Entamoeba spp. are anaerobic protists primarily inhabiting the digestive tracts of vertebrates, some of which are pathogenic or zoonotic. However, genetic information on Entamoeba spp. in rodents, including rats, remains limited. This study investigated the prevalence and molecular characterization of Entamoeba in pet fancy rats in China. Fecal samples were collected from a total of 152 fancy rats from four pet shops in two provinces of China. Microscopy and PCR targeting the small subunit ribosomal RNA (SSU rRNA) gene were used for detection. Lineage differentiation was evaluated using compensatory base change (CBC) analysis of the SSU rRNA secondary structure, with base-pair assignments anchored to the published Entamoeba muris AB445018 secondary-structure model. Phylogenetic analysis was applied to infer the evolutionary relationships of the identified lineages. The overall prevalence of Entamoeba was 8.6% (13/152). Three distinct conditional lineages (CLs) were identified, designated Entamoeba CL-Rat1 (n = 4), CL-Rat2 (n = 5), and CL-Rat3 (n = 4). At least one manually verified CBC was detected in each pairwise comparison among the three lineages and between each lineage and the included reference taxa, supporting their recognition as independent evolutionary lineages. Notably, six CBCs differentiated CL-Rat1 from its closest relative RL11, two CBCs separated CL-Rat3 from E. muris AB445018, and CL-Rat2 was separated from all other octonucleated reference taxa by one or more CBCs. Phylogenetic analyses further resolved their evolutionary positions: CL-Rat1 clustered closely with Entamoeba RL11 from a field vole, with 18.4-18.7% sequence divergence; CL-Rat2 represented a deeply divergent octonucleated lineage, showing >27.3% sequence divergence from all other octonucleated taxa; CL-Rat3 was placed as sister to the E. muris reference sequence AB445018, with 11.9-12.3% sequence divergence. The three lineages showed similar cyst morphology, although CL-Rat1 cysts were slightly larger and possessed a near-central karyosome. No zoonotic Entamoeba histolytica/Entamoeba dispar DNA was detected. This is the first report on molecular characterization of octonucleated Entamoeba in pet fancy rats in China. These results reveal considerable cryptic genetic diversity among rodent Entamoeba and support the evolutionary distinctness of the three identified lineages.

Balantioides coli is a significant zoonotic parasitic protozoan, with pigs serving as its reservoir host. The limited molecular typing tools for B. coli have hindered understanding of its transmission and genetic variability in intensive pig farming. In this study, 1251 pig fecal samples were collected in two batches from three intensive farms. A dual-locus molecular typing method based on the β-tubulin and ITS genes was developed and applied to analyze the epidemiological characteristics and genetic variability of B. coli. The results revealed an overall infection rate of 87.7% (1097/1251), with an age-dependent pattern where lower rates were observed in 1-2-month-old piglets, followed by a rapid increase at 3-4 months, and near 100% prevalence by 5-6 months. Phylogenetic analysis based on the β-tubulin gene delineated three haplotypes (I, II, and III), confirming the previously identified ITS sequence variants A and B, and further resolving the novel sequence variant C. The expression of haplotypes and sequence variants showed a marked geographical distribution pattern, with haplotype I and variant A predominantly concentrated in the Zhejiang region, while haplotype III and variant C were detected in pigs and exclusively found in this area. These findings indicate that B. coli transmission is highly prevalent under intensive, high-density rearing conditions, and that the distribution of certain sequence variants shows geographical clustering, which may reflect local ecological or husbandry factors. This study provides important dual-locus genotyping insights for elucidating the transmission mechanisms of B. coli within intensive farming systems.

