Infective Larvae Research Articles

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.

Zophobas morio larvae as a novel model for the study of Acinetobacter virulence and antimicrobial resistance.

The use of mammalian models for in vivo testing of bacterial virulence raises ethical concerns and is expensive and time-consuming. As an alternative, non-mammalian models are sought. Galleria mellonella larvae have been used as a model to study several bacterial pathogens. However, their maintenance is challenging, and commercial supply is low. In this study, we aimed to establish the Zophobas morio larvae as an alternative non-mammalian model for the evaluation of the pathogenicity and antimicrobial susceptibility of Acinetobacter baumannii. We infected Z. morio with Acinetobacter strains and determined the optimal temperature and inoculum. To visualize the bacterial distribution within the larvae, hematoxylin and eosin (H&E) staining was performed. Next, a survival model of infected larvae was established, and virulence was compared between strains. The effect of antimicrobial treatment in relation to antibiotic susceptibility was studied. Our results demonstrate that Z. morio can be used as a model system for in vivo studies of A. baumannii.

Immune priming modulates Galleria mellonella and Pseudomonas entomophila interaction. Antimicrobial properties of Kazal peptide Pr13a.

Galleria mellonella larvae repeatedly infected with Pseudomonas entomophila bacteria re-induced their immune response. Its parameters, i.e. the defence activities of cell-free hemolymph, the presence and activity of antimicrobial peptides, and the expression of immune-relevant genes were modulated after the re-challenge in comparison to non-primed infected larvae, resulting in better protection. No enhanced resistance was observed when the larvae were initially infected with other microorganisms, and larvae pre-infected with P. entomophila were not more resistant to further infection with other pathogens. Then, the peptide profiles of hemolymph from primed- and non-primed larvae infected with P. entomophila were compared by quantitative RP-HPLC (Reverse Phase - High Performance Liquid Chromatography). The level of carbonic anhydrase, anionic peptide-1, proline peptide-2, and finally, unknown so far, putative Kazal peptide Pr13a was higher in the primed infected animals than in the larvae infected with P. entomophila for the first time. The expression of the Pr13a gene increased two-fold after the infection, but only in the primed animals. To check whether the enhanced level of Pr13a could have physiological significance, the peptide was purified to homogeneity and checked for its defence properties. In fact, it had antibacterial activity: at the concentration of 15 µM and 7.5 µM it reduced the number of P. entomophila and Bacillus thuringiensis CFU, respectively, to about 40%. The antibacterial activity of Pr13a was correlated with changes observed on the surface of the peptide-treated bacteria, e.g. surface roughness and adhesion force. The presented results bring us closer to finding hemolymph constituents responsible for the effect of priming on the immune response in re-infected insects.

Virulence Evaluation of Aeromonas spp. KS-1 Isolated from Kitchen Sponge using Omphisa fuscidentalis Larvae

Aeromonas spp. causes the human diseases including diarrhea, gastroenteritis, and bacteremia. Aeromonas spp. can be found in kitchen sponge, one of the reservoirs for food-borne bacterial pathogens. Virulence study of Aeromonas spp. in vivo in animal model is important since the animal model can mimic manifestasions in human infections. Omphisa fuscidentalis was chosen for alternative virulence model, since they are in the same taxonomical order with the well-known infections model, Galleria mellonella. Bacterial isolation and selection of kitchen sponge used Brain Heart Infusion agar and Endo Agar, respectively. Bacterial virulence of KS-1 was injected into Omphisa fuscidentalis larvae. Survival percentage and melanization score of infected larvae were evaluated. Hemolymph of larvae with melanization score of 1 and 4 were stained with Giemsa method to observe the hemocyte changes. Bacterial identification of isolate KS-1 based on 16S rRNA sequence resulted in 96.9% identity to Aeromonas spp. strain VS7. Isolate KS-1 injection to O. fuscidentalis revealed higher bacterial dosage resulting more severe symptoms to the larvae according to survival percentage and melanization score. However, statistical analysis showed evaluation of melanization score could distinguish larvae with 106 and 107 CFU/larva dosage injection, while evaluation of survival percentage could not. Hemocyte of larvae with melanization score 1 had larger and more cytoplasmic vacuolization than the score 4 (healthy larvae). Omphisa fuscidentalis is an alternative of insect model for bacterial infections with survival percentage and melanization score as the evaluation. Cytoplasmic vacuolization of hemocyte can be used as larvae’s health indicator in a cellular level.

