Molecular Characterization and Phylogenetic Studies of Fungal Species in Piggeries and Implications for Mycotoxin Contamination and Food Safety

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Fungi such as Aspergillus, Fusarium, and Penicillium can produce mycotoxins, which impact livestock and human health. This study aimed to isolate and molecularly characterize fungal species in pig feed and faeces from registered and unregistered farms in Lagos State, Nigeria. Eighteen samples (6 feed and 12 feces) were collected and refrigerated before culturing on Sabouraud Dextrose Agar at 28°C for 5-7 days. Pure isolates were identified morphologically, and DNA was extracted. PCR amplification targeted ITS region of ribosomal DNA, with sequence comparisons made using GenBank references for species identification. Phylogenetic analysis assessed genetic similarity. Results Results showed that fungal contamination was more frequent in faeces collected from unregistered farms than registered farms. Microfungi belonging to the genera Aspergillus , Pichia , Rhizopus , and Trichoderma were isolated and purified from feed while Aspergillus and Candida genera were isolated and purified from faeces. Molecular analyses revealed high genetic similarity among isolates, with Aspergillus flavus and Rhizopus arrhizus showing 100% identity with known strains. All four evolutionary models produced consistent phylogenetic trees, grouping the fungal species into well-supported clades, with only minor differences in branch lengths and bootstrap values. No significant genetic divergence was observed between isolates from registered and unregistered farms. Conclusions The occurrence of mycotoxin-producing fungi in pig farms, emphasizes the need for strict monitoring to enhance food safety. Fungal contamination food safety Pig farms Mycotoxins Molecular characterization Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 BACKGROUND The fungal kingdom represents an extraordinary diversity of organisms with profound impacts across animal and plant species and ecosystem health (Fisher et al. ,2020). They exist freely or in symbiosis with bacteria, plants, or animals and are identified by their filamentous growth (hyphae) and external digestion (Moënne-Loccoz et al., 2015 ). Fungi colonize animal feeds through exposure to various environmental matrices such as soil, dust, and insects. The microbial diversity in feeds depends on factors like moisture, pH, oxygen levels, and nutrient content, with some moulds capable of growing even in dry, stored grains (Maciorowski et al., 2007 ). Certain fungi produce mycotoxins, toxic secondary metabolites found in agricultural products that are susceptible to mold infestation (Ráduly et al., 2020 ). In livestock, mycotoxins reduce productivity by impairing growth, decreasing milk and egg production, and causing immune suppression, reproductive issues, and organ damage. Environmentally, mycotoxins also contaminate soil and water, resulting in crop losses, economic damage, and disruptions to biodiversity. Their persistence in the food chain further amplifies these risks. Mycotoxigenic fungi that contaminate the human food chain primarily belong to three genera: Aspergillus, Fusarium , and Penicillium (Ráduly et al., 2020 ). Fusarium species are aggressive plant pathogens that produce mycotoxins either before harvest or soon after, while Penicillium and Aspergillus species are more commonly linked to food contamination during drying and storage (Ezekiel et al. , 2020). The susceptibility of crops to these fungi varies; for instance, Fusarium species frequently affect corn, whereas Aspergillus flavus is a major contaminant in peanuts (Dohlman et al ., 2011). Post-harvest handling plays a crucial role in determining mycotoxin contamination levels. Poor storage conditions, such as high moisture and inadequate ventilation, create an ideal environment for fungal growth and toxin production in stored grains (Pitt & Hocking, 2009). Furthermore, processing techniques like milling and grinding can concentrate mycotoxins in specific grain fractions, increasing potential health risks (Pickova et al ., 2021). In recent years, the incidence of mycotoxin contamination in swine feed has drawn considerable attention (Haque et al., 2020 ). Among livestock species, pigs are particularly vulnerable to mycotoxin exposure due to their dietary requirements (Yang et al, 2020 ). Sources of mycotoxin contamination in piggeries include consumption of moldy grains, improper feed storage, and exposure to contaminated water or bedding materials, all of which contribute to the spread of these harmful toxins. Humans are exposed to these toxins primarily by consuming contaminated pork and dairy products, which pose significant health risks. Additionally, occupational exposure among farmers, butchers, and meat processors, as well as environmental contamination from pig waste, further increases the likelihood of human contact. Ensuring food safety and preventing food-borne diseases in humans and animals is a growing priority for specialists in human and veterinary medicine worldwide. Mycotoxins have the potential to disrupt global food supply chains, making it a global concern that requires continuous monitoring, regulation, and control (FAO/WHO, 2019 ). While developed countries have well-developed infrastructures for monitoring food quality and ensuring high standards, weaker monitoring systems and limited enforcement of safety regulations increase contamination risks in developing regions (Khalid, 2016 ) . Molecular techniques such as PCR and phylogenetic analysis are widely used for fungal identification, but studies in developing countries remain limited. A key gap exists in the molecular characterization of mycotoxin-producing fungi in pig farms, especially in unregistered farms with poor management and inadequate regulatory oversight (Jayawardhana et al. , 2020; Liu et al. , 2023). Addressing these gaps will promote food safety monitoring, guide policy recommendations, and help mitigate mycotoxin risks in livestock and human food chains. This study therefore aimed to identify and molecularly characterize fungal species occurring in pig faeces and feed in registered and unregistered farms in Lagos State, Nigeria. MATERIALS AND METHODS Study area This study was conducted in four pig farms in Lagos State with GPS coordinates latitude 6.465422 and longitude 3.406448 (Figure 1). The farms were selected based on their registration status, geographical distribution, and willingness to participate in study. Two registered farms at Odogunyan (ST1) and Abule Oshorun (ST3) and two unregistered farms at Gberigbe (ST2) and Ebute-Meta (ST4) were included, along with a control site at the Federal College of Education, Akoka, Yaba. The registration status of the farms was verified through the Lagos State Ministry of Agriculture by reviewing farm registration certificates and consulting the Livestock Department. Registered farms adhered to government regulations, while unregistered farms operated without formal approval. Collection and preservation of samples Zipper storage bags were used to collect feed samples from the selected farms, labelled appropriately, and placed in well-labeled, clean plastic polythene bags. Fecal samples were collected at random from 3 pigs on each farm. They were collected directly into sterile sample bottles as soon as they were egested by each selected pig to avoid contamination from the piggery. The sample bottles were appropriately labelled and carefully placed in clean plastic polythene bags , each marked with its corresponding label. Subsequently , the samples were transported to the laboratory for analysis. To maintain their integrity , the samples were stored in a refrigerator regulated at -4°C until time for laboratory analyses. Microbial analysis of samples Agar preparation and sterilization Exactly 39-gram Potato Dextrose Agar (PDA) and 15 mg streptomycin were suspended into a 1000 ml conical flask filled with 1000 ml distilled water. The mixture was stirred with a stirring rod to dissolve the medium completely and the flask was corked with aluminum foil and tightened properly using paper tape. Nine milliliters of distilled water were pipetted into 96 test tubes and corked properly. In the autoclave, the medium and test tubes were placed and autoclaved at 121 o C (15 Ibs pressure) for 15 minutes and allowed to cool at 45 o C. Serial dilution Using one in eight serial dilution method, the test tubes were labelled A -1 to A -8 , B -1 to B -8 , C -1 to C -8 , D -I to D -8 , E -1 to E -8 , F -1 to F -8, G -1 to G -8 , H -1 to H -8 , I -1 to I -8 , J -1 to J -8 , K -1 to K -8 , L -1 to L -8 , M -1 to M -8 , N -1 to N -8 , O -1 to O -8 , P -1 to P -8 , Q -1 to Q -8 , and R -1 to R -8 . One gram each of the feed samples were poured into test tubes labelled A -1 , B -1 , C -1 , D -1 , E -1 and F -1 respectively while 1 gram of fecal sample was transferred into corresponding test tubes labeled G -1 , H -1 , I -1 , J -1 , K -1 , L -1 , M -1 , N -1 , O -1 , P -1 , Q -1 and R -1 . The suspensions were mixed thoroughly to obtain a homogenous suspension. From test tube A -1 , 1 ml of the suspension was pipetted into A -2 , 1 ml from A -2 was pipetted into A -3 , 1 ml from A -3 was pipetted into A - 4, 1 ml from A -4 was pipetted into A - 5, 1 ml from A -5 was pipetted into A -6 , 1 ml from A -6 was pipetted into A -7 and 1 ml from A -7 was pipetted into A -8 . The same procedure was done for B -1 to R -8 . Fungi culture The culture medium was mixed properly before pouring into labelled 162 sterile petri dishes. Each milliliter from test tubes labelled -4, -6, and -8 were poured into 54 petri dishes using the pour plate method and rocked gently. The culture was replicated giving a total of 162 petri dishes. The medium was allowed to solidify and incubated at 37 o C for 5 days alongside 3 control petri dishes containing PDA with no streak. Sub-culturing of samples Colonies grown on each medium were distinguished based on their surface characteristics such as texture, color, zonation, sporulation, and diameters. The distinguishable colonies were sub-cultured on a PDA slant and incubated at room temperature for 7 days. To obtain pure strains, from the sub-cultured plates, using an inoculating loop, each isolated colony was picked and transferred into already labeled MacConkey bottles filled with 9 ml Potato Dextrose Broth and placed in an incubator shaker for 4 days at 29 o C. The isolates were identified, and pure isolates were maintained in PDA slants and stored in the refrigerator for further identification. Fungi genomic DNA extraction The fungal tissues obtained were transferred into labeled 1.5 ml microcentrifuge tubes aseptically using a pair of sterile tweezers. These tissues were frozen by dipping in liquid nitrogen. The frozen tissues were ground using sterilized laboratory mortar and pestle to release the contents of the spore. Exactly 100 mg of the powdered tissues were weighed and transferred into new d microcentrifuge tubes. The manual procedures were followed. The quality of the extracted DNA was assessed by electrophoresis in 1% agarose gel (1-gram agarose in 100 ml Tris buffer). DNA concentration was measured at the optical density (OD) of 260 nm and purity was recorded at 260 and 280 nm wavelengths using a UV spectrophotometer before storing extracted DNA at -20°C for subsequent analyses. PCR amplification and gel electrophoresis Primers targeting the internal transcribed spacer DNA namely ITS1 5' (TCC GTA GGT GAA CCT GCG G) 3' (forward primer) and ITS4 5' (TCC TCC GCT TAT TGA TAT GC) 3' (reverse) with length of 600 