortality at 1 × 108 conidia/ml. Scanning electron microscopy was used to monitor morphological aspects of the infection process. When hyphal bodies (Hbs) of M. rileyi were injected into the hemocoel of host, they were not recognized by the host’s immune system as invaders. There were nonsignificant differences in the hemocytes and phenoloxidase activity between the infected and control larvae at the initial 30 h, indicating that fungus successfully avoids the attack from the immune system. However, it inhibited the enzyme activity at the latest stage when Hbs reached a high density. After infection with M. rileyi, the activity of glutathione-S-transferase (GST) of S. frugiperda larvae presented a significant upward trend, while the activity of acetylcholinesterase (AchE) initially increased, followed by a decrease, and eventually stabilized. However, the activity of both detoxification enzymes in the control group showed slight fluctuations in the initial stage, and then stabilized at a low level.ConclusionObtained results provided new insights into the virulence and potential physiological responses of M. rileyi. This provides ideas for further development of novel and efficient entomopathogenic fungi (EPF) for pest control.

Local terrestrial snails as natural intermediate hosts of the zoonotic parasite Angiostrongylus cantonensis in the new European endemic area of Valencia, Spain.

The rat lungworm, Angiostrongylus cantonensis, has recently been found in the city of Valencia, parasitizing rats, Rattus norvegicus and Rattus rattus, its natural definitive hosts. This is the first finding of this zoonotic nematode in continental Europe. After informing local and national health authorities, the collection of local terrestrial snails took place with the aim of elucidating their potential role as intermediate hosts of A. cantonensis. A total of 145 terrestrial snails, belonging to the species Cernuella virgata, Cornu aspersum, Eobania vermiculata, Otala punctata, Pseudotachea splendida, Rumina decollata and Theba pisana, were randomly collected between May and December 2022 in public gardens, parks and orchards in six districts of Valencia, in five of which A. cantonensis had been reported previously in rats. Once collected and identified, the snails were frozen at -20°C. Subsequently, the DNA was isolated and screened by PCR using specific primers targeting the A. cantonensis COI gene. Seven individual snails, belonging to the species C. virgata, C. aspersum and T. pisana, were positive, for an overall prevalence of 4.8%. The PCR product from one of them was sequenced by Sanger sequencing. The three positive terrestrial snail species are among the edible species that are frequently included in various dishes in Spain. C. virgata is reported as a previously unrecorded intermediate host and should be added to the list of more than 200 species of terrestrial snails that have been reported worldwide as intermediate hosts of the rat lungworm. Considering that these terrestrial snails may release infective larvae of A. cantonensis on leafy green vegetables on which they feed and during their handling and preparation for consumption, prophylactic measures to prevent human neuroangiostrongyliasis in Valencia and other regions to which this zoonotic parasite may spread are recommended.

Therapeutic Potential of Coriander (Coriandrum sativum) Seeds Extract Treatment on Hematological and Biochemical Parameters in Healthy and Trichinella spiralis Infected Mice

The smallest known human nematode parasite is Trichinella spiralis (T. spiralis). A parasitic zoonosis that can be found anywhere in the world is trichinellosis. When a host consumes raw meat contaminated with Trichinella larvae, the larvae mature into adults in the host's stomach in 5 to 6 days. The females subsequently give birth to additional larvae, some of which eventually make it to muscle cells after travelling through blood channels. These cells allow infective larvae to survive for years because, after two to three weeks, the larvae become infectious and the host cell transforms into a nurse cell. The current study used parasitological assessment to count adult worms and encysted larvae at 5 weeks after infection (5WPI) to determine the effect of coriander seeds extract (CSE) against encysted larvae further to hematological and biochemical tests. Current results revealed that; Trichinella spiralis experimentally infection (5WPI) induced significant elevation in white blood cells (WBCs) counts, neutrophil, monocyte, eosinophil percentage, alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), urea, creatinine and a significant depletion hemoglobin (Hb), red blood cell (RBC) count, hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), platelet count (PLT), lymphocytes percentage, albumin, total proteins as compared to control and CSE groups. Meanwhile, treatments of 5WPI with CSE (5WPI+CSE) revealed a significant reduction in the number of Trichinella spiralis encysted larvae and improvements in all hematological parameters, liver and kidney functions as compared to 5WPI. We can conclude that; CSE could be an alternative method for treatment against trichinellosis.