Entamoeba spp. are anaerobic protists primarily inhabiting vertebrate digestive tracts, some of which are pathogenic or zoonotic. However, genetic information on Entamoeba spp. in rodents, including gerbils, is scarce. This study aimed to investigate the prevalence and molecular characterization of Entamoeba in two pet gerbil species in China. A total of 120 fecal samples were collected from pet Mongolian gerbils and fat-tailed gerbils (60 each) purchased from six pet shops in three provinces. Microscopy, PCR, and sequence analysis of small subunit ribosomal RNA (SSU rRNA) gene were used for detection and identification. The overall Entamoeba infection rate was 10.0

Climate change is driving more frequent extreme rainfall events, which in turn trigger nutrient pulse events. These events can degrade water quality and reduce biodiversity in receiving water bodies. While submerged macrophytes are critical for stabilising aquatic ecosystems during such nutrient pulses, differences in macrophyte composition and coverage may affect their functional effectiveness. This study simulated nutrient pulse events by conducting in situ mesocosm experiments to investigate the effects of different macrophytes composition (single-species community [Vallisneria natans] vs. multi-species community [V. natans + Hydrilla verticillata + Potamogeton wrightii]) and coverage levels (0%, 30%, 40%, 50% and 60%) on system buffering capacity and stability. Additionally, we identified the coverage thresholds required for stabilising aquatic ecosystems under pulse loading conditions. Higher submerged macrophyte coverage significantly reduced TN, TP, Chl-a concentrations and water turbidity, with a critical threshold of 50% macrophyte coverage for optimal nutrient pulse mitigation. Compared with mixed communities, monocultures of V. natans showed better nutrient buffering capacity, with TN and TP removal efficiencies increased by 1.93 and 1.49 times at 50% coverage. Selecting appropriate submerged macrophyte coverage and suitable community compositions is crucial for improving ecological restoration efforts, especially in reducing nutrient disturbances and maintaining long-term aquatic ecosystem stability.

1. An understanding of how biodiversity confers ecosystem stability is crucial in managing ecosystems under major environmental changes. Multiple biodiversity drivers can stabilize ecosystem functions over time. However, we know little about how local environmental conditions can influence these biodiversity drivers, and consequently how they indirectly shape the ecological stability of ecosystems. 2. We hypothesized that environmental factors can have opposite influences (i.e., not necessarily either positive or negative) on the temporal stability of communities in different environmental ranges depending on the biodiversity drivers involved. We tested this novel hypothesis by using data from a 4-year-long field study of submerged macrophyte across a water depth gradient in 8 heterogeneous bays of Erhai lake (with total sample size of 30,071 quadrats), a large lentic system in China. 3. Results indicate that a unimodal pattern of stability in temporal biomass measurements occurred along the water-depth gradient, and that multiple biodiversity drivers (the asynchrony in species dynamics, and the stability of dominant species) generally increased the temporal stability of aquatic primary producers. However, the effect of water depth either increased or decreased the stability of biomass according to the environmental conditions associated with sites along the water depth gradient. 4. Synthesis. These results reveal the influence of local environmental conditions on the biodiversity drivers of stability may help predict the functional consequences of biodiversity change across different scenarios of environmental change.

Understanding how biodiversity and community functional traits preserve lake ecosystem multidimensional stability under global environmental changes is crucial for sustaining the vital ecosystem services we depend on. Based on sediment nutrient gradient experiments and three‐year seasonal monitoring of macrophyte communities in Erhai Lake, southwest China, spanning pre‐ and post‐algal bloom periods, we explored how species diversity and a key community functional trait (stoichiometric homeostasis) affect multiple dimensions (temporal stability, resistance, resilience and recovery) and facets (function, composition, diversity and functional trait) of stability of macrophyte communities following algal blooms. Generally, we found that species diversity and stoichiometric homeostasis of phosphorus ( H P ) had positive relationships with functional and compositional temporal stability, resistance and recovery, indicating that ecosystems with high species diversity and community H P are more resistant and stable in response to external algal bloom disturbances. However, species diversity and community H P had no positive or even negative relationships with resilience, suggesting that high biodiversity with high‐ H P species‐dominated ecosystems is not beneficial for the rapid recovery from disturbances, probably due to the slow growth and reproduction rate of high‐ H P species. In addition, we found strong positive correlations between functional and compositional stability across the four dimensions of stability, while the stability of species diversity and the key functional trait ( H P ) exhibited complex relationships, implying the difficulty of optimizing multiple dimensions and facets of stability simultaneously. Synthesis . Our work demonstrated that macrophyte species diversity and community H P are critical in determining the multiple dimensions and facets of stability in response to disturbances, which provides new insights for predicting the responses of macrophyte‐dominated lake ecosystems to the current increasing frequency of algal blooms.