bp were employed for amplification. Nine isolates with DNA purity levels between 1.80 - 2.20 ng/µl were used for PCR amplification ensuring all farms were represented. The reaction was performed in a 20 µL mixture as follows: 1 µL gDNA template, 0.2 µL DNA polymerase, 0.5 µL each of forward and reverse primers, 1 µL dNTPs and sterile double distilled water. The thermocycler was programed for the following PCR conditions: initial denaturation at 95 o C for 5 mins, 30 cycles of denaturation at 95 o C for 30 s, annealing at 52 o C for 60 s, and extension at 72 o C for 2 min, with a final extension at 72 o C for 10 mins. After amplification, the PCR products were subjected to gel electrophoresis using 1% agarose gel with ethidium bromide as the staining agent and PCR-specific bands visualized by UV trans-illumination. A segment of DNA of known size was used as positive control while molecular grade water was used as negative control. The size of the amplicons was estimated using a 1 kb DNA ladder. Sequence and phylogenetic analyses Several isolates produced multiple bands, each of which was purified for sequencing and analysis. The PCR amplicons from six isolates obtained from pig feces and nine isolates from feed were purified and sequenced by Inqaba Biotec West Africa (Ibadan, Nigeria). The raw sequences were then trimmed and edited using Sequencher 5.4.6 and SnapGene 7.0.1 . Sequence homology analysis was performed using the NCBI nucleotide BLAST tool , while sequence alignment was conducted using ClustalW in MEGA 11 . The evolutionary distances were computed using the Tamura-Nei method (Tamura and Nei, 1993) and Jukes-Cantor method (Jukes and Cantor, 1969) and are in the units of the number of base substitutions per site. These analyses involved 12 nucleotide sequences respectively. All ambiguous positions were removed for each sequence pair (pairwise deletion option). Evolutionary analyses were conducted in MEGA11 (Tamura et al., 2021). RESULTS Microfungal isolates The microbial plates showed cultural features (color/texture) and sporangiospores/conidia characteristic of the different microfungal genera identified (Figure 2). The occurrence of fungal contamination in feed and faeces are summarized in Table 1. Feed samples collected from registered and unregistered farms were all contaminated with fungi. Fungal contamination in faeces was more frequent in unregistered farms (ST2 - 100.0%; ST4 - 66.7%) than in registered farms (ST1 - 66.7%, ST3 - 33.3%). Microfungi belonging to the genera Aspergillus , Pichia , Rhizopus , and Trichoderma were isolated and purified from feed while Aspergillus and Candida genera were isolated and purified from faeces (Table 1). Aspergillus genus was detected in samples from registered and unregistered farms. Trichoderma sp. was found contaminating only samples from registered farms while Candida sp. was only found in unregistered farms. Table 1: Occurrence of fungi genera in feed and feaces collected from registered and unregistered farms in Lagos, Nigeria Farm Sample type No. collected No. of samples contaminated Percentage occurrence (%) Fungi genera ST1 Feed 1 1 100.0 Aspergillus sp., Pichia sp. Faeces 3 2 66.7 Aspergillus sp. ST2 Feed 2 2 100.0 Pichia sp., Rhizopus sp. Faeces 3 3 100.0 Candida sp., Aspergillus sp. ST3 Feed 2 2 100.0 Trichoderma sp., Aspergillus sp. Faeces 3 1 33.3 Aspergillus sp. ST4 Feed 1 1 100.0 Aspergillus sp., Rhizopus sp. Faeces 3 2 66.7% Candida sp. ST1 and ST3 = registered farms; ST2 and ST4 = unregistered farms Molecular characterization Agarose gel electrophoresis revealed amplicons with molecular weight between 353 and 650 bp. Some isolates returned multiple bands even after reamplification (Figure 3). PCR amplicons from 9 feed isolates and 6 faecal isolates were sequenced and deposited in NCBI-GenBank with accession numbers, OR652266-74, OR646806-11 as shown in Table 2. Table 2: NCBI-GenBank accession numbers and submission information NCBI-GenBank Code Accession no. Identity Farm Status Nucleotide sequences (bp) Feed sample AFS1 OR652266 Aspergillus flavus ST1 551 AFS2 OR652267 Pichia kudriavzevii ST2 445 AFS3 OR652268 Rhizopus oryzae ST2 581 AFS4 OR652269 Rhizopus microsporus ST2 650 AFS5 OR652270 Trichoderma longibrachiatum ST3 383 AFS6 OR652271 Pichia kudriavzevii ST1 463 AFS7 OR652272 Aspergillus niger ST3 484 AFS8 OR652273 Aspergillus flavus ST4 550 AFS9 OR652274 Rhizopus oryzae ST4 584 Fecal sample AFI1 OR646806 Aspergillus niger ST1 552 AFI2 OR646807 Candida akabanensis ST2 354 AFI3 OR646808 Aspergillus niger ST3 500 AFI4 OR646809 Aspergillus flavus ST3 542 AFI5 OR646810 Aspergillus flavus ST2 550 AFI6 OR646811 Candida akabanensis ST4 353 ST1 and ST3 = registered farms; ST2 and ST4 = unregistered farms; ‘AFS’ represents feed samples; ‘AFI’ represents faecal samples Table 3 shows a diverse range of fungal organisms isolated from feed and fecal samples, predominantly originating from China, Vietnam, and South Africa. Isolates from the feed samples exhibited perfect genetic matches with Aspergillus flavus LUOHE and Rhizopus arrhizus jx01 from China. Similarly, fungal isolate AFS5 had 100% identity match with an Egyptian strain, Trichoderma longibrachiatum KABOFT5. Table 3: Identity of Strains with Closest Homology from NCBI-BLAST Organism with Closest Homology Location % Identity Feed sample Aspergillus flavus LUOHE China 100 Pichia kudriavzevii er 2 ( Issatchenkia orientalis er 2) China 99.78 Rhizopus arrhizus jx01 China 100 Rhizopus microsporus DTO 402-G1 Italy 100 Trichoderma longibrachiatum KABOFT5 Egypt 100 Pichia kudriavzevii G1-10 China 99.78 Aspergillus niger G12 China 100 Aspergillus flavus 275N China 99.82 Rhizopus arrhizus ND4 ( Rhizopus oryzae ND4) Vietnam 99.83 Fecal sample Aspergillus niger NW-47 South Africa 100 Candida akabanensis TB1Y Vietnam 99.72 Aspergillus niger G12 China 99.60 Aspergillus flavus 275N China 99.82 Aspergillus flavus 7 South Africa 100 Candida akabanensis TB1Y Vietnam 100 Phylogenetic relationships The phylogenetic analysis of fungal isolates from pig feed and fecal samples was conducted using four evolutionary models Jukes-Cantor, Tamura-Nei, Maximum Composite Likelihood, and p-distance via the Neighbor-Joining method in MEGA11 (Figures 4-7). All four models produced consistent phylogenetic trees, grouping the fungal species into well-supported clades, with only minor differences in branch lengths and bootstrap values. Isolates of Aspergillus flavus , Aspergillus niger , Rhizopus arrhizus , Rhizopus microsporus , Pichia kudriavzevii , Trichoderma longibrachiatum , and Candida akabanensis consistently clustered with reference strains from regions such as China, South Africa, and Vietnam. There was no significant genetic divergence was observed between isolates from registered and unregistered farms. DISCUSSION This study explored the occurrence and genetic diversity of fungal species contaminating feed and fecal samples from registered and unregistered pig farms in Lagos State, Nigeria. The results underscore the widespread occurrence of fungal species, particularly from the Aspergillus genus, which have known implications for food safety and animal health due to their potential to produce harmful mycotoxins. This study showed high genetic similarity observed among fungal isolates from both regulated and unregulated farms, with several isolates showing 100% identity with known strains from other regions. The absence of genetic differentiation agrees with similar research conducted on fungal species in agricultural environments (Kepler et al ., 2015). Wokorach et al. (2021) who reported genetic characterization of fungal biodiversity in storage grains in Northern Uganda found that environmental factors such as humidity, temperature can influence the proliferation of genetically similar fungal species across different geographic regions. Similar pattern was observed among fecal isolates exhibiting 100% identity matches with Aspergillus niger NW-47 and Candida akabanensis TB1Y strains from South Africa and Vietnam respectively. Faecal isolates also showed at least 99% similarity with Aspergillus flavus 275N and Aspergillus niger G12 isolates in China. Studies carried out in Lagos pig farms also reveal that the environmental conditions in both regulated and unregulated farms do not exert significant selective pressures, allowing genetically stable fungal populations to thrive (Afolabi et al., 2023). The presence of Aspergillus species in both feed and fecal samples is similar to the findings from other studies that have highlighted the ubiquitous nature of these fungi in agricultural settings. Aspergillus flavus and Aspergillus niger , two species detected in this study, are well-known mycotoxin producers, particularly aflatoxins, which cause detrimental health risks to both livestock and humans (Zulkifli & Zakaria, 2017). The presence of these species in both regulated and unregulated farms emphasize the need for stringent mitigation and control measures. Poor storage conditions, particularly in unregulated farms, can enhance fungal growth, as seen in similar studies conducted in sub-Saharan Africa, where poor storage and handling practices were linked to increased fungal diversity and mycotoxin levels (Chilaka et al., 2022). Research findings from Liu et al. (2020) demonstrated that environments with proper storage conditions are more susceptible to fungal contamination, which often leads to food spoilage and the production of harmful mycotoxins. The identification of Pichia kudriavzevii in both feed and fecal samples highlights the widespread distribution of these species, which can be associated with fermentation processes (Zulkifli & Zakaria, 2017). While not typically considered a major mycotoxin producer, its presence in animal feed raises concerns about the potential for fermentation-related spoilage, especially in environments with high moisture content. This agrees with findings from studies on fungal contamination in livestock feed, which shows that the occurrence of such species is often linked to improper feed handling and storage conditions (Wokorach et al., 2021). In addition to Aspergillus and Pichia , Rhizopus arrhizus and Rhizopus microsporus were primarily detected in unregulated farms. These species are mostly associated with food spoilage, especially in environments where hygiene and storage conditions are below standard practice. Liu et al . (2020) demonstrated that Rhizopus species are prevalent in environments with poor storage practices, where they rapidly colonize and spoil food products, leading to significant economic losses. The detection of these species in unregulated farms further highlights the need for improved storage and handling practices to mitigate the risk of contamination. The fecal samples also revealed significant fungal contamination in unregulated farms, including the detection of Candida akabanensis . While typically associated with environmental sources, its presence in fecal samples suggests potential contamination through feed or poor hygiene practices on the farms. This raises biosecurity concerns, particularly regarding the potential for cross-contamination between animals and their environment, which could exacerbate the spread of fungal species capable of producing harmful mycotoxins. Frisvad et al . (2019) support this finding, who reported fungal contamination in feed and agricultural products shows integral role of hygiene and environmental factors in fungal proliferation. The phylogenetic analysis reported in this study shows the genetic stability of the fungal isolates, with many showing close genetic clustering with reference strains from other regions. This