Cerebral hydatid cyst masquerading as multiple cystic metastases

Hydatid disease is caused by encysted larvae of the dog tapeworm Echinococcus granulosus. Cerebral hydatidosis is a rare central nervous system larval infection affecting approximately 2–3% of patients. CT and MRI scans of the boy, described in our case, showed multiple cystic areas. Total surgical extirpation is the therapeutic approach, emphasizing the importance of early diagnosis for a favourable prognosis. Recurrence and unfavourable outcomes are commonly associated with incomplete excision and surgical rupture, necessitating careful removal to prevent anaphylaxis and widespread infection. Dowling's technique is the preferred surgical approach and postoperatively, patients are administered antiparasitic drugs such as Albendazole or Mebendazole for 6–12 months in most cases, with doses ranging from 10-15mg/kg in divided doses. The results of pharmacological treatment vary across series, with response rates ranging from 43.5 to 80%. Early diagnosis significantly contributes to a favourable prognosis by preventing neurological consequences.

Exposure of Cryptococcus neoformans to low nitrogen levels enhances virulence

Previous studies have shown a correlation between nitrogen levels and Cryptococcus neoformans pathogenicity. Here we report on the in vivo effects of cryptococcal pre-exposure to ecologically relevant nitrogen levels. C. neoformans H99 was cultured in yeast carbon base (YCB) supplemented with 0.53 g/L NH4Cl and 0.21 g/L NH4Cl, respectively, and used to infect larvae of the Greater Wax moth, Galleria mellonella. Cells cultured in low nitrogen YCB (LN) were more virulent compared to cells cultured in high nitrogen YCB (HN). Microscopic examination of haemolymph collected from infected larvae revealed that cells cultured in LN were larger than cells cultured in HN, with the majority of LN cells exceeding 10 µm and possibly entering titanisation. Additionally, compared to HN-cultured cells, fewer LN-cultured cells were engulfed by macrophages. The enhanced virulence of LN-cultured cells was attributed to the increased cell size in vivo. In contrast, reduced macrophage uptake was attributed to increased capsule thickness of in vitro cells. Not only do these findings demonstrate the effects of culture conditions, specifically nitrogen levels, on C. neoformans virulence, but they also highlight the importance of isolate background in the cryptococcal-host interaction.

Galleria mellonella in vitro model for chromoblastomycosis shows large differences in virulence between isolates

BackgroundChromoblastomycosis is the World Health Organization (WHO)-recognized fungal implantation disease that eventually leads to severe mutilation. Cladophialophora carrionii (C. carrionii) is one of the agents. However, the pathogenesis of C. carrionii is not fully investigated yet.MethodsWe investigated the pathogenic potential of the fungus in a Galleria mellonella (G. mellonella) larvae infection model. Six strains of C. carrionii, and three of its environmental relative C. yegresii were tested. The G. mellonella model was also applied to determine antifungal efficacy of amphotericin B, itraconazole, voriconazole, posaconazole, and terbinafine.ResultsAll strains were able to infect the larvae, but virulence potentials were strain-specific and showed no correlation with clinical background of the respective isolate. Survival of larvae also varied with infection dose, and with growth speed and melanization of the fungus. Posaconazole and voriconazole exhibited best activity against Cladophialophora, followed by itraconazole and terbinafine, while limited efficacy was seen for amphotericin B.ConclusionInfection behavior deviates significantly between strains. In vitro antifungal susceptibility of tested strains only partly explained the limited treatment efficacy in vivo.

