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Sympatric non-biting flies serve as potential vectors of zoonotic protozoan parasites on pig farms in China

Parasites & Vectors volume 18, Article number: 59 (2025) Cite this article

Abstract

Background

Cryptosporidium spp., Enterocytozoon bieneusi, and Blastocystis sp. are common enteric parasites in humans and pigs. Ascertaining whether non-biting flies (NBFs) serve as potential vectors of these parasites on pig farms is a crucial aspect of disease control.

Methods

Non-biting flies were collected and identified by morphology analysis together with sequence analysis of the mitochondrial cytochrome c oxidase I (CO1) gene as confirmation. In a cross-sectional study, the small-subunit ribosomal RNA (SSU rRNA) gene of Cryptosporidium spp., the internal transcribed spacer region (ITS) region of E. bieneusi and the SSU rRNA gene of Blastocystis sp. were investigated in fresh pig fecal samples and sympatric NBFs.

Results

The results revealed the occurrence of five species of NBFs (Musca domestica, 91.2%; Lucilia sericata, 5.8%; Chrysomya megacephala, 1.7%; Aldrichina grahami, 0.6%; Helicophagella melanura, 0.6%) in the collected pig fecal samples. The prevalence of Cryptosporidium spp., E. bieneusi and Blastocystis sp. on the body surface of NBFs was 0.6% (2/342), 4.4% (15/342) and 20.8% (71/342), respectively. Similarly, the prevalence of these parasites in the lysates of NBFs (= in vivo carriage) was 0% (0/342), 2.7% (9/342) and 10.5% (36/342), respectively. The prevalence of Cryptosporidium spp., E. bieneusi and Blastocystis sp. in pigs from which fly samples were collected was 2.3% (41/1794), 12.6% (226/1794) and 30.8% (553/1794), respectively. The zoonotic Cryposporidium suis/C. scrofarum, E. bieneusi ITS genotypes EbpA/EbpC and Blastocystis sp. subtypes ST1/ST3/ST5 were identified in both NBFs and pig feces. NBFs were found to carry E. bieneusi and Blastocystis sp. on their body surface as well as in the lysates.

Conclusions

These findings demonstrate the role of NBFs as potential vectors in the dissemination of these zoonotic parasites in pig farms, and also highlight the possibility of their transmission to humans.

Graphical Abstract

Background

Non-biting flies (NBFs) generally feed and breed in the field and are frequently found in areas where human and animal activities are common, including livestock and poultry farms [1]. Parasites can adhere to the body hairs, wings and appendages of NBFs, or even be ingested by these flies, and, therefore, NBFs can serve as vectors [2, 3]. NBFs are considered to be the carriers of hundreds of pathogens, including viruses, bacteria, fungi and parasites of both veterinary and medical importance [1, 3].

Cryptosporidium spp., Enterocytozoon bieneusi and Blastocystis sp. are important and common zoonotic protozoan parasites in humans, pigs and other animals [4]. They are transmitted mainly via the fecal–oral route (contaminated water or food), causing acute gastroenteritis, abdominal pain and diarrhea in infected hosts, particularly in immunocompromised hosts [5]. To date, more than 40 species and more than 120 genotypes of Cryptosporidium have been identified on the basis of the small subunit ribosomal RNA gene (SSU rRNA) and glycoprotein (gp60) genes [6, 7], of which Cryptosporidium suis and C. scrofarum are the most common Cryptosporidium species identified in pigs and reported in humans [8]. More than 500 E. bieneusi genotypes have been reported based on identification using the internal transcribed spacer (ITS) locus, of which more than 60 different genotypes have been identified in pigs, with the majority of them also found in humans [9,10,11]. Similarly, 32 subtypes of Blastocystis sp. have been identified on the basis of SSU rRNA, of which nine (ST1–ST8 and ST12) are zoonotic subtypes [12,13,14].