genetic similarity, particularly among Aspergillus flavus and Aspergillus niger isolates, which showed 100% identity with strains from China and South Africa, underscores the global distribution of these fungi (Frisvad et al., 2019). The global ubiquity of these species highlights the need for international collaboration in addressing mycotoxin contamination, as fungal species that thrive in one region may easily spread to others through global trade and agricultural practices (Fumagalli et al., 2021). In conclusion, there is need for improved monitoring and control measures in both regulated and unregulated farms to mitigate the risk of fungal contamination and mycotoxin production. The relatively high genetic similarity of fungal isolates from different sampling sites suggests that these species are well-adapted to several environmental conditions, which further emphasize its importance to global food safety. Therefore, implementing better storage and handling practices, particularly in unregulated farms is required to significantly reduce the risk of fungal contamination and ensure the safety of animal feed and food products in developing regions. Declarations Ethics approval and consent to participate All applicable international and institutional guidelines for the care and use of animals were strictly followed. All procedures performed in this study complied with the ethical standards outlined in the 1964 Declaration of Helsinki and its subsequent amendments. Additionally, the study adhered to the ethical guidelines established by the University of Lagos Committee on the Use of Animals in Scientific Research. Ethics approval and consent to participate Not applicable — this study did not involve human participants, personal data, or clinical images requiring consent to participate or publish. Conflict of Interest Statement The authors declare that they have no known financial or personal relationships that could have appeared to influence the work reported in this manuscript. All authors have read and approved the final manuscript and confirm that there are no competing interests. Animal Ethics Declaration Not applicable — this study did not involve any procedures that required approval by an animal ethics committee. Consent for publication Not applicable – the manuscript does not contain any individual person’s data in any form (including individual details, images, or videos). Availability of data and material The DNA sequence data generated and analyzed during this study are available in the NCBI GenBank repository under accession numbers OR652266 to OR652274 and OR646806 to OR646811. Competing interests The authors declare that they have no known competing financial or non-financial interests that could have influenced the work reported in this manuscript. Funding No funding was received to carry out this research but Institutional resources Clinical Trial Registration Not Applicable-This study did not involve any clinical trials. Acknowledgements The authors are grateful to the management and staff of the Federal Institute of Industrial Research, Oshodi, and the Department of Zoology, University of Lagos for access to laboratory facilities. Special thanks to Inqaba Biotech West Africa for sequencing support. CRediT authorship contribution statement IA: Conceptualization, Supervision, Resources, Project administration, Methodology, Investigation, Writing – original draft. TF: Resources, Investigation, Methodology, Writing – original draft. FD: Methodology, Investigation. FO : Methodology, Investigation. OA: Methodology, Investigation, Writing – review and editing . FDa : Methodology, Investigation, Writing. OI: Methodology, Resources, Investigation SA: Methodology, Investigation. MI: Methodology, Investigation. 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Microorganisms, 9 (2), 383. https://doi.org/10.3390/microorganisms9020383 Yang, C., Song, G., & Lim, W. (2020). Effects of mycotoxin-contaminated feed on farm animals. Journal of Hazardous Materials, 389 , 122087. https://doi.org/10.1016/j.jhazmat.2020.122087 Zulkifli, N. A., & Zakaria, L. (2017). Morphological and molecular diversity of Aspergillus from corn grain used as livestock feed. Hayati Journal of Biosciences, 24 (1), 26-34. Additional Declarations No competing interests reported. Supplementary Files UncroppedGelImagesSupplementaryFigure.doc Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6811857","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":471347761,"identity":"145bcfda-017a-465b-b189-12c0ab99da4d","order_by":0,"name":"Idowu Aneyo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYBACNghlw8DAzNwAYjE2EKklDaiFkUgtUHAYrpiwFj72swcf81ScT9zOztj48QuDjeyGA7yHX+B1GE9esjHPmduJO5sZm6VlGNKMNxzgS7PAq4Uhx0yat+124obDjA3SEgyHEzcc4DEzwKuF/435b962cyAtzb8lGP4ToUUix4yZt+0ASEub5AeGAyAtxg/wa3ljLDnnTLIxSIs1g0Gy8czDPGb4dDDI9+cYfnhTYSe74fzhwzd/ABl9x3uMP+DVAwRMPFAGMw/IE8xAqwlpYfyBxmAmaMsoGAWjYBSMKAAAqexKRzapMDkAAAAASUVORK5CYII=","orcid":"","institution":"University of Lagos","correspondingAuthor":true,"prefix":"","firstName":"Idowu","middleName":"","lastName":"Aneyo","suffix":""},{"id":471347762,"identity":"b159a0b0-4610-48b6-b285-42bb6ddae3f6","order_by":1,"name":"Temitope Fadipe","email":"","orcid":"","institution":"Federal Institute of Industrial Research","correspondingAuthor":false,"prefix":"","firstName":"Temitope","middleName":"","lastName":"Fadipe","suffix":""},{"id":471347763,"identity":"f6d453c8-0d85-4010-ae48-1da5e534f8ac","order_by":2,"name":"Funmilayo Doherty","email":"","orcid":"","institution":"Yaba College of Technology","correspondingAuthor":false,"prefix":"","firstName":"Funmilayo","middleName":"","lastName":"Doherty","suffix":""},{"id":471347764,"identity":"a67a48f8-1886-44fc-9e23-701e7c59054a","order_by":3,"name":"Funsho Oluwaloni","email":"","orcid":"","institution":"Federal Institute of Industrial Research","correspondingAuthor":false,"prefix":"","firstName":"Funsho","middleName":"","lastName":"Oluwaloni","suffix":""},{"id":471347765,"identity":"40e7a698-8125-4659-921c-6676189fadb8","order_by":4,"name":"Oluwayomi Adeyemi","email":"","orcid":"","institution":"University of Lagos","correspondingAuthor":false,"prefix":"","firstName":"Oluwayomi","middleName":"","lastName":"Adeyemi","suffix":""},{"id":471347766,"identity":"2583a8f1-1e07-4508-a0c0-6184f8cb9801","order_by":5,"name":"Funmilayo Dada","email":"","orcid":"","institution":"Federal Institute of Industrial Research","correspondingAuthor":false,"prefix":"","firstName":"Funmilayo","middleName":"","lastName":"Dada","suffix":""},{"id":471347767,"identity":"32ba4edb-6a87-499e-a11e-bfe84627626c","order_by":6,"name":"Olubunmi Ibidapo","email":"","orcid":"","institution":"Federal Institute of Industrial Research","correspondingAuthor":false,"prefix":"","firstName":"Olubunmi","middleName":"","lastName":"Ibidapo","suffix":""},{"id":471347768,"identity":"1345456b-5527-4446-bc1b-171d0acfcf4a","order_by":7,"name":"Sandra Ajaero","email":"","orcid":"","institution":"University of Lagos","correspondingAuthor":false,"prefix":"","firstName":"Sandra","middleName":"","lastName":"Ajaero","suffix":""},{"id":471347769,"identity":"d1713efa-69ef-4905-8f51-5fb55d7a2b9c","order_by":8,"name":"Muideen Ismail","email":"","orcid":"","institution":"University of Lagos","correspondingAuthor":false,"prefix":"","firstName":"Muideen","middleName":"","lastName":"Ismail","suffix":""},{"id":471347770,"identity":"de95dbbf-86c6-4c08-a93c-4bbc206b042d","order_by":9,"name":"Oluwagbemiga Igbagbosanmi","email":"","orcid":"","institution":"Yaba College of Technology","correspondingAuthor":false,"prefix":"","firstName":"Oluwagbemiga","middleName":"","lastName":"Igbagbosanmi","suffix":""},{"id":471347771,"identity":"6b440131-2eba-4d4c-9799-a112d08d923e","order_by":10,"name":"Abiodun Toku","email":"","orcid":"","institution":"Yaba College of Technology","correspondingAuthor":false,"prefix":"","firstName":"Abiodun","middleName":"","lastName":"Toku","suffix":""}],"badges":[],"createdAt":"2025-06-03 13:38:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6811857/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6811857/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84712866,"identity":"33034d4e-1eca-4137-a380-e234c821d3b2","added_by":"auto","created_at":"2025-06-16 13:43:38","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":583844,"visible":true,"origin":"","legend":"\u003cp\u003eMap of Study area showing the sampling sites\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6811857/v1/a564275e2af41cb68f469024.jpeg"},{"id":84714159,"identity":"c1b5f077-ff54-4d96-b646-62d9b06a593f","added_by":"auto","created_at":"2025-06-16 13:59:38","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":374536,"visible":true,"origin":"","legend":"\u003cp\u003eFungal cultures of feed and fecal isolates on PDA after 5 days of incubation: (A) \u003cem\u003ePichia spp.\u003c/em\u003e (B) \u003cem\u003eAspergillus\u003c/em\u003e \u003cem\u003espp.\u003c/em\u003e (C) \u003cem\u003eAspergillus\u003c/em\u003e \u003cem\u003espp.\u003c/em\u003e (D) \u003cem\u003eRhizopus\u003c/em\u003e \u003cem\u003espp. \u003c/em\u003e(E) \u003cem\u003eTrichoderma\u003c/em\u003e \u003cem\u003espp.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6811857/v1/835c1f085316f085710d965f.jpeg"},{"id":84713285,"identity":"250a1b05-7de2-4706-a550-64deb25af575","added_by":"auto","created_at":"2025-06-16 13:51:39","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":175092,"visible":true,"origin":"","legend":"\u003cp\u003eAgarose gel electrophoresis image of PCR-specific bands visualized by UV trans-illumination. Samples that produced multiple bands (wells L, M, O and S) were re-amplified, samples in wells L and O returned 551 and 650 bp respectively, while DNA in well M produced two bands that were 445 and 5581 bp in size. DNA sample in well S produced several bands even after re-amplification.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6811857/v1/a56f11856999cdf6ad0e4aed.jpeg"},{"id":84712869,"identity":"743bb6ef-44fa-48b7-b959-58502123ea3c","added_by":"auto","created_at":"2025-06-16 13:43:38","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":307769,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum likelihood phylogenetic tree showing the relationships between the examined fungal isolates from feed (AFS1-9) and faeces (AFI1-6) and reference strains from GenBank based on Jukes-Cantor method. Triangles represent registered farms; squares represent unregistered farms. Isolates from faeces are coloured red while isolates from feed are coloured blue. References strains from GenBank are represented with black circles.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6811857/v1/2563d4d7c1ec698cee47035e.jpeg"},{"id":84712871,"identity":"502efc61-247a-40ef-ae59-161038450995","added_by":"auto","created_at":"2025-06-16 13:43:38","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":484121,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum likelihood phylogenetic tree showing the relationships between the examined fungal isolates from feed (AFS1-9) and faeces (AFI1-6) and reference strains from GenBank based on Tamura-Nei method. Triangles represent registered farms; squares represent unregistered farms. Isolates from faeces are coloured red while isolates from feed are coloured blue. References strains from GenBank are represented with black circles.