First use of tissue exudate serology to identify Toxocara spp. infection in food animals

Toxocara canis and Toxocara cati are globally distributed, zoonotic roundworm parasites. Human infection can have serious clinical consequences including blindness and brain disorders. In addition to ingesting environmental eggs, humans can become infected by eating infective larvae in raw or undercooked meat products. To date, no studies have assessed the prevalence of Toxocara spp. larvae in meat from animals consumed as food in the UK or assessed tissue exudates for the presence of anti-Toxocara antibodies. This study aimed to assess the potential risk to consumers eating meat products from animals infected with Toxocara spp. Tissue samples were obtained from 155 different food producing animals in the south, southwest and east of England, UK. Tissue samples (n = 226), either muscle or liver, were processed by artificial digestion followed by microscopic sediment evaluation for Toxocara spp. larvae, and tissue exudate samples (n = 141) were tested for the presence of anti-Toxocara antibodies using a commercial ELISA kit. A logistic regression model was used to compare anti-Toxocara antibody prevalence by host species, tissue type and source. While no larvae were found by microscopic examination after tissue digestion, the overall prevalence of anti-Toxocara antibodies in tissue exudates was 27.7%. By species, 35.3% of cattle (n = 34), 15.0% of sheep (n = 60), 54.6% of goats (n = 11) and 61.1% of pigs (n = 18) had anti-Toxocara antibodies. Logistic regression analysis found pigs were more likely to be positive for anti-Toxocara antibodies (odds ration (OR) = 2.89, P = 0.0786) compared with the other species sampled but only at a 10% significance level. The high prevalence of anti-Toxocara antibodies in tissue exudates suggests that exposure of food animals to this parasite is common in England. Tissue exudate serology on meat products within the human food chain could be applied in support of food safety and to identify practices that increase risks of foodborne transmission of zoonotic toxocariasis.

Isolation of a Novel Pythium Species, P. thermoculicivorax, and Trichoderma sp. from Natural Enzootic Mosquito Larval Infections.

Only a handful of microbial mosquito larval pathogens have been described to date. Sampling several natural enzootic infections of mosquito larvae in southwestern Florida indicated the presence of microbial pathogens capable of extensive larval mortality. A microscopic analysis of one sample site revealed extensive apparent growth of a Pythium-like microbe on mosquito larvae, with the highest degree of infection observed in the siphon and head regions. Structures consistent with sporangia were seen on infected insects after lactophenol blue staining, and higher-resolution scanning electron microscopy (SEM) micrographs showed sporangia and encysted zoospores targeting the head and siphon regions. The isolate was single-colony purified, and molecular identification targeting the ITS and COX1 loci coupled to phylogenetic reconstruction indicated that the isolate belonged to the Pythium genus but was distinct from its closest characterized species, P. inflatum. Morphological features were characterized, with the isolate showing rapid growth on all mycological media tested and relatively high thermotolerance, capable of robust growth at 37 °C; hence, it was designated P. thermoculicivorax. Sampling from a second series of natural infections of mosquito larvae resulted in the molecular identification of three Trichoderma isolates, one with high similarity to T. strigosum and the other two clustering closely with T. asperellum. These data highlight the occurrence of natural enzootic infections of mosquito larvae, potentially as a resource for the identification of new mosquito pathogens.

