ABSTRACT _Duddingtonia flagrans_ is a nematode-trapping fungus that is widely used to control parasitic nematodes in livestock. After oral ingestion and passage through the digestive tract

of animals, this microorganism captures nematodes in feces. Although many researchers have examined the safety of this fungus for humans, animals, and the environment, few reports have

discussed the safety of nematode-trapping _D. flagrans_ biologics for animals. In this study, _D. flagrans_ safety was tested, while adverse effects and toxicities were examined in sheep.

First, the nematode killing effects in naturally parasitized sheep after administration of lyophilized _D. flagrans_ preparations were tested.lyophilized _D. flagrans_ preparations were

effects of naturally parasitized sheep after administration were tested. The results demonstrated that treatment with _D. flagrans_ isolates significantly reduced developing larvae numbers

in feces, with an efficiency of 92.99%. Lyophilized preparations had no observable effects on physiological parameters in sheep, thus indicating a wide safety range in target animals, with

potentially minimal risks in veterinary clinical practice. Overall, _D. flagrans_ freeze-dried biologics effectively helped to controlled parasitic infections, which are safe in animals like

sheep, and thus may provide a practical platform for nematode-trapping fungi in veterinary clinical settings. SIMILAR CONTENT BEING VIEWED BY OTHERS ASSESSING THE EFFICACY OF THE OVICIDAL

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Article Open access 10 August 2021 INTRODUCTION Currently, to biologically control parasites in livestock, anthelmintic drugs are widely applied but the misuse of these drugs has led to

serious resistance issues; therefore, new control measures must be researched and developed1. One of the most important approaches is the exploitation of natural enemies, including predatory

fungi that prevent and control parasites2. Predatory fungi are widely distributed in soils and exist in many ecosystems, from tropical areas to cold Antarctica and from terrestrial to

aquatic ecosystems3. Based on their discovery, considerable research efforts have been directed toward predatory fungi, their predatory properties, biochemical mechanisms, and clinical

applications4. For example, the nematode-predatory fungus _Duddingtonia flagrans_generates large numbers of thick-walled spores, which can pass through animal digestive tracts without

inactivation in clinical use, and then germinate in feces5. With recognized predatory roles, _D. flagrans_ then targets a variety of parasitic nematodes, such as _Osertagia spp._,_

Nematodirus_,_ and Strongyloides stercoralis._ More recently, Mendes et al. demonstrated that a solution of _D. flagrans_conidia was effective in vitro against gastrointestinal nematodes in

buffaloes when used alone and/or in association with ivermectin6. In Voinot et al., the daily administration of nutritional pellets enriched with _M. circinelloides_ and _D. flagrans_

spores, for a prolonged interval (2 years), prevented helminth infection (_C. daubneyi_and gastrointestinal nematodes) in dairy cattle under rotational grazing7. Critically, predatory

activities increase with increased larvae numbers, thus the fungus has broad application values8,9,10,11,12. However, there is a dearth of information on predatory fungi safety profiles in

target animals13. In a recent study, edible gelatins were desiccated and tested on captive bison maintained in a zoological garden under continuous pasturing. Lyophilized _D.

flagrans_preparations had no effects on physiological parameters in bison14. Recently, Braga et al.15 demonstrated the efficacy and safety of Bioverm®, a commercial product based on _D.

flagrans_ chlamydospores (AC001), available in Brazil for the integrated control of helminth infections in farm animals. Despite the scientific efficiency and proven safety of _D.

flagrans_(AC001) by oral administration, no reports have yet assessed possible harmful effects during gastrointestinal transit. Long-term clinical and anatomical-pathological studies on

heifers fed parasitic fungal pellets on a daily basis were performed and showed no side-effects or specific lesions. Moreover, the risk of heifers being infected with trematodes was reduced

and their health was improved16. _Duddingtonia flagrans _has been positively developed as a biological agent in the United States, Sweden, and New Zealand for controlling parasitic

nematodes15. Biological control with the predacious fungi _D. flagrans _still remains a promising free-living parasite regulator alternative for use in livestock17. To control ruminant