Malignant tumors are prevalent with high mortality rates in humans, dogs, and cats. Some microorganisms have been shown to inhibit cancer progression. The objective of this study is to evaluate the inhibitory effects of Neospora caninum, a livestock parasite, on three different tumor models in C57BL/6 mice, including Lewis subcutaneous tumors, Lewis and B16F10 melanoma lung metastasis. The results showed that a sufficient amount of N. caninum tachyzoites can significantly inhibit the development of subcutaneous tumors and lung metastasis (P < 0.001), and induce more than 50

Climate change intensifies nutrient pulses through extreme rainfall and agricultural runoff, yet the buffering capacity of submerged macrophytes against such disturbances remains unquantified. Through a large-scale enclosure experiment simulating ammonium pulses (1.24 mg/L NH4-N), we tested how submerged macrophytes coverage (SMC, 0-100%) modulates water quality, ecosystem resilience, and regime shifts (from clear to turbid). The system's buffering capacity and resilience stability increased significantly with SMC, whereas its recovery stability decreased. High SMC (>50%) accelerated NH4-N removal (96 h vs 168 h in controls), suppressed phytoplankton blooms (Chl-a increase: 102.5% vs 237.4%), and sustained clear water. Conversely, low and medium SMC (<50%) did not prevent transitions to algal-dominated states. Furthermore, NH4-N stress was inversely correlated with SMC, and persistently high NH4-N at low SMC increased macrophyte degradation risk. Structural equation modeling revealed that macrophytes-mediated nutrient competition and light stabilization underpinned these effects. Additionally, we identify a critical SMC threshold (39-51%) to mitigate pulse impacts─a finding urgently needed to guide lake restoration in a changing climate. This work bridges the gap between pulse ecology and adaptive management, offering actionable strategies for SDG 6 (Clean Water) and 13 (Climate Action).

Rebuilding a clear-water state dominated by submerged macrophytes is essential for addressing eutrophication, yet the impact of benthic fish on water quality is complex. We conducted two experiments to explore the interaction of submerged plants and benthic fish on the water quality. Experiment I investigated the water clearing effects of submerged macrophytes with varying coverage (from 0% to 40%) before and after the removal of benthic fish. Experiment II explored the impacts of benthic fish at different densities on aquatic ecosystems with and without submerged macrophytes. The results showed that an increase in submerged macrophytes coverage significantly enhanced the reduction of some major water quality parameters. We assert that the coverage of submerged macrophytes should not be lower than 40% to establish and sustain a clear-water state in shallow lakes. However, benthic fish significantly weaken the ability of submerged macrophytes to improve water quality. Surprisingly, the presence or absence of macrophytes may reverse the role of benthic fish in freshwater ecosystems. When macrophytes are present, benthic fish can cause water quality to deteriorate. Conversely, when macrophytes are absent, benthic fish with a density of ≤ 10 g/m3 can restrict the growth of phytoplankton by directly consuming algae or by disturbing sediments to increase turbidity, thereby potentially improving water quality. But the detrimental effects of benthic fish with higher densities may gradually outweigh their benefits to water clarity. Therefore, the percentage of submerged macrophyte cover in combination with the density of benthic fish play crucial roles in shaping the ecological effects of benthic fish and overall ecosystem dynamics. These findings underscore the importance of understanding ecosystem interactions and have practical implications for the management of shallow lakes.