50 Effects of cottonseed supplementation on the rumen microbiota of parasite-challenged meat goats

Abstract Cotton serves as a vital economic driver within the agricultural sector of Mississippi. The seeds of cotton are rich in fat and protein content, making them a cost-effective alternative to soybean meal and other expensive protein sources. Additionally, cottonseed is the primary source of gossypol, a phenolic compound with significant potential for natural parasite control. The rumen microbiota, a diverse community of microorganisms, has a pivotal role in nutrient digestion and detoxification. However, the impact of cottonseed on rumen microbial ecosystems remains largely unexplored. The aim of this study is to investigate the effects of cottonseed supplementation on the rumen microbiota of meat goats with and without the challenge of parasites. Forty female weaned Boer × Spanish crossed meat goats were assigned to one of the four treatments (n = 10 per treatment) in a 2 × 2 factorial design consisting of control group (CON), parasite challenge group (P), cottonseed supplementation (0.5% body weight) without parasite challenge group (CS-NP), and CS supplementation with parasite challenge group (CS-P). The parasite challenge was performed with a dose of 4,000 infective third-stage larvae of Haemonchus contortus per animal. After 25 d following the first dose, the treatments were applied for 10 wk. The goats were slaughtered at the end of the experiment, and rumen digesta samples were collected for microbiota analysis using high-resolution full-length 16S rRNA gene amplicon sequencing. The results revealed that the parasite challenge significantly reduced the number of bacterial species in the rumen (P = 0.032), regardless of cottonseed supplement. Anaeromassilibacillus senegalensis, which has been isolated from kwashiorkor (severe protein malnutrition) patient, Hungateiclostridium aldrichii, and Mogibacterium diversum were enriched in rumen of P group, while Anaerocella delicata and the rumen lipolytic organism Anaerovibrio lipolyticus were reduced compared with the CON group [linear discriminant analysis (LDA) scores > 3.6]. Cottonseed supplementation did not affect the number of bacterial species in the rumen of parasite-challenged goats, but it did lead to an increase of Anaerovibrio lipolyticus (P = 0.008) and a reduction of Anaeromassilibacillus senegalensis (P = 0.007). Moreover, cottonseed supplementation tended to increase the number of bacterial species in the rumen of meat goats without parasite challenge (P = 0.08) and increased the relative abundance of butyrate-producing bacteria such as Pseudobutyrivibrio xylanivorans and Intestinimonas butyriciproducens, along with the butanol-producing bacteria Clostridium saccharoperbutylacetonicum (LDA scores > 2). Conversely, cottonseed supplementation inhibited cellulolytic bacteria Fibrobacter succinogenes, polysaccharide-degrading bacteria Butyrivibrio proteoclasticus, and several Prevotella species, including P. oris, P. loescheii, P. copri, and P. albensis (LDA score > 2). In conclusion, parasite challenge affects the rumen microbial diversity and certain bacterial species that utilize protein and lipids, and cottonseed supplementation partially restores the altered rumen microbial ecosystem of the parasite-challenged meat goats.

74 Effect of grazing native warm season grasses on parasitism and growth in weaned Katahdin lambs

Abstract Parasitism in lambs during the post-weaning period has effects on growth, survival, and sheep enterprise profitability. Opportunities to minimize exposure to infective larvae during this period may reduce clinical effects and improve lamb performance. Native warm season grasses (NWSG) offer increased growth during summer months and taller plants that allow grazing at greater heights. The objective here was to evaluate NWSG as a forage for post-weaning development of lambs in spring lambing, forage-based systems. Over 2 yr, Katahdin lambs (Yr 1, n = 30; Yr 2, n = 31) were managed at a producer farm in the piedmont region of North Carolina. Beginning 2 wk post-weaning, lambs were randomly assigned to graze on either NWSG (Big Bluestem and Indiangrass) or cool season grass (CSG, Kentucky 31 Tall Fescue base) pasture. Lambs were continuously grazed for 70 d in Yr 1 and 55 d in Yr 2. Fecal egg count (FEC), FAMACHA score, and body weight (BW) were measured every two weeks. Lambs were dewormed at FAMACHA ≥ 3. Forage analysis was conducted at the start and end of the grazing period. Statistical analysis were performance using the PROC Mixed procedure of SAS. Lambs in each group were raised in similar litter sizes and sire and sex were evenly distributed between groups. Fecal egg count and growth estimated breeding values (EBV) were similar between NWSG and CSG groups. In Yr 1 and 2, no differences were observed in FEC between grazing groups (P > 0.05). However, FEC did increase in both groups throughout the grazing period (P < 0.05). In Yr 1, no differences were observed in total BW gain (P = 0.74). However, in Yr 2, lambs grazing CSG had greater total BW gain than the NWSG group (P < 0.05). In both years, there were no differences in the percentage of lambs requiring deworming (Yr 1, P = 0.36; Yr 2, P = 0.47) but lambs requiring treatment had greater FEC EBVs (P < 0.05). Nutrient quality of NWSG pasture was reduced compared with CSG pastures. Total digestible nutrients and crude protein were less in NWSG compared with CSG pastures (Yr 2: 61.5% vs. 63.9% and 8.9% vs. 13.6%, respectively) and neutral detergent fiber was greater (Yr 2: 74.1% vs. 65.2%, respectively) resulting in decreased nutrient availability for NWSG group. While NWSG may provide additional biomass for summer grazing, rapid maturity rates may limit nutrient availability for lambs resulting in decreased growth performance despite similar levels of parasitism. Lamb FEC EBV may have a role in parasite resistance and deworming requirements regardless of forage type. Thus, genetics must be considered along with grazing management as part of an integrated parasite management plan.