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6811857/v1/2fd4f26946077090264b0b74.jpeg"},{"id":84712875,"identity":"64513b34-0e67-428b-b3d8-7d41b787ccbd","added_by":"auto","created_at":"2025-06-16 13:43:38","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":25008,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum likelihood phylogenetic tree showing the relationships between the examined fungal isolates from feed (AFS1-9) and faeces (AFI1-6) and reference strains from GenBank based on Maximum Composite Likelihood method. Triangles represent registered farms; squares represent unregistered farms. Isolates from faeces are coloured red while isolates from feed are coloured blue. References strains from GenBank are represented with black circles.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6811857/v1/deaa1d006ffd083bd1a9dae1.png"},{"id":84714160,"identity":"1e158152-89f9-4da4-bec9-40ff56a168f6","added_by":"auto","created_at":"2025-06-16 13:59:38","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":483979,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum likelihood phylogenetic tree showing the relationships between the examined fungal isolates from feed (AFS1-9) and faeces (AFI1-6) and reference strains from GenBank based on p-distance method. Triangles represent registered farms; squares represent unregistered farms. Isolates from faeces are coloured red while isolates from feed are coloured blue. References strains from GenBank are represented with black circles.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6811857/v1/3ac52b0c7b952485bc28751a.jpeg"},{"id":86422568,"identity":"5d33bced-f0c9-4f5d-84c2-ec0440771358","added_by":"auto","created_at":"2025-07-10 13:01:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3688013,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6811857/v1/d106f8a6-c6bb-4886-a2ac-943084d6d6e8.pdf"},{"id":84713275,"identity":"d3deea0d-93a8-4001-991c-b812b652cb7f","added_by":"auto","created_at":"2025-06-16 13:51:38","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":101376,"visible":true,"origin":"","legend":"","description":"","filename":"UncroppedGelImagesSupplementaryFigure.doc","url":"https://assets-eu.researchsquare.com/files/rs-6811857/v1/5db34ce144878e7709f51612.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"Molecular Characterization and Phylogenetic Studies of Fungal Species in Piggeries and Implications for Mycotoxin Contamination and Food Safety","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003eThe fungal kingdom represents an extraordinary diversity of organisms with profound impacts across animal and plant species and ecosystem health (Fisher \u003cem\u003eet al.\u003c/em\u003e,2020). They exist freely or in symbiosis with bacteria, plants, or animals and are identified by their filamentous growth (hyphae) and external digestion (Mo\u0026euml;nne-Loccoz et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Fungi colonize animal feeds through exposure to various environmental matrices such as soil, dust, and insects. The microbial diversity in feeds depends on factors like moisture, pH, oxygen levels, and nutrient content, with some moulds capable of growing even in dry, stored grains (Maciorowski et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCertain fungi produce mycotoxins, toxic secondary metabolites found in agricultural products that are susceptible to mold infestation (R\u0026aacute;duly et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In livestock, mycotoxins reduce productivity by impairing growth, decreasing milk and egg production, and causing immune suppression, reproductive issues, and organ damage. Environmentally, mycotoxins also contaminate soil and water, resulting in crop losses, economic damage, and disruptions to biodiversity. Their persistence in the food chain further amplifies these risks.\u003c/p\u003e \u003cp\u003eMycotoxigenic fungi that contaminate the human food chain primarily belong to three genera: \u003cem\u003eAspergillus, Fusarium\u003c/em\u003e, and \u003cem\u003ePenicillium\u003c/em\u003e (R\u0026aacute;duly et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). \u003cem\u003eFusarium\u003c/em\u003e species are aggressive plant pathogens that produce mycotoxins either before harvest or soon after, while \u003cem\u003ePenicillium\u003c/em\u003e and \u003cem\u003eAspergillus\u003c/em\u003e species are more commonly linked to food contamination during drying and storage (Ezekiel \u003cem\u003eet al.\u003c/em\u003e, 2020). The susceptibility of crops to these fungi varies; for instance, \u003cem\u003eFusarium\u003c/em\u003e species frequently affect corn, whereas \u003cem\u003eAspergillus flavus\u003c/em\u003e is a major contaminant in peanuts (Dohlman \u003cem\u003eet al\u003c/em\u003e., 2011). Post-harvest handling plays a crucial role in determining mycotoxin contamination levels. Poor storage conditions, such as high moisture and inadequate ventilation, create an ideal environment for fungal growth and toxin production in stored grains (Pitt \u0026amp; Hocking, 2009). Furthermore, processing techniques like milling and grinding can concentrate mycotoxins in specific grain fractions, increasing potential health risks (Pickova \u003cem\u003eet al\u003c/em\u003e., 2021).\u003c/p\u003e \u003cp\u003eIn recent years, the incidence of mycotoxin contamination in swine feed has drawn considerable attention (Haque et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Among livestock species, pigs are particularly vulnerable to mycotoxin exposure due to their dietary requirements (Yang et al, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Sources of mycotoxin contamination in piggeries include consumption of moldy grains, improper feed storage, and exposure to contaminated water or bedding materials, all of which contribute to the spread of these harmful toxins. Humans are exposed to these toxins primarily by consuming contaminated pork and dairy products, which pose significant health risks. Additionally, occupational exposure among farmers, butchers, and meat processors, as well as environmental contamination from pig waste, further increases the likelihood of human contact. Ensuring food safety and preventing food-borne diseases in humans and animals is a growing priority for specialists in human and veterinary medicine worldwide.\u003c/p\u003e \u003cp\u003eMycotoxins have the potential to disrupt global food supply chains, making it a global concern that requires continuous monitoring, regulation, and control (FAO/WHO, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). While developed countries have well-developed infrastructures for monitoring food quality and ensuring high standards, weaker monitoring systems and limited enforcement of safety regulations increase contamination risks in developing regions (Khalid, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eMolecular techniques such as PCR and phylogenetic analysis are widely used for fungal identification, but studies in developing countries remain limited. A key gap exists in the molecular characterization of mycotoxin-producing fungi in pig farms, especially in unregistered farms with poor management and inadequate regulatory oversight (Jayawardhana \u003cem\u003eet al.\u003c/em\u003e, 2020; Liu \u003cem\u003eet al.\u003c/em\u003e, 2023). Addressing these gaps will promote food safety monitoring, guide policy recommendations, and help mitigate mycotoxin risks in livestock and human food chains. This study therefore aimed to identify and molecularly characterize fungal species occurring in pig faeces and feed in registered and unregistered farms in Lagos State, Nigeria.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003eStudy area\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted in four pig farms in Lagos State with GPS coordinates latitude 6.465422 and longitude 3.406448 (Figure 1). The farms were selected based on their registration status, geographical distribution, and willingness to participate in study. Two registered farms at Odogunyan (ST1) and Abule Oshorun (ST3) and two unregistered farms at Gberigbe (ST2) and Ebute-Meta (ST4) were included, along with a control site at the Federal College of Education, Akoka, Yaba. The registration status of the farms was verified through the Lagos State Ministry of Agriculture by reviewing farm registration certificates and consulting the Livestock Department. Registered farms adhered to government regulations, while unregistered farms operated without formal approval.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCollection and preservation of samples\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZipper storage bags were used to collect feed samples from the selected farms, labelled appropriately, and placed in well-labeled, clean plastic polythene bags. Fecal samples were collected at random from 3 pigs on each farm. They were collected directly into sterile sample bottles as soon as they were egested by each selected pig to avoid contamination from the piggery. The sample bottles were appropriately labelled and carefully placed in clean plastic polythene bags\u003cem\u003e,\u003c/em\u003e each marked with its corresponding label. Subsequently\u003cem\u003e,\u003c/em\u003e the samples were transported to the laboratory for analysis. To maintain their integrity\u003cem\u003e,\u003c/em\u003e the samples were stored in a refrigerator regulated at -4\u0026deg;C until time for laboratory analyses.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicrobial analysis of samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAgar preparation and sterilization\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExactly 39-gram Potato Dextrose Agar (PDA) and 15 mg streptomycin were suspended into a 1000 ml conical flask filled with 1000 ml distilled water. The mixture was stirred with a stirring rod to dissolve the medium completely and the flask was corked with aluminum foil and tightened properly using paper tape. Nine milliliters of distilled water were pipetted into 96 test tubes and corked properly. In the autoclave, the medium and test tubes were placed and autoclaved at 121\u003csup\u003eo\u003c/sup\u003eC (15 Ibs pressure) for 15 minutes and allowed to cool at 45\u003csup\u003eo\u003c/sup\u003eC.