Modified technique for recovery of nematode infective larvae in different grass genera

Most gastrointestinal nematodes are in the grazing environment. As an option, the study aimed to evaluate an alternative technique for recovery of infecting larvae in various grass genera: Tyfton 85 (Cynodon dactylon) Capim–Vencedor (Megathyrsus maximus) Capim–Gordura (Melinis minutiflora) Capim–Marandu (Brachiaria brizantha), Setaria (Setaria anceps) Capim-Humidicula (Brachiaria humidicula) and Andropogon (Andropogon gayanus). The collection of samples and procedures were developed at the Institute of Animal Science, Nova Odessa, Brazil. The grasses came from isolated paddocks. Three subsamples were used: 1-control sample (AC1); 2-control frozen sample (ACC2); 3-infested sample (AI). AC1, after the process of larvae recoveries, served as control, the rest was frozen. After de-freezing, it was divided into two samples (ACC2 and AI). In ACC2, no larvae were observed. AI was infested with 1600 ± 30 L3 larvae of trichostrongylids, recovered from sheep manure. The larvae recovery procedures were applied to the samples in the three periods. The overall mean of recovery (±SD) and percentage of success was estimated (SISVAR®) in 1544.63 ± 1.31 larvae, 96.54 ± 17% respectively. There were no differences between grass genera, as well as between weeks, with near recovery numbers (p&gt;0.05). The technique had satisfactory results in a short time.

A multidisciplinary study on Clinostomum infections in Nile tilapia: micro-morphology, oxidative stress, immunology, and histopathology

Yellow grub disease, caused by Clinostomum metacercaria, is an endemic zoonotic infection in freshwater fish, responsible for Halzoun syndrome transmitted through the consumption of raw infected fish. This study aimed to conduct a multidisciplinary investigation integrating detailed morphology, oxidative stress, immunology, and histopathology alteration to advance our understanding of Clinostomum infection. In this annual study, 400 Nile tilapia (Oreochromis niloticus) were collected from the Nile River at Al Bahr Al Aazam, Giza Governorate to assess Clinostomum infection prevalence. Of the examined fish, 160 individuals (40.0%) harboured larval Clinostomum infections. Clinostomum metacercariae were observed in various anatomical locations, with 135 fish (33.8%) in buccal cavities, 21 fish (5.25%) in gill chambers, and 4 fish (1.0%) on the skin. Infection intensity ranged from 2 to 12 cysts per fish, averaging 5 cysts, notably with skin infections characterized by a single cyst in each fish. Macroscopic encysted metacercariae were collected from buccal cavities, gills, and skin. Micro-morphology revealed distinct features in C. complanatum, including an elliptical oral sucker with collar-like rings and large sensory papilla-like structures, contrasting with the absence of these features in C. phalacrocoracis. Oxidative stress, assessed through malondialdehyde (MDA) and nitric oxide levels, revealed an elevation in MDA to 35.13 ± 6 nmol/g and nitric oxide to 25.80 ± 3.12 µmol/g in infected fish. In infected fish, MHC-I gene expression increased approximately 13-fold, MHC-II peaked at 19-fold, and IL-1β significantly upregulated by 17-fold, compared to control levels. Histopathology detailed associated lesions, such as cyst encapsulation and eosinophilic infiltration. Clinstomiasis and its impacts on fish hosts offer crucial insights to control this emerging fish-borne zoonotic disease, threatening wildlife, aquaculture, and human health.