parasitic nematodes, several studies examining _D. flagrans _as a biological control agent have shown excellent results18,19. The main advantages of _D. flagrans_ include fewer resistance

problems, a fully natural product, and environmentally friendly. To characterize potential primary and clinical applications, predatory _D. flagrans_safety profiles were examined in our

study in target animals, with a view to their becoming widely accepted lyophilized biological agents1. Our study lays the foundation for future clinical applications and commercial

production, and provides a reference for future in-depth predatory fungi studies. MATERIALS AND METHODS FUNGAL STRAIN A test strain of _D. flagrans_ strain CIM1 (NCBI bio-sample accession

number: SAMN05504105), was obtained from the Veterinary Parasite Laboratory of the Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China. _D. flagrans _was inoculated onto

Potato Dextrose Agar (PDA) (Beijing Landbridge, China) medium until the mycelium covered the dish surface16, then it was cut into 0.5 cm × 0.5 cm squares, added to a new dish, and incubated

for 1 week at 25 °C. Next, the mycelium was cut into 0.5 cm × 0.5 cm squares and transferred to corn kernel medium at 25 °C for 3 weeks. Spore eluate was prepared by adding 1 mL Tween-80 to

500 mL of water, and then heating it to 121℃ for 15 min. Spores were eluted several times from the medium using spore eluent and then filtered on an ultra-clean bench. Samples then underwent

vacuum freeze drying until a dry powder was obtained. We determined chlamydospore counts in the lyophilized formulation to be 2.13 × 108 chlamydospores/g. The Bottle were then sealed with

sealing film, and stored at 4℃. ANIMALS _D. FLAGRANS_ USE IN CONTROLLING HELMINTH PARASITES Experimental sheep (_n_ = 30) were naturally infected with gastrointestinal nematodes on farms in

Siziwang Banner, Ulanqab, Inner Mongolia, China, and had not been previously dewormed within 6 months of the trial. The Animal Experimentation Ethics Committee followed Chinese National

Standard Laboratory Animal-Guidelines for the ethical review of animal welfare (GB/T 35892 − 2018). All animal protocols were reviewed by the Animal Experiments Ethics Committee at Inner

Mongolia Agricultural University. ANIMAL SAFETY EXPERIMENTS The animal safety test site was located in the animal house of Inner Mongolia Agricultural University. 3–4 month old sheep were

selected (_n_ = 9). At 10 days before the trial, albendazole (99% purity; Kangmu Animal Pharmaceutical, China) was orally administered once. Ivermectin (99% purity; Kangmu Animal

Pharmaceutical, China). was also subcutaneously injected for 3 consecutive days according to body weight, to ensure the absence of helminth infections. METHODS IN VIVO EFFICACY OF _D.

FLAGRANS_ ON NEMATODE EGGS AND LARVAE IN SHEEP FECES Thirty Small Tailed Han Sheep, 6 ~ 8 months old, and approximately 50 kg were randomly selected. Rectal feces were collected and examined

for nematode infection. Ten sheep with a similar number of EPG infections were divided into groups, including ivermectin, _D. flagrans_ biologic, and blank control groups. The _D. flagrans_

group dose was 1 × 106chlamydospores/kg body weight (bw)20. Egg numbers per gram (EPG) and larvae per gram (LPG) of feces were measured and collected before and 1 week after the trial. Eggs

in feces were counted using a modified McMaster technique to calculate EPG. Fecal samples were incubated at a constant temperature of 25 °C for 15 days. After this period, third-stage

non-predatory larvae (L3) were isolated using the modified Baermann’s method21. Mean EPG and LPG values in groups were compared before and after dosing to determine _D. flagrans_

effectiveness in controlling parasites. IN VIVO SAFETY EXPERIMENTAL TESTS Nine sheep with an average body weight = 18 kg were randomly divided into 5-fold dose (5 × 106 chlamydospores/kg

bw), 10-fold dose (1 × 107 chlamydospores/ kg bw), and blank control groups. Each group consisted of three sheep, and corresponding doses were applied based on bw. Suspensions were

administered as a single dose on an empty stomach by perfusion with a syringe. CLINICAL OBSERVATIONS During trials, multiple key parameters were observed in sheep every day, including

behavioral disorders, food and drink appetite, jaundice, respiratory rate, cough, neurological signs, diarrhea, edema, and developmental status. Behavioral disorders referred to excitement,

depression, restlessness, and other abnormalities. Defecation represented constipation and diarrhea. During trials, sheep were fed high-quality alfalfa three times a day (morning, noon, and

night). BODY TEMPERATURE AND WEIGHT MEASUREMENTS Oral _D. flagrans_ biological effects on body mass and weight gain were examined by weighing sheep before the trial and at days3, 7, 15, and

