Detection of DNA sequences attributed to bovine meat and milk factors (BMMF/SPHINX) in food-related samples

Pohl, Sina; Habermann, Diana; Link, Ellen Kathrin; Fux, Robert; Boldt, Christine L.; Franz, Charles M. A. P.; Hölzel, Christina; Klempt, Martin

doi:10.1016/j.foodcont.2021.108779

Cited by 5 publications

(6 citation statements)

References 9 publications

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“…Our previously published results confirmed the existence of circular Rep-encoding DNA molecules in a broad spectrum of non-bovine foods, including meats, fish, seafood, vegetables, and fruits, as well as in animal samples from chicken and pig (saliva, feces), via a targeted PCR amplification and sequencing-based approach [12]. In this work, the full-length sequences of 16 circular Rep-encoding genomes could be successfully assembled and verified.…”

Section: Resultssupporting

confidence: 76%

“…Interestingly, analysis of the sampling source did not show any source-specific branching, indicating that the S/B molecules are broadly distributed in both plants and animals. This disproves the theory that S/B molecules are solely found in taurine-derived samples, supported by the previous findings of König et al [9,10], Khostovv et al [11], and Pohl et al [12].…”

Section: Discussionsupporting

confidence: 70%

“…First doubts regarding the latter theory arose after the molecules were detected in the milk of other Bovidae species, such as water buffalo, goat, and sheep [9,10]. In addition, it was previously also demonstrated that specific S/B sequences were also detectable in a broad range of food samples of plant and animal origin [12]. However, our previous investigation [12] reported only partial sequences; therefore, in this work, we describe for the first time full-length circular S/B group 1 and 2 sequences found in alfalfa, radish, and broccoli sprouts, as well as in apple, carrot, Alaska pollock, pangasius, black tiger shrimp, chicken, pork, and wild boar.…”

Section: Discussionmentioning

confidence: 99%

“…After cooling down to room temperature and adding 0.25 mM dNTPs and 10 U Phi29 polymerase, the reaction was incubated at 30 • C for 16 h and then terminated by heating to 60 • C for 10 min. After this step, 0.3 to 1 µL of the RCA reaction was used as a template for PCR amplification with group-specific primers [12]. The amplified fragments were separated on a 1% agarose gel by electrophoresis in 1 × TAE buffer.…”

Section: Dna Extraction and Pcrmentioning

confidence: 99%

“…Khvostov et al detected SPHINX/BMMF molecules in sheep meat by a real-time PCR approach [11]. Finally, these molecules were also found in a wide range of food products, including vegetables, fruits, fish, seafood, and meat of pork, chicken, and wild boar by PCR amplification [12]. Here, 16 novel full-length SPHINX/BMMF molecules from pork, wild boar, chicken, Alaska pollock, pangasius, black tiger shrimp, apple, carrot, and sprouts from alfalfa, radish, and broccoli are described.…”

Section: Introductionmentioning

confidence: 99%

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Identification and Characterization of Novel SPHINX/BMMF-like DNA Sequences Isolated from Non-Bovine Foods

Habermann

Klempt

Franz

2023

Genes

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Sixteen novel circular rep-encoding DNA sequences with high sequence homologies to previously described SPHINX and BMMF sequences were isolated for the first time from non-bovine foods (pork, wild boar, chicken meat, Alaska pollock, pangasius, black tiger shrimp, apple, carrot, and sprouts from alfalfa, radish, and broccoli). The phylogenetic analysis of the full-length circular genomes grouped these together with previously described representatives of SPHINX/BMMF group 1 and 2 sequences (eight in each group). The characterization of genome lengths, genes present, and conserved structures confirmed their relationship to the known SPHINX/BMMF sequences. Further analysis of iteron-like tandem repeats of SPHINX/BMMF group 1-related genomes revealed a correlation with both full-length sequence tree branches as well as Rep protein sequence tree branches and was able to differentiate subtypes of SPHINX/BMMF group 1 members. For the SPHINX/BMMF group 2 members, a distinct grouping of sequences into two clades (A and B) with subgroups could be detected. A deeper investigation of potential functional regions upstream of the rep gene of the new SPHINX/BMMF group 2 sequences revealed homologies to the dso and sso regions of known plasmid groups that replicate via the rolling circle mechanism. Phylogenetic analyses were accomplished by a Rep protein sequence analysis of different ssDNA viruses, pCRESS, and plasmids with the known replication mechanism, as this yielded deeper insights into the relationship of SPHINX/BMMF group 1 and 2 Rep proteins. A clear relation of these proteins to the Rep proteins of plasmids could be confirmed. Interestingly, for SPHINX/BMMF group 2 members, the relationship to rolling circle replication plasmids could also be verified. Furthermore, a relationship of SPHINX/BMMF group 1 Rep proteins to theta-replicating plasmid Reps is discussed.