Macrophytes with different growth forms exhibit diverse functional traits and ecological functions. In natural sub-deep lakes, there are often large differences in water quality between nearshore areas with macrophytes and open water areas. However, it remains unclear whether this phenomenon can be attributed to differences in plant growth forms. Therefore, we conducted continuous monitoring for four years, both before and after the implementation of an ecological restoration project, to explore whether the change in plant growth forms caused differences in water quality between the nearshore and open water areas. The results showed that implementing ecological restoration projects proved highly effective in improving the local environment, including water physicochemical properties and biological components, in the implementation area. First, the ecological restoration project greatly altered the plant community structure in the nearshore area before and after restoration. After restoration, there was a significant increase in the biomass and distribution area of noncanopy-forming plants (including erect and rosette-forming plants), while the opposite effect was observed for canopy-forming plants. Second, the transition of macrophyte community growth forms enhanced the stability of both macrophyte communities and water physicochemical parameters. Furthermore, the reduction in canopy-forming plants facilitated a more efficient water body exchange, resulting in greater homogeneity in water quality between the nearshore and open water areas. Overall, the presence of canopy-forming plants can hinder water body exchange due to large canopy formations on the water surface. In light of these findings, it is recommended that ecological restoration projects in natural lakes should consider the functional group composition of macrophytes.

Enterocytozoon bieneusi, the most common microsporidian species, has been detected in humans and a variety of animals worldwide. However, limited information is available on the prevalence and molecular characterization of this parasite in guinea pigs. In this study, we conducted the first investigation of E. bieneusi infection in hairless guinea pigs recently introduced into China as new exotic pets. A total of 324 fecal samples were collected from hairless guinea pigs from a pet market and four breeding facilities in China. Sequence alignment of the internal transcribed spacer (ITS) revealed an infection rate of 14.2% (46/324) and two known ITS genotypes, S7 and PGP. Genotype S7 was the dominant genotype in these animals (42/46, 91.3%). Due to significant ITS sequence divergence, four and two PGP isolates from hairless and regular guinea pigs, respectively were further identified by PCR and phylogenetic analysis based on the small subunit (SSU) rRNA gene, as well as phylogenetic analysis of the ITS locus using E. hepatopenaei and two related genera Enterospora and Nucleospora as the outgroup. Three out of the six PGP isolates were successfully sequenced and generated the same sequences. Phylogenetic analysis of SSU rRNA and ITS loci revealed that PGP isolates formed a separate clade that was distinct and far away from E. bieneusi, suggesting that they represent a new species of Enterocytozoon. These findings indicate the dominance of zoonotic E. bieneusi genotype S7 in hairless guinea pigs and the existence of a cryptic Enterocytozoon species in guinea pigs.

Submerged macrophytes can improve water quality and buffer the effects of external nutrient loading, which helps to maintain a clear-water state in shallow lakes. We constructed 12 large enclosures with contrasting coverages (treatments) of submerged macrophytes (SMC) to elucidate their buffering capacity and resilience to nutrient pulses. We found that aquatic ecosystems with high SMC had higher buffering capacity and resilience, vice versa, i. e, the enclosures with high SMC quickly buffered the nutrient pulse and rebounded to clear-water state after a short stay in turbid-water state dominated by algae, while the treatments with low SMC could not fully buffer the pulse and rebound to clear-water state, and they slowly entered the transitional state after staying in turbid-water state. This means that the enclosures with high SMC had a better water quality than those with low SMC, i.e., the levels of nutrients and Chl-a were lower in the treatments with high plant coverage. In addition, plant coverage had a significantly positive buffering effect against nitrogen and phosphorus pulses, i.e., the nutrient concentrations in the treatments with high SMC took shorter time to return to the pre-pulse level. Overall, our results evidenced that the higher that the SMCs is, the better is the water quality and buffering capacity against nutrient pulses, i.e. the more stable is the clear-water state. However, low SMC may not be able to resist the impact of such strong nutrient pulse. Our results provide reference and guidance for water pollution control and water ecological restoration.