30 after dosing. Measurements were taken between 9 am and 10 am. on an empty stomach. ROUTINE BLOOD TESTS Blood samples were collected for routine blood tests at 3 days before the trial and

then at days15 and 30 after dosing. The following biomarkers were measured using an automatic animal blood cell analyzer: WBC, Lym, Mon, Neu, Eo, Ba, RBC, MCV, Hct, MCHC, Hb, and MPV. ORGAN

HISTOMORPHOLOGY At study end, all test sheep were euthanized according to ethical regulations. Euthanasia was conducted by intravenously injecting animals with phenobarbital (90 mg/kg body

weight). This was formulated in accordance with Laboratory Animal-Guidelines for Euthanasia by the People’s Republic of China (GB/T39760-2021). The sheep dissected, and the abdominal cavity

cut open to check for fluids or blood in the cavity. All soft ribs on the left side were cut using bone cutters, and ribs on left and right sides were broken by hand to expose the entire

thoracic cavity to observe pleural colors and bleeding/adhesions. The following organs and tissues were checked by visual inspection: brain, cerebellum, kidney, heart, pancreas, liver,

spleen, lung, stomach, ileum, jejunum, colon, cecum, and lymph nodes. All gross pathological lesions were photographed and recorded. The heart, liver, spleen, lungs, and kidneys were then

taken fur further examinations. Excess connective tissue around organs was carefully peeled away and surface fluids blotted with filter paper before weighing and recording data. To calculate

organ coefficients, the following equation was used: organ weight (g)/body weight (g) × 100%. STATISTICAL ANALYSIS Experimental data were processed as the mean ± standard deviation, and

ANOVA was performed using IBM SPSS Statistics 22 software. Data were analyzed for normal distributions using Kolmogorov–Smirnov and Levene’ s tests. If variance was unevenly distributed,

equivalent non-parametric tests (primarily Kruskal–Wallis analysis of variance) were used. A _P_ < 0.05 value was considered significant. RESULTS NEMATODE PREDATION EFFICIENCY IN FECES

AFTER ORAL BIOLOGIC ADMINISTRATION Mean EPG and LPG values in group were compared before and after dosing to determine the parasite effects of biologic agents. After treatment, fungi

significantly reduced LPG development in feces. As shown (Table 1), results from ivermectin and _D. flagrans_ biologic groups were significantly different (_P_ < 0.05) when compared with

the control group, while the _D. flagrans_ group showed better effectiveness than the ivermectin group in terms of significant effects. CLINICAL OBSERVATIONS One sheep in the 10-fold dose

group had coughing symptoms, which was recovered within 48 h without treatment. Other sheep showed no abnormal changes in terms of respiration, spirit, appetite, or feces during the trial.

BODY TEMPERATURE AND WEIGHT MEASUREMENTS All body temperatures were within normal ranges except for one sheep, which was outside the range. However, no statistical differences were observed

between dose groups at 3, 7, 15 and 30 days when compared with the pre-dose period (Table 2). Body weights in all groups increased, however, weight gain differences in groups before the

trial and at days 7, 15, and 30 after the trial were not statistically significant (Table 3). PATHOLOGICAL CHANGES IN SHEEP No fluids were found in thoracic or abdominal cavities in any

sheep. Similarly, no abnormal contents, abnormal position, or organ shape were discovered; and no dislocation, adhesion, torsion, or rupture changes were recorded. The shape, color, size,

and texture of the heart, liver, spleen, lungs, kidneys, brain, and other organs were normal and were without bleeding, scar formation, nodules, or necrosis. Pancreas size, texture, and

color were normal. Intestinal duct plasma membrane surfaces were normal in color, without adhesion, parasitic nodules, tumors, or hemorrhages. All pathological sections were normal (Fig. 1).