Swine feces and associated waste are the preferred feeding and breeding sites for NBFs, and many types of NBFs have been documented on and around livestock farms [1]. The transmission of NBFs is remarkable, as they carry many zoonotic protozoan parasites in the environment [15,16,17]. Therefore, we investigated the diversity of the sympatric NBFs in pig farms and their potential role in the transmission of zoonotic Cryptosporidium spp., E. bieneusi and Blastocystis sp. This study provides insight into the potential role of NBFs as carriers (vectors) of protozoan pathogens, and the importance of their control in pig farms as a means to strengthen public health safety.

Methods

Sample collection

In this cross-sectional study conducted from May 2021 to November 2022, a total of 3420 NBFs were captured from 10 small-scale farms (< 100 pigs) and seven large-scale farms (> 100 pigs) situated in six provinces of China (Fig. 1), using fly trap paper and/or fly-fishing nets. The fly traps were placed on the floor of the farm building (2 m away from the areas where pigs were held) and on the inner and outer surface of the farm walls (1 m above from the ground), at locations where pigs could not disturb them. Traps were also set close to the windows and doors, especially near manure and also hung on the ceiling where flies generally rest. Likewise, fly-trap nets were swept in the air in and around the farm to collect flying NBFs. The captured NBFs were picked out of the traps and directly transferred into the zip lock bags in dry condition. At the same time, 1794 fresh fecal samples from pigs were collected from the same pig farms (Additional file 1: Table S1). To avoid cross contamination, only freshly passed fecal samples were collected via rectal sampling or immediately after defecation. All collected samples were kept in a refrigerated ice foam box, immediately transported to the International Joint Research Laboratory for Zoonotic Diseases of Henan within 48 h and then stored in a freezer at 4 °C.

Fig. 1
figure 1

Location map of the study area. Six provinces from where the pig fecal samples and the sympatric non-biting flies (NBFs) were collected are depicted with the respective legends. The pie chart indicates the species-specific proportion of collected HBFs in the six provinces. The study area map was created using ArcGIS Pro 3.1.6

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Identification of sympatric NBF species

Each sympatric NBF was examined under a stereo microscope and classified to the species level on the basis of morpho-taxonomical characteristics such as body size, body color and thoracic and abdominal structures [2, 18]. The identity of the NBF species was confirmed by a molecular method, as follows. The flies were pooled into 342 pools, each containing 10 flies of the same species on the basis of a similar morphology. Each pool of NBFs was crushed in a tissue grinder, and DNA was extracted from the tissue using the TlANamp Genomic DNA Kit (China) following the manufacturer’s instructions. The mitochondrial cytochrome c oxidase I (COl) gene of NBFs was amplified using primers reported in a previous study [19] (Additional file: Table S2).

Isolation of parasites from the body surface of the NBFs

All collected NBFs were divided into 342 sample pools, where one pool was defined as a collection of 10 flies with similar morphology and therefore considered to be a single sample. An appropriate amount of physiological saline (0.9% NaCl) was added to each pool, and the mixture was then placed in a shaker for 5 min without damaging the NBFs body [20, 21]. All eluants (body washes) were transferred to new tubes, followed by centrifugation. The supernatant was discarded, and the precipitate was used separately for parasite identification. This procedure was repeated 3 times to ensure the recovery of parasites attached to the body surface of the NBFs.

Isolation of parasites in the lysate of NBFs

After the body surface of each pool of flies had been washed to isolate the parasites from their external body surface, each pool of flies was transferred into a new tube. Then, each pool of NBFs was crushed with a tissue mortar so that the parasites they had ingested into the gut could be released into the respective lysates (homogenates) [20]. In the present study, the term ‘lysate’ mainly refers to the gut contents of the NBFs which contain the parasites the NBFs have ingested. The lysates were then immediately filtered through a 198-μm sieve, and the filtrates were subsequently centrifuged at 1500 g for 5 min at room temperature [20]. The supernatant was discarded after centrifugation, and the precipitates were retained for parasite identification [2, 22]. This procedure was repeated 3 times for each pool to ensure the recovery of parasites from the NBFs in lysates.