Impact of Saussurea lappa against foodborne parasite Trichinella spiralis experimental infections induced variation in DNA damage, oxidative stress and PCNA expression in rat skeletal muscles.

Trichinellosis is a parasite zoonosis that is spread through ingesting raw or undercooked meat that contains the Trichinella spiralis (T. spiralis) infective larvae. It has three clinical phases: intestinal, migratory, and muscular. Kuth root, also known as Costus (Saussurea lappa) roots, is used in many traditional medical systems all over the world to treat a variety of illnesses, such as dyspepsia, diarrhoea, vomiting, and inflammation. Current study assessed the therapeutic Potential of costus roots extract (CRE) treatment on experimental trichinellosis induce changes in DNA damage, oxidative stress and Proliferating cell nuclear antigen (PCNA) expression in muscle fibers in male rats. A total of 60 male Sprague Dawley rats were divided into 6 groups (Gps) [Gp1, Negative control; Gp2, Costus (CRE); Gp3, Positive control or Infected rats with T. spiralis, Gp4; Pre-treated infection with CRE; Gp5 & Gp6, Post treated infection with CRE for one and two weeks respectively]. Current results revealed that; Trichinella spiralis experimentally infection induced significant elevation in tissue malondialdehyde (MDA), DNA damage, PCNA expression and significant depletion in tissue glutathione (GSH), superoxide dismutase (SOD) and catalase (Cat) activities. Pre or/and post CRE treated infected rats with T. spiralis (Gp4-Gp6) induced improvements and depletion in DNA damage, PCNA expression, MDA and elevation in GSH, SOD, catalase as compared to infected rats with T. spiralis (Gp3) with best results for the pretreatments (Gp4). Trichinella spiralis experimental infection induced DNA damage and oxidative stress in rat skeletal muscles and treatments with costus roots extract modulates these changes.

Minor impact of probiotic bacteria and egg white on Tenebrio molitor growth, microbial composition, and pathogen infection.

The industrial rearing of the yellow mealworm (Tenebrio molitor) for feed and food purposes on agricultural by-products may expose larvae and adults to entomopathogens used as biocontrol agents in crop production. Bacterial spores/toxins or fungal conidia from species such as Bacillus thuringiensis or Metarhizium brunneum could affect the survival and growth of insects. Therefore, the aim of this study was to investigate the potential benefits of a wheat bran diet supplemented with probiotic bacteria and dried egg white on larval development and survival and its effects on the gut microbiome composition. Two probiotic bacterial species, Pediococcus pentosaceus KVL B19-01 and Lactiplantibacillus plantarum WJB, were added to wheat bran feed with and without dried egg white, as an additional protein source, directly from neonate larval hatching until reaching a body mass of 20 mg. Subsequently, larvae from the various diets were exposed for 72 h to B. thuringiensis, M. brunneum, or their combination. Larval survival and growth were recorded for 14 days, and the bacterial microbiota composition was analyzed using 16S rDNA sequencing prior to pathogen exposure and on days 3 and 11 after inoculation with the pathogens. The results showed increased survival for T. molitor larvae reared on feed supplemented with P. pentosaceus in the case of co-infection. Larval growth was also impacted in the co-infection treatment. No significant impact of egg white or of P. pentosaceus on larval growth was recorded, while the addition of Lb. plantarum resulted in a minor increase in individual mass gain compared with infected larvae without the latter probiotic. On day 14, B. thuringiensis was no longer detected and the overall bacterial community composition of the larvae was similar in all treatments. On the other hand, the relative operational taxonomic unit (OTU) abundance was dependent on day, diet, and probiotic. Interestingly, P. pentosaceus was present throughout the experiments, while Lb. plantarum was not found at a detectable level, although its transient presence slightly improved larval performance. Overall, this study confirms the potential benefits of some probiotics during the development of T. molitor while underlining the complexity of the relationship between the host and its microbiome.