ROUTINE BLOOD TEST RESULTS Routine blood biomarkers from the dose groups were within normal ranges before the trial and at days 15 and 30 after dosing. Differences between groups were not

significant (Table 4). BLOOD BIOCHEMISTRY RESULTS Blood biochemical parameters were within normal ranges for each dose group before the trial and at days 15 and 30 after dosing. Differences

between groups were not significant (Table 5). ORGAN COEFFICIENTS The heart, liver, spleen, lungs, and kidney organs were weighed at autopsy and organ coefficients calculated for all sheep.

There was no significant difference in organ coefficients between the experimental groups (Table 6). DISCUSSION In this work, _D. flagrans_ was shown to exert considerable predatory effect

on fecal larvae in in vivo experiments. In 2011, Paz-Silva et al. examined _D. flagrans_ predation efficiency on infective roundworm cyathostomin larvae and identified a 94% reduction in

larvae, while _D. flagrans_activity increased with egg and larvae numbers in co-culture11. Ferreira et al. also examined the same predation efficiency on L3 after passing through the

digestive tracts of rabbits pigs, and showed that L3 reductions in feces were 81.33%8. Wang showed a reduction of up to 100% in larvae numbers in feces when lyophilized _D. flagrans_biologic

preparations were used in combination with anthelmintic drugs22. The number of L3 fecal co-cultures decreased substantially in sheep grazing in The Netherlands after treatment with _D_.

_flagrans_spores, but no differences were observed between EPGs, but severe haemonchosis occurs also in treated group23. Similarly, in a field study on three farms in Switzerland,

co-cultured larval development was significantly inhibited but fecal EPGs were not significantly affected during _D. flagrans_feeding24. The fungal treatment group in this study

significantly reduced LPG development in feces, but EPG did not show an effect, similar to our results. When _D. flagrans_came into contact with larval worms, they adhered to and immobilized

them, allowing the fungus to penetrate cell walls and feed on the contents4. _D. flagrans_can also produce chlamydospores, which are a type of thick-walled resting spore25. This allows the

fungus to maintain high environmental tolerance and pass through gastrointestinal tracts of grazing livestock without reducing germination rates or predation efficiency. The literature has

reported that _D. flagrans_chlamydospores can withstand gastrointestinal transportation and other undesirable environments to germinate, forming a predator-based, three-dimensional network

structure that captures living larvae in feces15. Therefore, the continuous use of _D. flagrans_ reduces infective larvae in pastures and has great potential to control reinfection by

nematodes. These studies also suggested that _D. flagrans_ had good validity in in vitro predatory activity, consistent with our observations. While the effectiveness of _D. flagrans_

predation efficiency and its clinical application is undoubted, its safety profiles in target animals have been rarely reported. In 2020, at the request of the European Commission, Bampidis

et al. examined _D. flagrans_safety and predation efficiency (as a feed) and stated that it did not irritate the skin or eyes, but was sensitive to the respiratory tract26. No conclusions

were drawn on its skin-sensitizing potential. In our study, after oral 5- and 10-fold _D. flagrans_ administrative doses to sheep, we observed that preparations had no effects on key

clinical traits, such as respiration, mental status, appetite, and fecal excretion in groups. After extensive measurements, only one sheep had a body temperature of up to 40℃, while the

remainder were within normal ranges. The normal body temperature of a sheep is in the 38.3–39.9 °C range, and capture stress can cause a 0.5–1 °C increase in temperature during measurements.

One sheep had coughing symptoms at blood collection, which were fully recovered after 48 h without treatments. Moreover, heavy rain occurred in Hohhot the day before symptom onset and the

temperature dropped dramatically. Coughing symptoms were probably related to the cool weather, and no abnormalities were found after hematological analysis, which was not sufficient to

indicate that the preparation influenced the normal physiological indicators of the sheep. Necropsy techniques allowed for a more thorough visual inspection of lesions in different organs

and tissues in animals, which are vital when studying different drug effects on an organism. Autopsy results showed that no abnormal changes had occurred in organs and tissues and that

pathological sections were normal. Organ coefficient values are commonly used in toxicological studies and in our study, organ coefficient differences between groups were not significant

when compared with the control group. The safety profiles of lyophilized _D. flagrans_ in sheep have shown that this agent has the potential for use in clinical and commercial applications.