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSerial dilution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing one in eight serial dilution method, the test tubes were labelled A\u003csup\u003e-1\u003c/sup\u003e to A\u003csup\u003e-8\u003c/sup\u003e, B\u003csup\u003e-1\u003c/sup\u003e to B\u003csup\u003e-8\u003c/sup\u003e, C\u003csup\u003e-1\u003c/sup\u003e to C\u003csup\u003e-8\u003c/sup\u003e, D\u003csup\u003e-I\u003c/sup\u003e to D\u003csup\u003e-8\u003c/sup\u003e, E\u003csup\u003e-1\u003c/sup\u003e to E\u003csup\u003e-8\u003c/sup\u003e, F\u003csup\u003e-1\u003c/sup\u003e to F\u003csup\u003e-8,\u0026nbsp;\u003c/sup\u003eG\u003csup\u003e-1\u003c/sup\u003e to G\u003csup\u003e-8\u003c/sup\u003e\u003cem\u003e,\u003c/em\u003e H\u003csup\u003e-1\u003c/sup\u003e to H\u003csup\u003e-8\u003c/sup\u003e\u003cem\u003e,\u003c/em\u003e I\u003csup\u003e-1\u003c/sup\u003e to I\u003csup\u003e-8\u003c/sup\u003e\u003cem\u003e,\u003c/em\u003eJ\u003csup\u003e-1\u003c/sup\u003e to J\u003csup\u003e-8\u003c/sup\u003e\u003cem\u003e,\u003c/em\u003eK\u003csup\u003e-1\u003c/sup\u003e to K\u003csup\u003e-8\u003c/sup\u003e\u003cem\u003e,\u003c/em\u003eL\u003csup\u003e-1\u003c/sup\u003e to L\u003csup\u003e-8\u003c/sup\u003e\u003cem\u003e,\u003c/em\u003e M\u003csup\u003e-1\u003c/sup\u003e to M\u003csup\u003e-8\u003c/sup\u003e\u003cem\u003e,\u003c/em\u003e N\u003csup\u003e-1\u003c/sup\u003e to N\u003csup\u003e-8\u003c/sup\u003e\u003cem\u003e,\u003c/em\u003e O\u003csup\u003e-1\u003c/sup\u003e to O\u003csup\u003e-8\u003c/sup\u003e\u003cem\u003e,\u003c/em\u003eP\u003csup\u003e-1\u003c/sup\u003e to P\u003csup\u003e-8\u003c/sup\u003e\u003cem\u003e,\u003c/em\u003eQ\u003csup\u003e-1\u003c/sup\u003e to Q\u003csup\u003e-8\u003c/sup\u003e\u003cem\u003e,\u003c/em\u003e and R\u003csup\u003e-1\u003c/sup\u003e to R\u003csup\u003e-8\u003c/sup\u003e. \u0026nbsp; One gram each of the feed samples were poured into test tubes labelled A\u003csup\u003e-1\u003c/sup\u003e, B\u003csup\u003e-1\u003c/sup\u003e, C\u003csup\u003e-1\u003c/sup\u003e, D\u003csup\u003e-1\u003c/sup\u003e, E\u003csup\u003e-1\u003c/sup\u003e and F\u003csup\u003e-1\u003c/sup\u003e respectively while 1 gram of fecal sample was transferred into corresponding test tubes\u003cem\u003e\u0026nbsp;\u003c/em\u003elabeled G\u003csup\u003e-1\u003c/sup\u003e\u003cem\u003e,\u003c/em\u003e H\u003csup\u003e-1\u003c/sup\u003e\u003cem\u003e,\u003c/em\u003e I\u003csup\u003e-1\u003c/sup\u003e\u003cem\u003e,\u003c/em\u003e J\u003csup\u003e-1\u003c/sup\u003e\u003cem\u003e,\u003c/em\u003e K\u003csup\u003e-1\u003c/sup\u003e\u003cem\u003e,\u003c/em\u003e L\u003csup\u003e-1\u003c/sup\u003e\u003cem\u003e,\u003c/em\u003e M\u003csup\u003e-1\u003c/sup\u003e\u003cem\u003e,\u003c/em\u003e N\u003csup\u003e-1\u003c/sup\u003e\u003cem\u003e,\u003c/em\u003e O\u003csup\u003e-1\u003c/sup\u003e\u003cem\u003e,\u003c/em\u003e P\u003csup\u003e-1\u003c/sup\u003e\u003cem\u003e,\u003c/em\u003e Q\u003csup\u003e-1\u003c/sup\u003e and R\u003csup\u003e-1\u003c/sup\u003e. \u0026nbsp; The suspensions were mixed thoroughly to obtain a homogenous suspension. From test tube A\u003csup\u003e-1\u003c/sup\u003e, 1 ml of the suspension was pipetted into A\u003csup\u003e-2\u003c/sup\u003e, 1 ml from A\u003csup\u003e-2\u0026nbsp;\u003c/sup\u003ewas pipetted into A\u003csup\u003e-3\u003c/sup\u003e, 1 ml from A\u003csup\u003e-3\u0026nbsp;\u003c/sup\u003ewas pipetted into A\u003csup\u003e-\u003c/sup\u003e4, 1 ml from A\u003csup\u003e-4\u0026nbsp;\u003c/sup\u003ewas pipetted into A\u003csup\u003e-\u003c/sup\u003e5, 1 ml from A\u003csup\u003e-5\u0026nbsp;\u003c/sup\u003ewas pipetted into A\u003csup\u003e-6\u003c/sup\u003e,\u003csup\u003e\u0026nbsp;\u003c/sup\u003e1 ml from A\u003csup\u003e-6\u0026nbsp;\u003c/sup\u003ewas pipetted into A\u003csup\u003e-7\u003c/sup\u003e and 1 ml from A\u003csup\u003e-7\u0026nbsp;\u003c/sup\u003ewas pipetted into A\u003csup\u003e-8\u003c/sup\u003e. The same procedure was done for B\u003csup\u003e-1\u003c/sup\u003e to R\u003csup\u003e-8\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFungi culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe culture medium was mixed properly before pouring into labelled 162 sterile petri dishes. Each milliliter from test tubes labelled -4, -6, and -8 were poured into 54 petri dishes using the pour plate method and rocked gently. The culture was replicated giving a total of 162 petri dishes. The medium was allowed to solidify and incubated at 37\u003csup\u003eo\u003c/sup\u003eC for 5 days alongside 3 control petri dishes containing PDA with no streak.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSub-culturing of samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eColonies grown on each medium were distinguished based on their surface characteristics such as texture, color, zonation, sporulation, and diameters. The distinguishable colonies were sub-cultured on a PDA slant and incubated at room temperature for 7\u0026nbsp;days. To obtain pure strains, from the sub-cultured plates, using an inoculating loop, each isolated colony was picked and transferred into already labeled MacConkey bottles filled with 9 ml Potato Dextrose Broth and placed in an incubator shaker for 4 days at 29\u003csup\u003eo\u003c/sup\u003eC. The isolates were identified, and pure isolates were maintained in PDA slants and stored in the refrigerator for further identification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFungi genomic DNA extraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe fungal tissues obtained were transferred into labeled 1.5 ml microcentrifuge tubes aseptically using a pair of sterile tweezers. These tissues were frozen by dipping in liquid nitrogen. The frozen tissues were ground using sterilized laboratory mortar and pestle to release the contents of the spore. Exactly 100 mg of the powdered tissues were weighed and transferred into new d microcentrifuge tubes. The manual procedures were followed. The quality of the extracted DNA was assessed by electrophoresis in 1% agarose gel (1-gram agarose in 100 ml Tris buffer). DNA concentration was measured at the optical density (OD) of 260 nm and purity was recorded at 260 and 280 nm wavelengths using a UV spectrophotometer before storing extracted DNA at -20\u0026deg;C for subsequent analyses.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePCR amplification and gel electrophoresis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrimers targeting the internal transcribed spacer DNA namely ITS1 5\u0026apos; (TCC GTA GGT GAA CCT GCG G) 3\u0026apos; (forward primer) and ITS4 5\u0026apos; (TCC TCC GCT TAT TGA TAT GC) 3\u0026apos; (reverse) with length of 600 bp were employed for amplification. Nine isolates with DNA purity levels between 1.80 - 2.20 ng/\u0026micro;l were used for PCR amplification ensuring all farms were represented. The reaction was performed in a 20 \u0026micro;L mixture as follows: 1 \u0026micro;L gDNA template, 0.2 \u0026micro;L DNA polymerase, 0.5 \u0026micro;L each of forward and reverse primers, 1 \u0026micro;L dNTPs and sterile double distilled water. The thermocycler was programed for the following PCR conditions: initial denaturation at 95 \u003csup\u003eo\u003c/sup\u003eC for 5 mins, 30 cycles of denaturation at 95 \u003csup\u003eo\u003c/sup\u003eC for 30 s, annealing at 52 \u003csup\u003eo\u003c/sup\u003eC for 60 s, and extension at 72 \u003csup\u003eo\u003c/sup\u003eC for 2 min, with a final extension at 72 \u003csup\u003eo\u003c/sup\u003eC for 10 mins. After amplification, the PCR products were subjected to gel electrophoresis using 1% agarose gel with ethidium bromide as the staining agent and PCR-specific bands visualized by UV trans-illumination. A segment of DNA of known size was used as positive control while molecular grade water was used as negative control. The size of the amplicons was estimated using a 1\u0026nbsp;kb\u0026nbsp;DNA\u0026nbsp;ladder.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSequence and phylogenetic analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeveral isolates produced multiple bands, each of which was purified for sequencing and analysis. The PCR amplicons from \u003cstrong\u003esix\u003c/strong\u003e isolates obtained from pig feces and \u003cstrong\u003enine\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eisolates from feed were purified and sequenced by Inqaba Biotec West Africa (Ibadan, Nigeria). The raw sequences were then trimmed and edited using \u003cstrong\u003eSequencher 5.4.6\u003c/strong\u003e and \u003cstrong\u003eSnapGene 7.0.1\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e Sequence homology analysis was performed using the \u003cstrong\u003eNCBI nucleotide BLAST tool\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e while sequence alignment was conducted using \u003cstrong\u003eClustalW\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ein\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eMEGA 11\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e The evolutionary distances were computed using the Tamura-Nei method (Tamura and Nei, 1993) and Jukes-Cantor method (Jukes and Cantor, 1969) and are in the units of the number of base substitutions per site. These analyses involved 12 nucleotide sequences respectively. All ambiguous positions were removed for each sequence pair (pairwise deletion option). Evolutionary analyses were conducted in MEGA11 (Tamura \u003cem\u003eet al.,\u003c/em\u003e 2021).\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eMicrofungal isolates\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe microbial plates showed cultural features (color/texture) and sporangiospores/conidia characteristic of the different microfungal genera identified (Figure 2). The occurrence of fungal contamination in feed and faeces are summarized in Table 1. Feed samples collected from registered and unregistered farms were all contaminated with fungi. Fungal contamination in faeces was more frequent in unregistered farms (ST2 - 100.0%; ST4 - 66.7%) than in registered farms (ST1 - 66.7%, ST3 - 33.3%). Microfungi belonging to the genera \u003cem\u003eAspergillus\u003c/em\u003e, \u003cem\u003ePichia\u003c/em\u003e, \u003cem\u003eRhizopus\u003c/em\u003e, and \u003cem\u003eTrichoderma\u003c/em\u003e were isolated and purified from feed while\u003cem\u003e\u0026nbsp;Aspergillus\u003c/em\u003e and \u003cem\u003eCandida\u003c/em\u003e genera were isolated and purified from faeces (Table 1). \u003cem\u003eAspergillus\u003c/em\u003e genus was detected in samples from registered and unregistered farms. \u003cem\u003eTrichoderma\u003c/em\u003e sp. was found contaminating only samples from registered farms while \u003cem\u003eCandida\u003c/em\u003e sp. was only found in unregistered farms.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1:\u003c/strong\u003e Occurrence of fungi genera in feed and feaces collected from registered and unregistered farms in Lagos, Nigeria\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"669\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFarm\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSample type\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo. collected\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo. of samples contaminated\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePercentage occurrence (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 234px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFungi genera\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003eST1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003eFeed\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e100.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 234px;\"\u003e\n \u003cp\u003e\u003cem\u003eAspergillus\u003c/em\u003e sp., \u003cem\u003ePichia\u003c/em\u003e sp.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003eFaeces\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e2\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e66.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 234px;\"\u003e\n \u003cp\u003e\u003cem\u003eAspergillus\u003c/em\u003e sp.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003eST2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003eFeed\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e2\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e100.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 234px;\"\u003e\n \u003cp\u003e\u003cem\u003ePichia\u003c/em\u003e sp., \u003cem\u003eRhizopus\u003c/em\u003e sp.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003eFaeces\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e100.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 234px;\"\u003e\n \u003cp\u003e\u003cem\u003eCandida\u003c/em\u003e sp., \u003cem\u003eAspergillus\u003c/em\u003e sp.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003eST3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003eFeed\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e2\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e100.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 234px;\"\u003e\n \u003cp\u003e\u003cem\u003eTrichoderma\u003c/em\u003e sp., \u003cem\u003eAspergillus\u003c/em\u003e sp.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003eFaeces\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e33.