Abstract 1430 MALDI Spatial Imaging to Characterize the Competition for Metabolic Resources During Parasitoid Wasp Infection of Fly Larvae

Abstract 1430 MALDI Spatial Imaging to Characterize the Competition for Metabolic Resources During Parasitoid Wasp Infection of Fly Larvae

Phytochemical analysis and anthelmintic activity of Combretum mucronatum leaf extract against infective larvae of soil-transmitted helminths including ruminant gastrointestinal nematodes

BackgroundSoil-transmitted helminths (STH) infect more than a quarter of the world’s human population. In the absence of vaccines for most animal and human gastrointestinal nematodes (GIN), treatment of infections primarily relies on anthelmintic drugs, while resistance is a growing threat. Therefore, there is a need to find alternatives to current anthelmintic drugs, especially those with novel modes of action. The present work aimed to study the composition and anthelmintic activity of Combretum mucronatum leaf extract (CMLE) by phytochemical analysis and larval migration inhibition assays, respectively.MethodsCombretum mucronatum leaves were defatted with petroleum ether and the residue was extracted by ethanol/water (1/1) followed by freeze-drying. The proanthocyanidins and flavonoids were characterized by thin layer chromatography (TLC) and ultra-high performance liquid chromatography (UPLC). To evaluate the inhibitory activity of this extract, larval migration assays with STH and GIN were performed. For this purpose, infective larvae of the helminths were, if necessary, exsheathed (Ancylostoma caninum, GIN) and incubated with different concentrations of CMLE.ResultsCMLE was found to be rich in flavonoids and proanthocyanidins; catechin and epicatechin were therefore quantified for standardization of the extract. Data indicate that CMLE had a significant effect on larval migration. The effect was dose-dependent and higher concentrations (1000 µg/mL) exerted significantly higher larvicidal effect (P < 0.001) compared with the negative control (1% dimethyl sulfoxide, DMSO) and lower concentrations (≤ 100 µg/ml). Infective larvae of Ascaris suum [half-maximal inhibitory concentration (IC50) = 5.5 µg/mL], Trichuris suis (IC50 = 7.4 µg/mL), and A. caninum (IC50 = 18.9 µg/mL) were more sensitive to CMLE than that of Toxocara canis (IC50 = 310.0 µg/mL), while infective larvae of Toxocara cati were largely unaffected (IC50 > 1000 µg/mL). Likewise, CMLE was active against most infective larvae of soil-transmitted ruminant GIN, except for Cooperia punctata. Trichostrongylus colubriformis was most sensitive to CMLE (IC50 = 2.1 µg/mL) followed by Cooperia oncophora (IC50 = 27.6 µg/mL), Ostertagia ostertagi (IC50 = 48.5 µg/mL), Trichostrongylus axei (IC50 = 54.7 µg/mL), Haemonchus contortus (IC50 = 145.6 µg/mL), and Cooperia curticei (IC50 = 156.6 µg/mL).ConclusionsThese results indicate that CMLE exhibits promising anthelmintic properties against infective larvae of a large variety of soil-transmitted nematodes.Graphical