The following study limitations were encountered. First, _D. flagrans_strains have enzymatic activity and can produce serine proteases27 which help improve feed digestibility and utilization

in animals, and fungi have been found to be effective in degrading complex compounds in several studies28. But this needs to be further verified. Second, we did not assess whether _D.

flagrans_secondary metabolites were carried into animal products and exerted effects on consumers. Third, in clinical applications, researchers have hypothesized that nematode-predatory

fungi are not harmful to the environment and are safe. However, in recent years, these fungi have been reported to affect rotifer populations and density levels (rotifers improve wastewater

quality during biological treatment)29,30. Similarly, high numbers of nematode-predatory fungi can endanger rotifer populations and therefore it is vital that in-depth studies examining

nematode-predatory fungi effects on certain organisms in other environments are conducted. We observed that _D. flagrans_ had some nematode repellent effects and were better than control

drug effects. Our results provide a new strategy to control veterinary parasites in China, and also new reference data for studying plant-derived insecticidal active substances. DATA

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Google Scholar  Download references AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, People’s

Republic of China Yuan Ma, Luyao Hao, Zhengyi Li, Yanni Zhang, Qiannan Li & Rui Wang * Key Laboratory of Clinical Diagnosis and Treatment of Animal Diseases, Ministry of Agriculture,

National Animal Medicine Experimental Teaching Center, Beijing, People’s Republic of China Yuan Ma, Luyao Hao, Zhengyi Li, Yanni Zhang, Qiannan Li & Rui Wang * College of Pharmacy Heze

University, University Road 2269, Heze, 274015, People’s Republic of China Lili Jiang & Zhaobin Fan * Rui Pu Agricultural Technology Co., Ltd, Hohhot, Inner Mongolia, People’s Republic

of China Hongliang Luo Authors * Yuan Ma View author publications You can also search for this author inPubMed Google Scholar * Lili Jiang View author publications You can also search for

this author inPubMed Google Scholar * Zhaobin Fan View author publications You can also search for this author inPubMed Google Scholar * Luyao Hao View author publications You can also

search for this author inPubMed Google Scholar * Zhengyi Li View author publications You can also search for this author inPubMed Google Scholar * Yanni Zhang View author publications You

can also search for this author inPubMed Google Scholar * Qiannan Li View author publications You can also search for this author inPubMed Google Scholar * Rui Wang View author publications

You can also search for this author inPubMed Google Scholar * Hongliang Luo View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS Y.M., L.J. and

Z.F. conceived the study. Y.M., Z.L., Y.Z. and Q.L. performed the experiments. Y.M., Z.L., L.H., H.L. and R.W. analyzed the data and wrote the manuscript. All authors have read and approved

thefinal version of the manuscript. CORRESPONDING AUTHORS Correspondence to Rui Wang or Hongliang Luo. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests.

ETHICS APPROVAL The studies involving animals were reviewed and approved by Inner Mongolia Agricultural University of Ethics Committee. All animal protocols followed Animal Experimentation

Ethics Committee regulations of the Inner Mongolia Agricultural University. INFORMED CONSENT Informed consent was not required as the study did not involve human participants. ADDITIONAL

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effects and safety tests of _Duddingtonia flagrans_ biological preparation in sheep. _Sci Rep_ 15, 1843 (2025). https://doi.org/10.1038/s41598-025-85844-z Download citation * Received: 26

September 2024 * Accepted: 06 January 2025 * Published: 13 January 2025 * DOI: https://doi.org/10.1038/s41598-025-85844-z SHARE THIS ARTICLE Anyone you share the following link with will be

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initiative KEYWORDS * Lyophilized biologic * Safety profiles * Killing effects * Nematode-trapping fungi