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 234px;\"\u003e\n \u003cp\u003e\u003cem\u003eAspergillus\u003c/em\u003e sp.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003eST4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003eFeed\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e100.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 234px;\"\u003e\n \u003cp\u003e\u003cem\u003eAspergillus\u003c/em\u003e sp., \u003cem\u003eRhizopus\u003c/em\u003e sp.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003eFaeces\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e2\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e66.7%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 234px;\"\u003e\n \u003cp\u003e\u003cem\u003eCandida\u003c/em\u003e sp.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eST1 and ST3 = registered farms; ST2 and ST4 = unregistered farms\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular characterization\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAgarose gel electrophoresis revealed amplicons with molecular weight between 353 and 650 bp. Some isolates returned multiple bands even after reamplification (Figure 3). PCR amplicons from 9 feed isolates and 6 faecal isolates were sequenced and deposited in NCBI-GenBank with accession numbers, OR652266-74,\u0026nbsp;OR646806-11 as shown in Table 2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2:\u0026nbsp;\u003c/strong\u003eNCBI-GenBank accession numbers and submission information\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"625\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNCBI-GenBank Code\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAccession no.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 176px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIdentity\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFarm Status\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNucleotide sequences (bp)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFeed sample\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 176px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\u0026nbsp;\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003eAFS1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003eOR652266\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 176px;\"\u003e\n \u003cp\u003e\u003cem\u003eAspergillus flavus\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eST1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e551\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\u0026nbsp;\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003eAFS2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003eOR652267\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 176px;\"\u003e\n \u003cp\u003e\u003cem\u003ePichia kudriavzevii\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eST2\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e445\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\u0026nbsp;\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003eAFS3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003eOR652268\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 176px;\"\u003e\n \u003cp\u003e\u003cem\u003eRhizopus oryzae\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eST2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e581\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\u0026nbsp;\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003eAFS4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003eOR652269\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 176px;\"\u003e\n \u003cp\u003e\u003cem\u003eRhizopus microsporus\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eST2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e650\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\u0026nbsp;\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003eAFS5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003eOR652270\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 176px;\"\u003e\n \u003cp\u003e\u003cem\u003eTrichoderma longibrachiatum\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eST3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e383\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\u0026nbsp;\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003eAFS6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003eOR652271\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 176px;\"\u003e\n \u003cp\u003e\u003cem\u003ePichia kudriavzevii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eST1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e463\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\u0026nbsp;\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003eAFS7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003eOR652272\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 176px;\"\u003e\n \u003cp\u003e\u003cem\u003eAspergillus niger\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eST3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e484\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\u0026nbsp;\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003eAFS8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003eOR652273\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 176px;\"\u003e\n \u003cp\u003e\u003cem\u003eAspergillus flavus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eST4\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e550\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\u0026nbsp;\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003eAFS9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003eOR652274\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 176px;\"\u003e\n \u003cp\u003e\u003cem\u003eRhizopus oryzae\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eST4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e584\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eFecal sample\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 176px;\"\u003e\u0026nbsp;\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\u0026nbsp;\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003eAFI1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003eOR646806\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 176px;\"\u003e\n \u003cp\u003e\u003cem\u003eAspergillus niger\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eST1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e552\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\u0026nbsp;\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003eAFI2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003eOR646807\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 176px;\"\u003e\n \u003cp\u003e\u003cem\u003eCandida akabanensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eST2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e354\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\u0026nbsp;\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003eAFI3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003eOR646808\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 176px;\"\u003e\n \u003cp\u003e\u003cem\u003eAspergillus niger\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eST3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\u0026nbsp;\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003eAFI4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003eOR646809\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 176px;\"\u003e\n \u003cp\u003e\u003cem\u003eAspergillus flavus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eST3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e542\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\u0026nbsp;\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003eAFI5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003eOR646810\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 176px;\"\u003e\n \u003cp\u003e\u003cem\u003eAspergillus flavus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eST2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e550\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\u0026nbsp;\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003eAFI6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003eOR646811\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 176px;\"\u003e\n \u003cp\u003e\u003cem\u003eCandida akabanensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eST4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e353\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eST1 and ST3 = registered farms; ST2 and ST4 = unregistered farms; \u0026lsquo;AFS\u0026rsquo; represents feed samples; \u0026lsquo;AFI\u0026rsquo; represents faecal samples\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable 3 shows a diverse range of fungal organisms isolated from feed and fecal samples, predominantly originating from China, Vietnam, and South Africa. Isolates from the feed samples exhibited perfect genetic matches with \u003cstrong\u003e\u003cem\u003eAspergillus flavus\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;LUOHE\u003c/strong\u003e and \u003cstrong\u003e\u003cem\u003eRhizopus arrhizus\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;jx01\u003c/strong\u003e from China. Similarly, fungal isolate AFS5 had 100% identity match with an Egyptian strain, \u003cstrong\u003e\u003cem\u003eTrichoderma longibrachiatum\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;KABOFT5.\u003c/strong\u003e \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3: Identity of Strains with Closest Homology from NCBI-BLAST\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"620\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 337px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOrganism with Closest Homology\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLocation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e% Identity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFeed sample\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 337px;\"\u003e\n \u003cp\u003e\u003cem\u003eAspergillus flavus\u0026nbsp;\u003c/em\u003eLUOHE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eChina\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\u0026nbsp;\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 337px;\"\u003e\n \u003cp\u003e\u003cem\u003ePichia kudriavzevii\u0026nbsp;\u003c/em\u003eer 2\u003cem\u003e\u0026nbsp;\u003c/em\u003e(\u003cem\u003eIssatchenkia orientalis\u003c/em\u003e er 2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eChina\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e99.78\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\u0026nbsp;\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 337px;\"\u003e\n \u003cp\u003e\u003cem\u003eRhizopus arrhizus\u0026nbsp;\u003c/em\u003ejx01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eChina\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\u0026nbsp;\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 337px;\"\u003e\n \u003cp\u003e\u003cem\u003eRhizopus microsporus\u0026nbsp;\u003c/em\u003eDTO 402-G1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eItaly\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\u0026nbsp;\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 337px;\"\u003e\n \u003cp\u003e\u003cem\u003eTrichoderma longibrachiatum\u0026nbsp;\u003c/em\u003e KABOFT5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eEgypt\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\u0026nbsp;\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 337px;\"\u003e\n \u003cp\u003e\u003cem\u003ePichia