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Section: Resultssupporting

confidence: 76%

Section: Discussionsupporting

confidence: 70%

Section: Discussionmentioning

confidence: 99%

Section: Dna Extraction and Pcrmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Identification and Characterization of Novel SPHINX/BMMF-like DNA Sequences Isolated from Non-Bovine Foods

Habermann

Klempt

Franz

2023

Genes

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Bovine meat and milk factor protein expression in tumor‐free mucosa of colorectal cancer patients coincides with macrophages and might interfere with patient survival

et al. 2023

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Bovine milk and meat factors (BMMFs) are plasmid‐like DNA molecules isolated from bovine milk and serum, as well as the peritumor of colorectal cancer (CRC) patients. BMMFs have been proposed as zoonotic infectious agents and drivers of indirect carcinogenesis of CRC, inducing chronic tissue inflammation, radical formation and increased levels of DNA damage. Data on expression of BMMFs in large clinical cohorts to test an association with co‐markers and clinical parameters were not previously available and were therefore assessed in this study. Tissue sections with paired tumor‐adjacent mucosa and tumor tissues of CRC patients [individual cohorts and tissue microarrays (TMAs) (n = 246)], low‐/high‐grade dysplasia (LGD/HGD) and mucosa of healthy donors were used for immunohistochemical quantification of the expression of BMMF replication protein (Rep) and CD68/CD163 (macrophages) by co‐immunofluorescence microscopy and immunohistochemical scoring (TMA). Rep was expressed in the tumor‐adjacent mucosa of 99% of CRC patients (TMA), was histologically associated with CD68+/CD163+ macrophages and was increased in CRC patients when compared to healthy controls. Tumor tissues showed only low stromal Rep expression. Rep was expressed in LGD and less in HGD but was strongly expressed in LGD/HGD‐adjacent tissues. Albeit not reaching statistical significance, incidence curves for CRC‐specific death were increased for higher Rep expression (TMA), with high tumor‐adjacent Rep expression being linked to the highest incidence of death. BMMF Rep expression might represent a marker and early risk factor for CRC. The correlation between Rep and CD68 expression supports a previous hypothesis that BMMF‐specific inflammatory regulations, including macrophages, are involved in the pathogenesis of CRC.

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First Insights into the Occurrence of Circular Single-Stranded DNA Genomes in Asian and African Cattle

König

Frölich²,

Jandowsky³

et al. 2023

Animals

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Circular replicase-encoding single-stranded (CRESS) DNA viruses and other circular DNA agents are increasingly found in various samples and animals. A specific class of these agents—termed bovine meat and milk factors (BMMF)—has been supposed to act as a factor in indirect carcinogenesis in humans. Initial observations attributed the BMMF to European cattle breeds and foodstuffs produced thereof. In the present study, blood and fecal samples from African and Asian cattle were examined. BMMF molecules and genomoviruses were detected in all bovids under study. The majority (79%) of the 29 circular elements could be assigned to BMMF groups 1 and 2, whereas CRESS viruses of the family Genomoviridae accounted for the smaller part (21%). Two genomoviruses belong to the genus Gemykibivirus and one to the genus Gemykrogvirus. The remaining three might be considered as novel species within the genus Gemycircularvirus. The majority of all isolated molecules originated from fecal samples, whereas only three derived from blood. The results from this study expand our knowledge on the diversity and presence of circular DNA in different ruminants that serve for food production in many countries over the world.

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Detection of DNA sequences attributed to bovine meat and milk factors (BMMF/SPHINX) in food-related samples

Cited by 5 publications

References 9 publications

Identification and Characterization of Novel SPHINX/BMMF-like DNA Sequences Isolated from Non-Bovine Foods

Identification and Characterization of Novel SPHINX/BMMF-like DNA Sequences Isolated from Non-Bovine Foods

Bovine meat and milk factor protein expression in tumor‐free mucosa of colorectal cancer patients coincides with macrophages and might interfere with patient survival

First Insights into the Occurrence of Circular Single-Stranded DNA Genomes in Asian and African Cattle

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