Introduction Macrophytes are essential for maintaining the health of shallow lake ecosystems, however, the driving and responsive relationship between ecological factors (such as seasonal changes and nutrition, etc.) and plant communities is not yet clear. Methods In this study, we conducted seasonal surveys of macrophyte community composition in lakes with different nutrient states, aiming to understand the incidence relation between macrophyte community diversity, seasonal changes and environmental factors. Results According to the classification criteria of comprehensive nutritional index, there were significant differences in the trophic status of the three lakes. Among them, the Xihu Lake has reached mild eutrophication with a TLI value of 56.33, both Cibi Lake and Haixihai Lake are mesotrophic with TLI value of 36.03 and 33.48, respectively. The results of diversity analysis showed a significant negative correlation between α-diversity (include Species richness, Shannon-Wiener index, Simpson index and Pielou index) and lake nutrient status. Among them, Xihu Lake showed the lowest α-diversity in all seasons, Haixihai Lake exhibited the middle α-diversity, Cibi Lake indicated the highest α-diversity. Non-metric multidimensional ordination showed that there were obvious spatial structures differences among the macrophyte communities in the three lakes. Macrophyte community composition in the three lakes was more similar in summer and autumn, but there was a wider gap in spring and winter. The redundancy analysis indicated distinct differences between diversity index and ecological factors, the eigenvalues of Axis 1 and Axis 2 being, respectively, 36.13% and 8.15%. Environmental factors could explain 44.8% of the total variation in macrophyte communities structure. Among these, nitrogen, phosphorus, water transparency and water temperature contributed 50.2%, 3.5%, 3.8% and 27.5%, respectively. Conclusions In summary, the community structure of macrophytes in plateau shallow lakes is co-regulated by seasons and nutrients.

To understand the infection situation of coccidia, a total of 384 fecal samples from an intensive sheep farm in Luoning county, Henan province were collected and examined by using saturated saline floating method and the McMaster’s method to detect the oocysts of Eimeria.The results showed that the total infection rate was 69.5%（267/384）,while the infection rates of suckling lambs, weaned lambs, lactating ewes and pregnant ewes were 52.6%（61/116）,94.5%（104/110）,65.8%（52/79） and 63.3%（50/79）,respectively.Oocysts of eleven species of Eimeria were identified in the sheep fecal samples as the following with individual infection rates: E.parva,27.1%; E.ovinoidalis,40.6%; E.pallida,25.5%;E.faurei,16.7%; E.crandallis,33.1%; E.bakuensis,27.9%; E.ahsata,18.5%;E.weybridgensis,16.4%;E.intricata,1.8%;E.marsica,3.6%;E.granulosa,6.8%.Most of sheep were co-infected by 2-5 specices of Eimeria with a mixed infection rate of 77.9%（208/267）.E.ovinoidalis and E.crandallis are the dominant species.In positive samples, the oocysts per gram feces（OPG） of suckling and weaned lambs were significantly higher than that of lactating and pregnant ewes（P＜0.01）.These findings showed that Eimeria infections are common in intensive sheep farms, and great attention should be paid to the control of coccidiosis in both suckling and weaned lambs.

Global climate changes are affecting organisms and their interactions in terrestrial and aquatic ecosystems, such as the increase in temperature and CO2 concentration. Herbivory interaction is a very important part of nutrient cycle and energy flow in freshwater ecosystem, and climate changes may directly or indirectly affect aquatic plants, aquatic herbivores and their interactions. In this study, we explored the effects of the rising temperature, elevated CO2 concentrations and herbivory by an herbivorous snail (Radix auricularia L.) on a submerged plant (Vallisneria natans L.). Our results showed that herbivory, temperature, and CO2 had specific effects on snail and plant growth, statistically there was only one interaction-a reduction in leaf number. Under different experimental conditions, snail herbivory always has negative effects on biomass accumulation and growth of V. natans. Moreover, the increases in temperature also inhibited its growth. Snail herbivory reduced the content of total carbon and total nitrogen of V. natans in all treatments, while the total phenols content increased. Our findings indicate that the rising temperature, elevated CO2 concentrations and herbivory have interactive effects on the growth and stoichiometry of submerged macrophytes, but further research is needed between aquatic plants and aquatic herbivores to aid prediction the impact of climate change on freshwater ecosystems.