Hygiene activity of bees in certain territories of Ukraine

The main results of the evaluation of the hygienic activity of bees are highlighted and it is assumed that pollution affects the behavioral patterns of bees and the general protective immune response of the body to the penetration of infection. After all, in Kharkiv, Sumy and Mykolaiv regions, an increased level of pollution was noted in connection with the approach to the zone of hostilities, in particular, the recording of flights to the territory of the apiary, shelling and the movement of heavy military equipment. We assumed that the weakening of the resistance of honey bees to diseases and the deterioration of the general immune response of the body may occur as a result of contamination of the natural environment, including feed, pollen and nectar, with various toxic substances. These substances can include heavy metals, radioactive elements, pesticides, herbicides, fungicides, antibiotics and other chemical compounds that negatively affect the health of bees and the quality of beekeeping products. It is shown that the best indicators of the activity of the hygienic behavior of bees were noted for the territory with the least anthropogenic load and the most distant from the zone of hostilities (Vinnytsia region). The hygienic behavior of bees is that they identify and remove infected or dead larvae from the hive, which can be a source of disease and parasites. This behavior is an important factor in the survival of bee colonies and maintaining productivity. Further studies include the selection of bees and beekeeping products (honey, honey) for laboratory studies to assess the possibility of their use in bioindication of environmental pollution. Bioindication is a method of measuring the quality of the environment using living organisms that respond to changes in environmental conditions. Bees are sensitive bioindicators because they forage over a large area and reflect the level of contamination in their products. Analyzing the research results, it can be concluded that in one group of bees, the indicators of the time of complete removal of dead brood for 2022–2023 had an uneven character. Fairly low indicators of hygienic activity over the two-year period were noted in the 2nd and 3rd groups of bees, as the average value of the time of complete removal of dead larvae was 22.7 and 23.5 hours, respectively. The lowest indicators of the time of removal of dead brood over the entire period of observation were noted in the 5th group of bees. However, the average indicators of this group are 6.5% lower than in the control group. When studying the hygienic behavior of bee colonies, we found that the efficiency of removing dead brood depends on the strength of the colony. A correlation between the values of hygienic activity of bees and family strength obtained in 2022 was found in the 2nd (R=0.95) and 3rd (R=0.78) groups. In 2023, a strong relationship between the indicators of the time of removal of 100% of larvae and the strength of the family was noted in the 5th (R=0.74), 1st groups (R=0.72). This indicates that stronger bee colonies have a greater ability to self-cleanse from dangerous factors that may threaten their health and productivity.

Equine enteroid-derived monolayers recapitulate key features of parasitic intestinal nematode infection

Stem cell-derived organoid cultures have emerged as attractive experimental models for infection biology research regarding various types of gastro-intestinal pathogens and host species. However, the large size of infectious nematode larvae and the closed structure of 3-dimensional organoids often hinder studies of the natural route of infection. To enable easy administration to the apical surface of the epithelium, organoids from the equine small intestine, i.e. enteroids, were used in the present study to establish epithelial monolayer cultures. These monolayers were functionally tested by stimulation with IL-4 and IL-13, and/or exposure to infectious stage larvae of the equine nematodes Parascaris univalens, cyathostominae and/or Strongylus vulgaris. Effects were recorded using transcriptional analysis combined with histochemistry, immunofluorescence-, live-cell- and scanning electron microscopy. These analyses revealed heterogeneous monolayers containing both immature and differentiated cells including tuft cells and mucus-producing goblet cells. Stimulation with IL-4/IL-13 increased tuft- and goblet cell differentiation as demonstrated by the expression of DCLK1 and MUC2. In these cytokine-primed monolayers, the expression of MUC2 was further promoted by co-culture with P. univalens. Moreover, live-cell imaging revealed morphological alterations of the epithelial cells following exposure to larvae even in the absence of cytokine stimulation. Thus, the present work describes the design, characterization and usability of an experimental model representing the equine nematode-infected small intestinal epithelium. The presence of tuft cells and goblet cells whose mucus production is affected by Th2 cytokines and/or the presence of larvae opens up for mechanistic studies of the physical interactions between nematodes and the equine intestinal mucosa.