DNA extraction and PCR amplification of parasites from NBFs

Genomic DNA from the body surface washings of the NBFs and in lysate eluants was extracted using the DNeasy® PowerSoil® ProKit following the manufacturer’s instructions (QIAGEN, Hilden, Germnay), and the extracted DNA from each pool was stored at – 20 °C. The PCR amplification of extracted DNA was carried out using species-specific primers targeting the 18S rRNA gene of Cryptosporidium spp., the ITS region of E. bieneusi and the SSU rRNA gene of Blastocystis sp., as described previously [23,24,25] (Additional file 2: Table S2). The PCR products were electrophoresed in a 1.5% agarose gel to identify the positive amplicons.

DNA extraction and PCR amplification of parasites from pig fecal samples

The genomic DNA of the pathogens was extracted from freshly collected pig fecal samples (20–200 mg) using E.Z.N.A. Stool DNA Kits (Omega Biotek Inc., Norcross, GA, USA). The PCR amplification conditions and identification process were the same as those described above for parasites extracted from NBF samples.

DNA sequence analysis

All positive PCR amplicons were submitted to a commercial sequencing company (SinoGenoMax Biotechnology Co., Ltd., Beijing, China) for bidirectional sequencing on an ABI-PRISM™ 3730XL DNA Analyzer using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA). The alignments were performed using MEGA 7.0 (https://megasoftware.net), followed by a Basic Local Alignment Search Tool (BLAST) search of the homology sequence in the GenBank database to determine the genotype and subtype of the parasites.

Statistical analysis

Data were statistically analyzed using IBM SPSS Statistics version 22 software (SPSS IBM, Armonk, NY, USA), and the Chi-square (χ2) test was used to analyze the prevalence of NBF species and the intestinal protozoan parasites carried by NBFs in different sampling regions. In addition, the prevalence of identified parasites in pigs was compared on the basis of the host’s age groups and sampling sites. Statistical significance was set at P < 0.05.

Results

Characteristics of the study population of pigs and NBFs

A total of 3420 NBFs were collected from the sampled pig farms, among which 1020 (29.8%) were from large-scale farms and 2400 (70.2%) were from small-scale farms (Fig. 1). Similarly, of the 1794 pig fecal samples collected in total, samples from pigs at various developmental stages, such as suckling piglets (< 21 days; n = 248, 13.8%), weaning pigs (1–2 months; n = 444, 24.7%), finishing pigs (3–6 months; n = 670, 37.3%) and sows (> 6 months; n = 432, 24.1%), were included. Regarding pig farm types, 53.6% (n = 961) of the fecal samples were collected from small-scale farms, and 46.4% (n = 833) were collected from large-scale farms. The overall sociodemographic characteristics of the NBFs and pigs included in this study are shown in Table 1.

Table 1 Overall sociodemographic characteristics of the non-biting flies and pigs included in the study
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Identification of NBF species

On the basis of morphological features and molecular methods, five NBF species (Musca domestica, Aldrichina grahami, Chrysomya megacephala, Helicophagella melanura and Lucilia sericata) were identified. Among the total NBFs collected, M. domestica was collected from farms in all the provinces and comprised the highest proportion of NBFs (91.2%; 3120/3420), followed by L. sericata (5.8%; n = 200), C. megacephala (1.8%; n = 60) and equal proportions (0.6%) of A. grahami (n = 20) and H. melanura (n = 20) (Table 1).