Studies on the embryonic development and larval infection potential of the stomach bot flies, Gasterophilus pecorum

Endangered Przewalski's horses have faced severe infections from Gasterophilus pecorum (Diptera, Gastrophilidae) in Xinjiang's Kalamaili Nature Reserve (KNR). This study examines G. pecorum's development and infection patterns in embryonic and larval stages, crucial for understanding horse botfly disease in desert grasslands. For the incubation of G. pecorum fertilized eggs, we established the six distinct temperature gradients: 16 °C, 20 °C, 24 °C, 28 °C, 30 °C, and 32 °C. Using the least squares method, we calculated the correlation between the developmental threshold temperature of the eggs and their cumulative effective temperature. Furthermore, we meticulously recorded the survival duration of the larvae across a spectrum of temperature gradients (−20 °C, −10 °C, 4 °C, 10 °C, 20 °C, and 30 °C) and under varying conditions (dark and light). This method allows us to analyze and interpret the impact of these environmental factors on larval survival durations. 1) The formula for predicting the embryonic development period of G. pecorum was N = (182.7 ± 12.03)/[T-(3.191 ± 1.48)], where the developmental threshold temperature was 3.191 ± 1.48 °C, and the effective accumulated temperature was 182.7 ± 12.03 d°C 2) The model describing the relationship between the embryonic development rate and temperature was: y = 0.0001x2+0.0007x+0.0378, demonstrating a positive correlation between the development rate and temperature (R-sq = 0.989, p < 0.001). 3) Larvae in the dark group exhibited a longer survival time, with the longest being 9 months at 4 °C. The adaptation of G. pecorum's embryonic development to cold temperature, combined with the extended survival period of larvae in the egg state, significantly increases the infection potential of G. pecorum in colder climates. This discovery offers essential insights into the predominance of G. pecorum in the KNR region and provides a crucial biological basis for the prevention of myiasis and the conservation of vulnerable species, such as Przewalski's horses.

Repurposing Mitomycin C in Combination with Pentamidine or Gentamicin to Treat Infections with Multi-Drug-Resistant (MDR) Pseudomonas aeruginosa.

The aims of this study were (i) to determine if the combination of mitomycin C with pentamidine or existing antibiotics resulted in enhanced efficacy versus infections with MDR P. aeruginosa in vivo; and (ii) to determine if the doses of mitomycin C and pentamidine in combination can be reduced to levels that are non-toxic in humans but still retain antibacterial activity. Resistant clinical isolates of P. aeruginosa, a mutant strain over-expressing the MexAB-OprM resistance nodulation division (RND) efflux pump and a strain with three RND pumps deleted, were used. MIC assays indicated that all strains were sensitive to mitomycin C, but deletion of three RND pumps resulted in hypersensitivity and over-expression of MexAB-OprM caused some resistance. These results imply that mitomycin C is a substrate of the RND efflux pumps. Mitomycin C monotherapy successfully treated infected Galleria mellonella larvae, albeit at doses too high for human administration. Checkerboard and time-kill assays showed that the combination of mitomycin C with pentamidine, or the antibiotic gentamicin, resulted in synergistic inhibition of most P. aeruginosa strains in vitro. In vivo, administration of a combination therapy of mitomycin C with pentamidine, or gentamicin, to G. mellonella larvae infected with P. aeruginosa resulted in enhanced efficacy compared with monotherapies for the majority of MDR clinical isolates. Notably, the therapeutic benefit conferred by the combination therapy occurred with doses of mitomycin C close to those used in human medicine. Thus, repurposing mitomycin C in combination therapies to target MDR P. aeruginosa infections merits further investigation.

Host gastric corpus microenvironment facilitates Ascaris suum larval hatching and infection in a murine model.