Aquatic plants in lakeshore zone play an important role in maintaining the health of lake ecosystem.In order to understand the current status of aquatic plants in the lakeshore zone of Lake Erhai,this study investigated the aquatic plants quarterly in the lakeshore zone from 2020 to 2021.The results showed that there were 206 species of aquatic plants belonging to 56 families and 156 genera in the lakeshore zone of Lake Erhai,including 149 species of hygrophytes,24 species of emergent plants,21 species of submerged plants,7 species of floating plants and 5 species of floating leaf plants.Among all these species,Cynodon dactylon,Zizania latifolia,Vallisneria natans and Trapa bispinosa,etc.were common species,Lonicera japonica,Elymus dahuricus,etc.were occasional species.From the distribution of floristic regions,the species in the lakeshore zone of Lake Erhai were mainly world distribution（83 species） and tropical distribution（55 species）,accounting for 40.28% and 26.71% of the total species,respectively.In terms of plant communities,there were 18 main plant community types in the lakeshore zone of Lake Erhai,including 4 types of hyophytes communities,3 types of emergent plant communities,9 types of submerged plant communities and 2 types of floating leaf plant communities.Among all the communities,the main dominant communities were Cynodon dactylon communities,Zizania latifolia communities,Vallisneria natans communities and Trapa bispinosa communities.Compared with previous investigations,it was concluded that the diversity of aquatic plants in the lakeshore zone of Lake Erhai had been significantly improved in recent years.However,there are some problems such as the small area of lakeshore zone and the single of emergent plant community.Therefore,the construction of lakeshore zone should be further strengthened to improve the habitats and increase species diversity.

随着养鸟文化的逐渐普及,越来越多的宠物鸟出现在人们的生活中.而鸟类可感染多种肠道寄生虫,影响鸟类健康,引起鸟类发病甚至死亡.另外,某些肠道寄生虫为人兽共患病原,可以通过直接接触或污染的食物或水传播给人,从而威胁人类健康.国内有关宠物鸟类肠道寄生虫感染情况的报道有限.齐萌等曾于2008年对郑州市宠物鸟的肠道寄生虫感染情况做过调查;近年来也有关于宠物鸟贾第虫和毕氏肠微孢子虫等特定病原的调查研究.本研究对洛阳地区部分宠物鸟类进行调查,旨在了解宠物鸟肠道寄生虫感染的近况,为宠物鸟寄生虫病防治和人兽共患寄生虫病防控提供参考依据.

To reveal the species of Fasciola fluke in yellow cattle,adult parasites of Fasciola were isolated from the liver of a yellow cattle in a beef cattle slaughterhouse in Jiyuan of Henan province and observed morphologically.Then the parasites were further identified by sequence analysis based on the ribosomal ITS1 and ITS2 regions and mitochondrial nad1 and cox1 genes.Sequence analysis based on ITS1 and ITS2 regions showed that the isolates JY003 and JY004 from the affected yellow cattle in this study were the Fh/Fg hybrid type of Fasciola.The nad1 and cox1 sequences of JY003 and JY004 were more similar to that of F.gigantica than F.hepatica,especieally the nad1 sequences of the two isolates were 100％identi-cal to that of the F.gigantica isolates from Yunnan of China,Japan and Korea.In the phylogenetic trees based on the nad1 and cox1 genes,the two isolates in this study were clustered into the same branch with the reference strains of F.gigantica.This is the first report of the Fh/Fg hybrid type of Fasciola infection in a yellow cattle from Henan province,providing important basic data for the prevention and control of fasciolosis in animals and humans.