Prevalence and genotypes of parasites on the body surface of NBFs

Among the 342 pools of NBFs, the prevalence rates of Cryptosporidium spp., E. bieneusi and Blastocystis sp. on the body surface of NBFs were 0.6% (2/342), 4.4% (15/342) and 20.8% (71/342), respectively (Table 2). Cryptosporidium spp. (0.6%, 2/342) identified on the body surface of NBFs were confirmed to be C. suis (n = 1) and C. scrofarum (n = 1). Cryptosporidium suis was identified on the body surface of Musca domestica collected from large-scale pig farms in Henan Province, whereas Cryptosporidium scrofarum was identified on the body surface of H. melanura captured from large-scale pig farms in Jiangsu Province. Regarding E. bieneusi (1.7%, 15/342), only the EbpC (n = 13) and EbpA (n = 2) genotypes were identified, with EbpC the dominant genotype. Similarly, among the Blastocystis sp. (20.8%, 71/342), only the ST3 (n = 56) and ST5 (n = 15) genotypes were identified, with ST3 the dominant genotype.

Table 2 Prevalence and genotypes of intestinal parasites on the body surface of non-biting flies)
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Prevalence and genotypes of parasites in lysates of NBFs

The in vivo identified parasites in NBF lysates were E. bieneusi and Blastocystis sp., with respective prevalence rates of 2.7% (9/342) and 10.5% (36/342), whereas Cryptosporidium spp. were not detected (Table 3) in the NBF lysates. With respect to E. bieneusi (n = 9), only two genotypes, EbpC (n = 8) and EbpA (n = 1), were identified. Among the total number of detected Blastocystis sp. (n = 36), three genotypes, namely, ST3 (n = 20), ST5 (n = 14) and ST1 (n = 2), were identified.

Table 3 Prevalence and genotypes of intestinal parasites in the lysates of non-biting flies
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Infection prevalence and genotypes of intestinal protozoan parasites in pigs

Our investigation on the co-occurrence of the three protozoan parasites in 1794 pig fecal samples showed that the infection rates of Cryptosporidium spp., E. bieneusi and Blastocystis sp. were 2.3% (41/1794), 12.6% (226/1794) and 30.8% (553/1794), respectively (Table 4). A total of 41 (2.3%, 41/1794) fecal samples tested positive for the 18S rRNA gene of Cryptosporidium spp., with C. suis and C. scrofarum identified in 21 and 20 of these fecal samples, respectively; the results confirmed by DNA sequencing. A total of 226 fecal samples (12.6%, 226/1794) tested positive for the ITS sequence of E. bieneusi. The genotypes identified were EbpC (n = 141), EbpA (n = 51), PigEBITS5 (n = 12), CHG19 (n = 5), HIJIV (n = 4), Henan-IV (n = 4), CHS5 (n = 4), O (n = 3), BEB6 (n = 1) and H (n = 1), among which EbpC was the dominant genotype. Similarly, 553 fecal samples (30.8%, 553/1794) tested positive for the SSU rRNA gene of Blastocystis sp., with three gene subtypes, namely ST5 (n = 508), ST3 (n = 24) and ST1 (n = 21) confirmed; the dominant subtype was ST5 (Table 4). Statistical analysis revealed highly significant differences in the prevalence of these three intestinal protozoan infections in pigs with respect to both different regions and different age groups of pigs (P < 0.01) (Table 4). A comparative analysis of these parasites in relation to the developmental stages of pigs revealed that all developmental categories were infected with C. scrofarum, whereas weaning, finishing and sows were infected with both C. scrofarum and C. suis. With respect to the E. bieneusi genotypes, the EbpC, EbpA and PigEBITS5 subtypes were detected in pigs of all age categories; the HIJ-IV, CHG19 and H subtypes were detected only at weaning; the Henan-IV subtype was detected at weaning and finishing; the CHS5 subtype was detected at weaning and in sows; and the BEB6 and O subtypes were detected only in sows. Similarly, Blastocystis subtypes ST1, ST3 and ST5 were found in all age categories of pigs, whereas suckling piglets had only subtype ST5.