Ascariasis (roundworm) is the most common parasitic helminth infection globally and can lead to significant morbidity in children including chronic lung disease. Children become infected with Ascaris spp. via oral ingestion of eggs. It has long been assumed that Ascaris egg hatching and larval translocation across the gastrointestinal mucosa to initiate infection occurs in the small intestine. Here, we show that A. suum larvae hatched in the host stomach in a murine model. Larvae utilize acidic mammalian chitinase (AMCase; acid chitinase; Chia) from chief cells and acid pumped by parietal cells to emerge from eggs on the surface of gastric epithelium. Furthermore, antagonizing AMCase and gastric acid in the stomach decreases parasitic burden in the liver and lungs and attenuates lung disease. Given Ascaris eggs are chitin-coated, the gastric corpus would logically be the most likely organ for egg hatching, though this is the first study directly evincing the essential role of the host gastric corpus microenvironment. These findings point towards potential novel mechanisms for therapeutic targets to prevent ascariasis and identify a new biomedical significance of AMCase in mammals.

Targeting the Antifungal Activity of Carbon Dots against Candida albicans Biofilm Formation by Tailoring Their Surface Functional Groups.

Carbon dots (CDs) are an emerging class of carbon nanoparticles, which for their characteristics have found applications in many fields such as catalysis, materials and biomedicine. Within this context, the application of CDs as antibacterial agents has received much attention in very recent years, while their use as antifungal nanoparticles has been scarcely investigated. Here we report a systematic investigation of the surface functional groups of CDs to study their influence on these nanoparticles' against Candida albicans. Three classes of CDs have been synthesised and fully characterized. A thorough in vitro and in vivo biological screening against C. albicans was performed to test their antifungal, antiadhesion and antibiofilm formation activities. Moreover, the interaction with C. albicans cells was investigated by microscopic analysis. Our results evidence how the presence of a positively polarised surface results crucial for the internalization into COS-7 cells. Positively charged nanoparticles were also able to inhibit adhesion and biofilm formation, to interact with the cellular membrane of C. albicans, and to increase the survival of G. mellonella infected larvae after the injection with positive nanoparticles. The antifungal activity of CDs and their extremely low toxicity may represent a new strategy to combat infections sustained by C.albicans.

Phospholipase A2 activity is required for immune defense of European (Apis mellifera) and Asian (Apis cerana) honeybees against American foulbrood pathogen, Paenibacillus larvae.

Honeybees require an efficient immune system to defend against microbial pathogens. The American foulbrood pathogen, Paenibacillus larvae, is lethal to honeybees and one of the main causes of colony collapse. This study investigated the immune responses of Apis mellifera and Apis cerana honeybees against the bacterial pathogen P. larvae. Both species of honeybee larvae exhibited significant mortality even at 102 103 cfu/mL of P. larvae by diet-feeding, although A. mellifera appeared to be more tolerant to the bacterial pathogen than A. cerana. Upon bacterial infection, the two honeybee species expressed both cellular and humoral immune responses. Hemocytes of both species exhibited characteristic spreading behaviors, accompanied by cytoskeletal extension along with F-actin growth, and formed nodules. Larvae of both species also expressed an antimicrobial peptide called apolipophorin III (ApoLpIII) in response to bacterial infection. However, these immune responses were significantly suppressed by a specific inhibitor to phospholipase A2 (PLA2). Each honeybee genome encodes four PLA2 genes (PLA2A ~ PLA2D), representing four orthologous combinations between the two species. In response to P. larvae infection, both species significantly up-regulated PLA2 enzyme activities and the expression of all four PLA2 genes. To determine the roles of the four PLA2s in the immune responses, RNA interference (RNAi) was performed by injecting gene-specific double stranded RNAs (dsRNAs). All four RNAi treatments significantly suppressed the immune responses, and specific inhibition of the two secretory PLA2s (PLA2A and PLA2B) potently suppressed nodule formation and ApoLpIII expression. These results demonstrate the cellular and humoral immune responses of A. mellifera and A. cerana against P. larvae. This study suggests that eicosanoids play a crucial role in mediating common immune responses in two closely related honeybees.