kudriavzevii\u0026nbsp;\u003c/em\u003eG1-10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eChina\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e99.78\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\u0026nbsp;\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 337px;\"\u003e\n \u003cp\u003e\u003cem\u003eAspergillus niger\u0026nbsp;\u003c/em\u003eG12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eChina\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\u0026nbsp;\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 337px;\"\u003e\n \u003cp\u003e\u003cem\u003eAspergillus flavus\u0026nbsp;\u003c/em\u003e275N\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eChina\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e99.82\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\u0026nbsp;\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 337px;\"\u003e\n \u003cp\u003e\u003cem\u003eRhizopus arrhizus\u0026nbsp;\u003c/em\u003eND4\u003cem\u003e\u0026nbsp;\u0026nbsp;\u003c/em\u003e(\u003cem\u003eRhizopus oryzae \u0026nbsp;\u003c/em\u003eND4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eVietnam\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e99.83\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFecal sample\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 337px;\"\u003e\n \u003cp\u003e\u003cem\u003eAspergillus niger\u0026nbsp;\u003c/em\u003eNW-47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eSouth Africa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\u0026nbsp;\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 337px;\"\u003e\n \u003cp\u003e\u003cem\u003eCandida akabanensis\u0026nbsp;\u003c/em\u003eTB1Y\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eVietnam\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e99.72\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\u0026nbsp;\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 337px;\"\u003e\n \u003cp\u003e\u003cem\u003eAspergillus niger\u003c/em\u003e G12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eChina\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e99.60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\u0026nbsp;\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 337px;\"\u003e\n \u003cp\u003e\u003cem\u003eAspergillus flavus\u0026nbsp;\u003c/em\u003e275N\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eChina\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e99.82\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\u0026nbsp;\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 337px;\"\u003e\n \u003cp\u003e\u003cem\u003eAspergillus flavus\u0026nbsp;\u003c/em\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eSouth Africa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\u0026nbsp;\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 337px;\"\u003e\n \u003cp\u003e\u003cem\u003eCandida akabanensis\u0026nbsp;\u003c/em\u003eTB1Y\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eVietnam\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic relationships\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe phylogenetic analysis of fungal isolates from pig feed and fecal samples was conducted using four evolutionary models Jukes-Cantor, Tamura-Nei, Maximum Composite Likelihood, and p-distance via the Neighbor-Joining method in MEGA11 (Figures 4-7). All four models produced consistent phylogenetic trees, grouping the fungal species into well-supported clades, with only minor differences in branch lengths and bootstrap values. Isolates of \u003cem\u003eAspergillus flavus\u003c/em\u003e, \u003cem\u003eAspergillus niger\u003c/em\u003e, \u003cem\u003eRhizopus arrhizus\u003c/em\u003e, \u003cem\u003eRhizopus microsporus\u003c/em\u003e, \u003cem\u003ePichia kudriavzevii\u003c/em\u003e, \u003cem\u003eTrichoderma longibrachiatum\u003c/em\u003e, and \u003cem\u003eCandida akabanensis\u003c/em\u003e consistently clustered with reference strains from regions such as China, South Africa, and Vietnam. There was no significant genetic divergence was observed between isolates from registered and unregistered farms. \u0026nbsp;\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study explored the occurrence and genetic diversity of fungal species contaminating feed and fecal samples from registered and unregistered pig farms in Lagos State, Nigeria. The results underscore the widespread occurrence of fungal species, particularly from the \u003cem\u003eAspergillus\u003c/em\u003e genus, which have known implications for food safety and animal health due to their potential to produce harmful mycotoxins.\u003c/p\u003e\n\u003cp\u003eThis study showed high genetic similarity observed among fungal isolates from both regulated and unregulated farms, with several isolates showing 100% identity with known strains from other regions. The absence of genetic differentiation agrees with similar research conducted on fungal species in agricultural environments (Kepler \u003cem\u003eet al\u003c/em\u003e., 2015). \u0026nbsp; Wokorach \u003cem\u003eet al.\u003c/em\u003e (2021) who reported genetic characterization of fungal biodiversity in storage grains in Northern Uganda found that environmental factors such as humidity, temperature can influence the proliferation of genetically similar fungal species across different geographic regions. Similar pattern was observed among fecal isolates exhibiting 100% identity matches with \u003cstrong\u003e\u003cem\u003eAspergillus niger\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;NW-47\u003c/strong\u003e and \u003cstrong\u003e\u003cem\u003eCandida akabanensis\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;TB1Y\u003c/strong\u003e strains from South Africa and Vietnam respectively. Faecal isolates also showed at least 99% similarity with \u003cstrong\u003e\u003cem\u003eAspergillus flavus\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;275N\u003c/strong\u003e and \u003cstrong\u003e\u003cem\u003eAspergillus niger\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;G12\u003c/strong\u003e isolates in China.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eStudies carried out in Lagos pig farms also reveal that the environmental conditions in both regulated and unregulated farms do not exert significant selective pressures, allowing genetically stable fungal populations to thrive (Afolabi \u003cem\u003eet al.,\u003c/em\u003e 2023). The presence of \u003cem\u003eAspergillus\u003c/em\u003e species in both feed and fecal samples is similar to the findings from other studies that have highlighted the ubiquitous nature of these fungi in agricultural settings. \u003cem\u003eAspergillus flavus\u003c/em\u003e and \u003cem\u003eAspergillus niger\u003c/em\u003e, two species detected in this study, are well-known mycotoxin producers, particularly aflatoxins, which cause detrimental health risks to both livestock and humans (Zulkifli \u0026amp; Zakaria, 2017). The presence of these species in both regulated and unregulated farms emphasize the need for stringent mitigation and control measures. Poor storage conditions, particularly in unregulated farms, can enhance fungal growth, as seen in similar studies conducted in sub-Saharan Africa, where poor storage and handling practices were linked to increased fungal diversity and mycotoxin levels (Chilaka \u003cem\u003eet al.,\u003c/em\u003e 2022). Research findings from Liu \u003cem\u003eet al.\u003c/em\u003e (2020) demonstrated that environments with proper storage conditions are more susceptible to fungal contamination, which often leads to food spoilage and the production of harmful mycotoxins.\u003c/p\u003e\n\u003cp\u003eThe identification of \u003cem\u003ePichia kudriavzevii\u003c/em\u003e in both feed and fecal samples highlights the widespread distribution of these species, which can be associated with fermentation processes (Zulkifli \u0026amp; Zakaria, 2017). While not typically considered a major mycotoxin producer, its presence in animal feed raises concerns about the potential for fermentation-related spoilage, especially in environments with high moisture content. This agrees with findings from studies on fungal contamination in livestock feed, which shows that the occurrence of such species is often linked to improper feed handling and storage conditions (Wokorach \u003cem\u003eet al.,\u003c/em\u003e 2021).\u003c/p\u003e\n\u003cp\u003eIn addition to \u003cem\u003eAspergillus\u003c/em\u003e and \u003cem\u003ePichia\u003c/em\u003e, \u003cem\u003eRhizopus arrhizus\u003c/em\u003e and \u003cem\u003eRhizopus microsporus\u003c/em\u003e were primarily detected in unregulated farms. These species are mostly associated with food spoilage, especially in environments where hygiene and storage conditions are below standard practice. Liu \u003cem\u003eet al\u003c/em\u003e. (2020) demonstrated that \u003cem\u003eRhizopus\u003c/em\u003e species are prevalent in environments with poor storage practices, where they rapidly colonize and spoil food products, leading to significant economic losses. The detection of these species in unregulated farms further highlights the need for improved storage and handling practices to mitigate the risk of contamination.\u003c/p\u003e\n\u003cp\u003eThe fecal samples also revealed significant fungal contamination in unregulated farms, including the detection of \u003cem\u003eCandida akabanensis\u003c/em\u003e. While typically associated with environmental sources, its presence in fecal samples suggests potential contamination through feed or poor hygiene practices on the farms. This raises biosecurity concerns, particularly regarding the potential for cross-contamination between animals and their environment, which could exacerbate the spread of fungal species capable of producing harmful mycotoxins. Frisvad \u003cem\u003eet al\u003c/em\u003e. (2019) support this finding, who reported fungal contamination in feed and agricultural products shows integral role of hygiene and environmental factors in fungal proliferation.\u003c/p\u003e\n\u003cp\u003eThe phylogenetic analysis reported in this study shows the genetic stability of the fungal isolates, with many showing close genetic clustering with reference strains from other regions. This genetic similarity, particularly among \u003cem\u003eAspergillus flavus\u003c/em\u003e and \u003cem\u003eAspergillus niger\u003c/em\u003e isolates, which showed 100% identity with strains from China and South Africa, underscores the global distribution of these fungi (Frisvad \u003cem\u003eet al.,\u003c/em\u003e 2019). The global ubiquity of these species highlights the need for international collaboration in addressing mycotoxin contamination, as fungal species that thrive in one region may easily spread to others through global trade and agricultural practices (Fumagalli \u003cem\u003eet al.,\u003c/em\u003e 2021).\u003c/p\u003e\n\u003cp\u003eIn conclusion, there is need for improved monitoring and control measures in both regulated and unregulated farms to mitigate the risk of fungal contamination and mycotoxin production. The relatively high genetic similarity of fungal isolates from different sampling sites suggests that these species are well-adapted to several environmental conditions, which further emphasize its importance to global food safety. Therefore, implementing better storage and handling practices, particularly in unregulated farms is required to significantly reduce the risk of fungal contamination and ensure the safety of animal feed and food products in developing regions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll applicable international and institutional guidelines for the care and use of animals were strictly followed. All procedures performed in this study complied with the ethical standards outlined in the 1964 Declaration of Helsinki and its subsequent amendments. Additionally, the study adhered to the ethical guidelines established by the University of Lagos Committee on the Use of Animals in Scientific Research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable \u0026mdash; this study did not involve human participants, personal data, or clinical images requiring consent to participate or publish.