Table 4 Infections and genotypes of intestinal parasites in pigs
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Possible transmission of Cryptosporidium spp., E. bieneusi and Blastocystis sp. between pigs and NBFs

In the present study, the prevalence rates of Cryptosporidium spp., E. bieneusi, and Blastocystis sp. were 2.3% (41/1794), 12.6% (226/1794) and 30.8% (553/1794) in pig feces; 0.6% (2/342), 4.4% (15/342) and 20.8% (71/342) on the body surface of NBFs; and 0% (0/342), 2.7% (9/342) and 10.5% (36/342) in NBF lysates as in vivo carriage (Fig. 2). One interesting observation in this study was that the co-prevalence of Cryptosporidium spp., genotypes of E. bieneusi and subtypes of Blastocystis sp. in pig feces and on the body surface and in lysates of the NBFs was found to be statistically significant (Chi-squared test, p < 0.05), suggesting that NBFs could play crucial roles in the distribution of these three protozoan parasites in pigs. The difference in the infection prevalence rates of the three parasites (Cryptosporidium spp., E. bieneusi and Blastocystis sp.) in pigs and on the body surface and in lysates of NBFs were compared using the Chi-squared test (p value < 0.05 was considered to be significant). The results showed a significant difference in the prevalence of these parasites in these groups of samples.

Fig. 2
figure 2

Proportion of Cryptosporidium spp., genotypes of Enterocytozoon bieneusi and subtypes of Blastocystis sp. detected on the body surface and in lysates of the sympatric non-biting flies (NBFs) collected from the pig farms

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Discussion

Non-biting flies naturally dwell in areas of filth, including pig farms, for feeding and breeding. Parasite cysts/oocysts readily adhere to the external body parts of NBFs, such as the proboscis, appendages, wings and bristles, while the flies are rest and moving around animal farms [1]. These flies not only facilitate the spread of parasites from one place to another but also lead to the contamination of food and water for animal consumption [15]. In the present study, morphological and molecular analyses revealed that 3420 flies (divided into 342 sample pools) collected from pig farms in six provinces in China were NBFs, with M. domestica being the dominant fly species. NBFs were found to carry E. bieneusi and Blastocystis sp. both on their body surface and in lysates, while Cryptosporidium spp. were detected only on the body surface. Our results confirmed that M. domestica from the same pig farms could use pig farms as abodes and contribute to the dissemination of Cryptosporidium spp., E. bieneusi and Blastocystis sp. In this context, we therefore suggest that different fly species can carry different species or genotypes of these intestinal protozoan parasites.

Different fly species can carry multiple parasites, and even a single fly species can serve as a vector for more than two parasite species [1]. NBFs can contaminate water, fruits, vegetables and animal feed mainly through surface contact and through their intestinal excretions [26]. Previous studies have shown that after the infection cycle, Cryptosporidium spp. infection by M. domestica can last up to 3 weeks, confirming the potential risk of mechanical transmission by M. domestica [27]. It has also been reported that the sequences of the SSU rRNA gene and gp60 locus of C. parvum from NBFs are 100% homologous to those of C. parvum from humans, suggesting that NBFs may be potential vectors of C. parvum [20].

NBFs are active mechanical vectors for the transmission of protozoan parasites [15, 28, 29]. In the present study, we identified E. bieneusi genotype EbpA from M. domestica, which is the first report of this genotype in this fly. Genotypes EbpA and EbpC both belong to Group 1 and are highly zoonotic. Although E. bieneusi genotype BEB6 belongs to Group 2, its detection in Swedish lambs [30], sheep in Inner Mongolia [31], red deer and Siberian roe deer [32], rodents, flies and cattle [21] and laboratory rodents [33] suggests zoonotic potential. EbpC is a genotype isolated only from NBFs associated with a wide range of infections, has been found in many mammals, including humans, and is classified as a Group 1 zoonotic [10]. The Cryptosporidium species identified in this study, C. suis and C. scrofarum, which were previously reported in humans, may pose a zoonotic risk. Screening for cryptosporidiosis in rural Madagascar identified cases of C. suis infection, suggesting the this species is highly likely to infect humans through contact with pigs or vector transmission [34]. The EbpC genotype of E. bieneusi has a wide host range and has been reported in humans and other animals [10].