Increasing eutrophication poses a considerable threat to freshwater ecosystems, which are closely associated with human well-being. As important functional entities for freshwater ecosystems, submerged macrophytes have suffered rapidly decline with eutrophication. However, it is unclear whether and how submerged macrophytes maintain their ecological functions under increasing eutrophication stress and the underlying patterns in the process. In the current study, we conducted an extensive survey of submerged macrophytes in 49 lakes and reservoirs (67% of them are eutrophic) on the Yunnan-Guizhou Plateau of southwestern China to reveal the relationship between submerged macrophyte biodiversity and ecosystem functioning (BEF) under eutrophication stress. Results showed that submerged macrophytes species richness, functional diversity (FD), and β diversity had positive effects on ecosystem functioning, even under eutrophication. Functional diversity was a stronger predictor of community biomass than species richness and β diversity, while species richness explained higher coverage variability than FD and β diversity. This suggests that species richness was a reliable indicator when valid functional traits cannot be collected in considering specific ecological process. With increasing eutrophication in water bodies, the mechanisms underlying biodiversity-ecosystem functioning evolved from “niche complementarity” to “selection effects”, as evidenced by decreased species turnover and increased nestedness. Furthermore, the relative growth rate, specific leaf area, and ramet size in trade-off of community functional composition became smaller along eutrophication while flowering duration and shoot height became longer. This study contributes to a better understanding of positive BEF in freshwater ecosystems, despite increasing anthropogenic impacts. Protecting the environment remained the effective way to protect biodiversity and corresponding ecological functions and services. It will be important to consider different facets of biodiversity on ecosystem functioning in future studies to improve effective management plans.

Abstract Background Melanoma is a malignant tumor with a high mortality rate. Some microorganisms have been shown to activate the immune system and limit cancer progression. The objective of this study is to evaluate the anti-melanoma effect of Neospora caninum, a livestock pathogen with no pathogenic activity in humans. Methods Neospora caninum tachyzoites were inoculated into a C57BL/6 mouse melanoma model by intratumoral and distal subcutaneous injections. Tumor volumes were measured, and cell death areas were visualized by hematoxylin and eosin staining and quantified. Apoptosis in cell cultures and whole tumors was detected by propidium iodide (PI) and TUNEL staining, respectively. Cytokine and tumor-associated factor levels in tumors and spleens were detected by real-time quantitative polymerase chain reaction. Infiltration of macrophages and CD8+ T cells in the tumor microenvironment (TME) were detected by immunohistochemistry with anti-CD68 and anti-CD8 antibodies, respectively. Finally, 16S rRNA sequencing of mice cecal contents was performed to evaluate the effect of N. caninum on gut microbial diversity. Results Intratumoral and distal subcutaneous injections of N. caninum resulted in significant inhibition of tumor growth (P < 0.001), and more than 50% of tumor cells were dead without signs of apoptosis. Neospora caninum treatment significantly increased the mRNA expression levels of IL-12, IFN-γ, IL-2, IL-10, TNF-α, and PD-L1 in the TME, and IL-12 and IFN-γ in the spleen of tumor-bearing mice (P < 0.05). An increase in the infiltration of CD8+ T cells and macrophages in the TME was observed with these cytokine changes. Neospora caninum also restored the abundance of gut microbiota Lactobacillus, Lachnospiraceae, Adlercreutzia, and Prevotellaceae associated with tumor growth, but the changes were not significant. Conclusion Neospora caninum inhibits B16F10 melanoma by activating potent immune responses and directly destroying the cancer cells. The stable, non-toxic, and efficacious properties of N. caninum demonstrate the potential for its use as a cancer treatment. Graphical Abstract