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known financial or personal relationships that could have appeared to influence the work reported in this manuscript. All authors have read and approved the final manuscript and confirm that there are no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal Ethics Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable \u0026mdash; this study did not involve any procedures that required approval by an animal ethics committee.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable \u0026ndash; the manuscript does not contain any individual person\u0026rsquo;s data in any form (including individual details, images, or videos).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe DNA sequence data generated and analyzed during this study are available in the NCBI GenBank repository under accession numbers OR652266 to OR652274 and OR646806 to OR646811.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial or non-financial interests that could have influenced the work reported in this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding was received to carry out this research but Institutional resources\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical Trial Registration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable-This study did not involve any clinical trials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are grateful to the management and staff of the Federal Institute of Industrial Research, Oshodi, and the Department of Zoology, University of Lagos for access to laboratory facilities. Special thanks to Inqaba Biotech West Africa for sequencing support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIA:\u003c/strong\u003e Conceptualization, Supervision, Resources, Project administration, Methodology, Investigation, Writing \u0026ndash; original draft. \u003cstrong\u003eTF:\u003c/strong\u003e Resources, Investigation, Methodology, Writing \u0026ndash; original draft. \u003cstrong\u003eFD:\u003c/strong\u003e Methodology, Investigation. \u003cstrong\u003eFO\u003c/strong\u003e: Methodology, Investigation. \u003cstrong\u003eOA:\u003c/strong\u003e Methodology, Investigation, Writing \u0026ndash; review and editing\u003cstrong\u003e. FDa\u003c/strong\u003e: Methodology, Investigation, Writing. \u003cstrong\u003eOI:\u003c/strong\u003e Methodology, Resources, Investigation \u003cstrong\u003eSA:\u003c/strong\u003e Methodology, Investigation. \u003cstrong\u003eMI:\u003c/strong\u003e Methodology, Investigation. \u003cstrong\u003eOIg:\u003c/strong\u003e Methodology, Investigation. \u003cstrong\u003eAT:\u003c/strong\u003e Formal analysis, Data curation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors have read and approved the final version of the manuscript. Each author has agreed to be personally accountable for their own contributions and to ensure that any questions related to the accuracy or integrity of any part of the work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eChilaka, C. A., Obidiegwu, J. E., Chilaka, A. C., Atanda, O. O., \u0026amp; Mally, A. (2022). 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Relationships between food and diseases: What to know to ensure food safety. \u003cem\u003eFood Research International, 137\u003c/em\u003e, 109414. https://doi.org/10.1016/j.foodres.2020.109414\u003c/li\u003e\n\u003cli\u003eHaque, M. A., Wang, Y., Shen, Z., Li, X., Saleemi, M. K., \u0026amp; He, C. (2020). Mycotoxin contamination and control strategy in human, domestic animal and poultry: A review. \u003cem\u003eMicrobial Pathogenesis, 142\u003c/em\u003e, 104095. https://doi.org/10.1016/j.micpath.2020.104095\u003c/li\u003e\n\u003cli\u003eHarman, G. E. (2019). 50 years of development of beneficial microbes for sustainable agriculture and society: Progress and challenges still to be met\u0026mdash;Part of the solution to global warming and \u0026quot;Hothouse Earth\u0026quot;. In S. M. Sarma, A. P. Singh, \u0026amp; R. Prasad (Eds.), \u003cem\u003eMicrobial interventions in agriculture and environment: Volume 1: Research trends, priorities and prospects\u003c/em\u003e (pp. 1-28). Springer. https://doi.org/10.1007/978-3-030-18975-4_1\u003c/li\u003e\n\u003cli\u003eKepler, R. M., Ugine, T. A., Maul, J. E., Cavigelli, M. A., \u0026amp; Rehner, S. A. (2015). Community composition and population genetics of insect pathogenic fungi in the genus \u003cem\u003eMetarhizium\u003c/em\u003e from soils of a long‐term agricultural research system. \u003cem\u003eEnvironmental Microbiology, 17\u003c/em\u003e(8), 2791-2804.\u003c/li\u003e\n\u003cli\u003eKhalid, S. M. N. (2016). Food safety and quality management regulatory systems in Afghanistan: Policy gaps, governance and barriers to success. \u003cem\u003eFood Control, 68\u003c/em\u003e, 192-199. https://doi.org/10.1016/j.foodcont.2016.03.028\u003c/li\u003e\n\u003cli\u003eLevin, R. E. (2012). PCR detection of aflatoxin producing fungi and its limitations. \u003cem\u003eInternational Journal of Food Microbiology, 156\u003c/em\u003e, 1-6. https://doi.org/10.1016/j.ijfoodmicro.2012.03.032\u003c/li\u003e\n\u003cli\u003eLiu, Q., Zhang, Y., \u0026amp; Zhang, J. (2020). The effect of storage conditions on the proliferation of \u003cem\u003eRhizopus\u003c/em\u003e species. \u003cem\u003eJournal of Food Safety, 40\u003c/em\u003e(1), 1081-1091.\u003c/li\u003e\n\u003cli\u003eLiu, Y., Galani Yamdeu, J. H., Gong, Y. Y., \u0026amp; Orfila, C. (2020). A review of postharvest approaches to reduce fungal and mycotoxin contamination of foods. \u003cem\u003eComprehensive Reviews in Food Science and Food Safety, 19\u003c/em\u003e(4), 1521-1560. https://doi.org/10.1111/1541-4337.12564\u003c/li\u003e\n\u003cli\u003eMaciorowski, K. G., Herrera, P., Jones, F. T., Pillai, S. D., \u0026amp; Ricke, S. C. (2007). Effects on poultry and livestock of feed contamination with bacteria and fungi. \u003cem\u003eAnimal Feed Science and Technology\u003c/em\u003e, \u003cem\u003e133\u003c/em\u003e(1-2), 109-136.\u003c/li\u003e\n\u003cli\u003eMo\u0026euml;nne-Loccoz, Y., Mavingui, P., Combes, C., Normand, P., \u0026amp; Steinberg, C. (2015). Microorganisms and biotic interactions. \u003cem\u003eEnvironmental microbiology: fundamentals and applications: microbial ecology\u003c/em\u003e, 395-444.\u003c/li\u003e\n\u003cli\u003ePanghal, A., Chhikara, N., Sindhu, N., \u0026amp; Jaglan, S. (2018). Role of food safety management systems in safe food production: A review. \u003cem\u003eJournal of Food Safety, 38\u003c/em\u003e(4), e12464. https://doi.org/10.1111/jfs.12464\u003c/li\u003e\n\u003cli\u003ePierron, A., Alassane-Kpembi, I., \u0026amp; Oswald, I. P. (2016). Impact of mycotoxin on immune response and consequences for pig health. \u003cem\u003eAnimal Nutrition, 2\u003c/em\u003e(2), 63-68. https://doi.org/10.1016/j.aninu.2016.03.001\u003c/li\u003e\n\u003cli\u003eR\u0026aacute;duly, Z., Szab\u0026oacute;, L., Madar, A., P\u0026oacute;csi, I., \u0026amp; Csernoch, L. (2020). Toxicological and medical aspects of \u003cem\u003eAspergillus\u003c/em\u003e-derived mycotoxins entering the feed and food chain. \u003cem\u003eFrontiers in Microbiology, 10\u003c/em\u003e, 482547. https://doi.org/10.3389/fmicb.2020.00482\u003c/li\u003e\n\u003cli\u003eWokorach, G., Landschoot, S., Audenaert, K., Echodu, R., \u0026amp; Haesaert, G. (2021). Genetic characterization of fungal biodiversity in storage grains: Towards enhancing food safety in Northern Uganda. \u003cem\u003eMicroorganisms, 9\u003c/em\u003e(2), 383. https://doi.org/10.3390/microorganisms9020383\u003c/li\u003e\n\u003cli\u003eYang, C., Song, G., \u0026amp; Lim, W. (2020). Effects of mycotoxin-contaminated feed on farm animals. \u003cem\u003eJournal of Hazardous Materials, 389\u003c/em\u003e, 122087. https://doi.org/10.1016/j.jhazmat.2020.122087\u003c/li\u003e\n\u003cli\u003eZulkifli, N. A., \u0026amp; Zakaria, L. (2017). Morphological and molecular diversity of \u003cem\u003eAspergillus\u003c/em\u003e from corn grain used as livestock feed. \u003cem\u003eHayati Journal of Biosciences, 24\u003c/em\u003e(1), 26-34.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Fungal contamination, food safety, Pig farms, Mycotoxins, Molecular characterization","lastPublishedDoi":"10.21203/rs.3.rs-6811857/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6811857/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFungal contamination in animal feed poses serious risks to animal health, food safety, and public health. Fungi such as \u003cem\u003eAspergillus, Fusarium,\u003c/em\u003e and \u003cem\u003ePenicillium \u003c/em\u003ecan produce mycotoxins, which impact livestock and human health. This study aimed to isolate and molecularly characterize fungal species in pig feed and faeces from registered and unregistered farms in Lagos State, Nigeria. Eighteen samples (6 feed and 12 feces) were collected and refrigerated before culturing on Sabouraud Dextrose Agar at 28°C for 5-7 days. Pure isolates were identified morphologically, and DNA was extracted. PCR amplification targeted ITS region of ribosomal DNA, with sequence comparisons made using GenBank references for species identification. Phylogenetic analysis assessed genetic similarity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResults showed that fungal contamination was more frequent in faeces collected from unregistered farms than registered farms. Microfungi belonging to the genera \u003cem\u003eAspergillus\u003c/em\u003e, \u003cem\u003ePichia\u003c/em\u003e, \u003cem\u003eRhizopus\u003c/em\u003e, and \u003cem\u003eTrichoderma\u003c/em\u003e were isolated and purified from feed while\u003cem\u003e Aspergillus\u003c/em\u003e and \u003cem\u003eCandida\u003c/em\u003e genera were isolated and purified from faeces. Molecular analyses revealed high genetic similarity among isolates, with \u003cem\u003eAspergillus flavus\u003c/em\u003e and \u003cem\u003eRhizopus arrhizus\u003c/em\u003e showing 100% identity with known strains. All four evolutionary models produced consistent phylogenetic trees, grouping the fungal species into well-supported clades, with only minor differences in branch lengths and bootstrap values. No significant genetic divergence was observed between isolates from registered and unregistered farms.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe occurrence of mycotoxin-producing fungi in pig farms, emphasizes the need for strict monitoring to enhance food safety.\u003c/p\u003e","manuscriptTitle":"Molecular Characterization and Phylogenetic Studies of Fungal Species in Piggeries and Implications for Mycotoxin Contamination and Food Safety","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-16 13:43:33","doi":"10.21203/rs.3.rs-6811857/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"de2f9231-67c1-4b7e-bb55-a3c53f003509","owner":[],"postedDate":"June 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-07T18:38:13+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-16 13:43:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6811857","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6811857","identity":"rs-6811857","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}