In the present study, Cryptosporidium species C. suis and C. scrofarum were both identified in pig feces and on the body surface of the NBFs, which suggests possible transmission by NBFs as mechanical carriers (Fig. 3). Comparative genotype analysis revealed the co-occurrence of the E. bieneusi genotypes EbpC and EbpA and the Blastocystis species genotypes ST5 and ST3 in pig feces and on the body surface and in lysates of the NBFs, indicating a potential vector. Likewise, the Blastocystis species subtype ST1 was identified in pig feces and in lysates of NBFs, thus suggesting that the NBFs might deserve vectorial capacity in transmitting this parasite as well. Notably, certain proportions of the E. bieneusi genotypes EbpC (n = 5) and EbpA (n = 1) and the Blastocystis species genotypes ST3 (n = 9) and ST5 (n = 8) were identified both on the body surface and in lysates of NBFs. A critical consideration of this scenario implies that the sympatric NBFs deserve to be classified as potential vectors for zoonotic protozoan parasites on pig farms in China, as depicted in this study (Fig. 3). However, the exact transmission dynamics of these species/genotypes between NBFs and pigs on farms need to be confirmed by further detailed investigations.

Fig. 3
figure 3

Illustration of possible transmission dynamics of zoonotic intestinal parasites by non-biting flies (NBFs)

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A survey of hospitalized children in Shanghai revealed that EbpC was the predominant genotype associated with high transmission risk [35]. In addition, when the occurrence of E. bieneusi in river water contaminated by dead pigs in Shanghai was investigated, EbpC was found to be the most common genotype and to have a high potential to cause health threats to humans [36]. Although the same EbpA genotype was not identified in NBFs or pigs in the same locality in the present study, this genotype may represent a threat to swine health owing to the behavioral characteristics of NBFs. The absence of certain parasite genotypes in NBFs, especially on their body surface, might be attributed to the grooming behavior of the flies, as this behavior could remove attached parasites from their bodies [37]. Moreover, we suggest that the feeding habits of different species of flies and the different climates of the study region could be another factor for the disparity in the occurrence of same genotype of parasites in pigs and NBFs. The most common subtype of Blastocystis species, ST1, is highly zoonotic, whereas ST3 is a subtype of human infection reported in many animal hosts and humans [38]. Subtype ST5 has been detected in pigs and farm workers, indicating that this subtype has the potential to cause zoonosis [39]. The zoonotic risk of Blastocystis sp. transmission in pigs was demonstrated in a study in Queensland, Australia, where zoonotic subtype ST5 was detected in pigs and pig farm workers [36]. Subtype ST5 has also been identified in humans and pigs in rural areas of Jiangxi owing to the frequent contact between humans and pigs and mutual transmission, thus indicating its potential for zoonotic transmission [38].

In summary, NBFs are mechanical vectors for various parasites, most of which are zoonotic, since NBFs may travel as vectors from humans to humans and from humans to animals, and vice versa. Acquiring data on monthly and seasonal variations in relation to the prevalence of parasites transmitted by NBFs would be an important focus of research. However, whether the extent to which these NBFs are vectors of the aforementioned parasites remains one of the important factors to be thoroughly investigated in the future studies. Regular inspection of pig farms for the presence of these NBFs and implementation of control measures are crucial aspects in mitigating the spread of zoonotic parasites among pigs, possibly to humans, and for standardizing the biosecurity with regard to One Health measures.

Conclusions

Non-biting flies were found to carry E. bieneusi and Blastocystis sp. both on their body surface and in lysates, whereas Cryptosporidium spp. were detected only on the body surface. Notably, M. domestica, present at all pig farms as the dominant NBF, was found to serve as a potential vector for all the detected protozoan parasites. Furthermore, C. suis, C. scrofarum, E. bieneusi genotype EbpC and Blastocystis sp. subtypes ST1, ST5 and ST3 were identified in both of these vector NBFs and in host pigs on the same pig farms and all are zoonotic. Our results indicate that NBFs play a possible role as vectors in disseminating zoonotic Cryptosporidium spp., E. bieneusi and Blastocystis sp. between pigs. This finding highlights the importance of stringent control of NBFs on pig farms as a crucial biosecurity measure for protecting susceptible hosts.

