A specific class of infectious agents isolated from bovine serum and dairy products and peritumoral colon cancer tissue

Abstract

The in silico analyses of 109 replication-competent genomic DNA sequences isolated from cow milk and its products (97 in the bovine meat and milk factors 2 group - BMMF2, and additional 4 in BMMF1) seems to place these in a specific class of infectious agents spanning between bacterial plasmid and circular ssDNA viruses. Satellite-type small plasmids with partial homology to larger genomes, were also isolated in both groups. A member of the BMMF1 group H1MBS.1 was recovered in a distinctly modified form from colon tissue by laser microdissection. Although the evolutionary origin is unknown, it draws the attention to the existence of a hitherto unrecognized, broad spectrum of potential pathogens. Indirect hints to the origin and structure of our isolates, as well as to their replicative behaviour, result from parallels drawn to the Hepatitis deltavirus genome structure and replication.

Keywords: Infectious bovine meat milk factors; colon tissue; new class; pathogenesis; plasmid DNA.

Figure 1.

Molecular phylogenetic analysis of the overlapping region of all Rep proteins of both BMMF1 and BMMF2. The evolutionary history was inferred by using the Maximum Likelihood method and JTT matrix-based model [28]. The bootstrap consensus tree inferred from 500 replicates [29] is taken to represent the evolutionary history of the taxa analysed [29]. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches [29]. Initial tree(s) for the heuristic search were obtained by applying the Neighbour-Joining method to a matrix of pairwise distances estimated using a JTT model. This analysis involved 104 amino acid sequences. There were a total of 319 positions in the final dataset. Evolutionary analyses were conducted in MEGA X [26].

Figure 2.

Phylogenetic tree of the nucleotide sequences of representatives of each BMMF2 isolate in comparison to the plasmids of A. baumanii p4ABAYE, pA85-1 and pTS236, genomic isolates from rat gut pRGRH0103 (RH103) and pRGRH0636 (RH0636) in clade A, as well as Sphinx2.36 and our previous BMMF1 isolates C2MI1 and C2HB7 in clade B. The recently isolated C2MI.5A.1 represents the full-length genome of C2HB2. The star indicate BMMF2 isolates of which the nucleotide sequence is almost identical in the overlapping nucleotide sequence of the respective “small” BMMF2 isolate. The evolutionary history was inferred by using the Maximum Likelihood method and Tamura-Nei model [38]. The bootstrap consensus tree inferred from 500 replicates [29] is taken to represent the evolutionary history of the taxa analysed [29]. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches [29]. Initial tree(s) for the heuristic search were obtained by applying the Neighbour-Joining method to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach. This analysis involved 48 nucleotide sequences. There were a total of 3170 positions in the final dataset. Evolutionary analyses were conducted in MEGA X [26].

Figure 3.

Schematic presentation of the ORF maps of BMMF2 isolates displaying truncated ORFs which encode for putative Rep proteins (arrows in red) in comparison to closely related isolates with intact Rep ORFs (blue arrows). The ORF map of p4ABAYE is shown in the middle. Arrows in same colour (green or yellow) represent ORFs which encode putative proteins showing high similarity between isolates. The GENESCAN analyses indicating the resulting splicing transcriptional events are indicated in purple. The large purple dot represents the poly A signal. In some cases no poly A signal is present for the respective transcript.

Figure 4.

Modified DNA genomes of BMMF1 H1MSB.1 were recovered by laser microdissection from the lamina propria cells of the colon from colon cancer patients. A – area dissected from the peritumoral colon tissue. Rep-stained areas are marked (yellow). Bar 100 μm. B – Clustal analysis of the modified genomes LD10.154 and LD10.158 with the original H1MSB.1 genome indicated a number of C/T transitions (light grey shaded) and fewer A/G modifications (dark grey shaded). C – Schematic presentation of the putative gene organization of these DNA genomes. The modifications in the LD10.154 genome led to a modified gene structure (red arrow) resulting in a 62 amino acid putative protein which could be involved in pathogenesis.