Availability of data and materials

No datasets were generated or analysed during the current study.

Abbreviations

COI:

Cytochrome c oxidase 1

ITS:

Internally transcribed spacer

NBFs:

Non-biting flies

SSU rRNA:

Small subunit ribosomal ribonucleic acid gene

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Acknowledgements

Not applicable.

Funding

This work was supported by the Key Research and Development Projects of Henan, China (231111111600), the National Key Research and Development Program of China (2023YFD1801201), the National Natural Science Foundation of China (32102698) and the National Pig Industry Technology System (CARS-35). The sponsors played no role in the study design; collection, analysis or interpretation of the data; writing of the report; or decision to submit the report for publication.

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Author notes

  1. Yufeng Liu, Pitambar Dhakal and Wenyan Hou contributed equally to this work.

Authors and Affiliations

  1. College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, 450046, Henan, China

    Yufeng Liu, Pitambar Dhakal, Wenyan Hou, Fa Shan, Nanhao Wang, Huikai Qin, Xiaoying Li, Rongjun Wang, Longxian Zhang, Sumei Zhang & Junqiang Li

  2. International Joint Research Laboratory for Zoonotic Diseases of Henan, Zhengzhou, 450046, China

    Yufeng Liu, Pitambar Dhakal, Wenyan Hou, Fa Shan, Nanhao Wang, Huikai Qin, Xiaoying Li, Rongjun Wang, Longxian Zhang, Sumei Zhang & Junqiang Li

  3. Key Laboratory of Quality and Safety Control of Poultry Products, Ministry of Agriculture and Rural Affairs, Zhengzhou, 450046, China

    Yufeng Liu, Pitambar Dhakal, Wenyan Hou, Fa Shan, Nanhao Wang, Huikai Qin, Xiaoying Li, Rongjun Wang, Longxian Zhang, Sumei Zhang & Junqiang Li

  4. Rural Industry Development Center of Shangqiu City, Shangqiu, 476100, China

    Bin Yang

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Contributions

Conceptualization: JL and SZ. Methodology: YL, WH and FS. Investigation: YL, WH, NW, BY and HQ. Writing– original draft: PD and JL. Writing–review and editing: SZ, JL, XL and LZ. Funding acquisition: JL and SZ. Resources: RW. Supervision: SZ and LZ. All authors have read and approved the final version of the manuscript.

Corresponding authors

Correspondence to Sumei Zhang or Junqiang Li.

Ethics declarations

Ethics approval and consent to participate

The Research Ethics Committee of Henan Agricultural University reviewed and approved the experimental protocol. The collection of samples was performed following the Guide for the Care and Use of Laboratory Animals (Ministry of Health, China). All samples tested in this experiment were collected with permission, while no special permission was required for the sampling locations.

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The authors declare no competing interests.

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Supplementary Information

13071_2025_6686_MOESM1_ESM.xls

Additional file 1: Table S1. Sociodemographic characteristics of the study population of non-biting flies (NBFs) and pigs

13071_2025_6686_MOESM2_ESM.docx

Additional file 2: Table S2. Primers used to amplify cytochrome c oxidase I (CO1) gene of NBFs and three intestinal protozoa (Cryptosporidium spp., Enterocytozoon bieneusi and Blastocystis sp.)

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Liu, Y., Dhakal, P., Hou, W. et al. Sympatric non-biting flies serve as potential vectors of zoonotic protozoan parasites on pig farms in China. Parasites Vectors 18, 59 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13071-025-06686-2

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Keywords

Parasites & Vectors

ISSN: 1756-3305

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