Whereas the capability of DNA uptake has been well established for numerous species and strains of bacteria grown in vitro, the broader distribution of natural transformability within bacterial communities remains largely unexplored. Here, we investigate the ability of bacterial isolates from the gut of grass grub larvae (Costelytra zealandica (White); Coleoptera: Scarabaeidae) to develop natural genetic competence in vitro. A total of 37 mostly species-divergent strains isolated from the gut of grass grub larvae were selected for spontaneous rifampicin-resistance. Genomic DNA was subsequently isolated from the resistant strains and exposed to sensitive strains grown individually using established filter transformation protocols. DNA isolated from wild-type strains was used as a control. None of the 37 isolates tested exhibited a frequency of conversion to rifampicin-resistance in the presence of DNA at rates that were significantly higher than the rate of spontaneous mutation to rifampicin-resistance in the presence of wild-type DNA (the limit of detection was approximately < 1 culturable transformant per 109 exposed bacteria). To further examine if conditions were conducive to bacterial DNA uptake in the grass grubs gut, we employed the competent bacterium Acinetobacter baylyi strain BD413 as a recipient species for in vivo studies. However, no transformants could be detected above the detection limit of 1 transformant per 103 cells, possibly due to low population density and limited growth of A. baylyi cells in grass grub guts. PCR analysis indicated that chromosomal AcinetobacterDNA remains detectable by PCR for up to 3 days after direct inoculation into the alimentary tract of grass grub larvae. Nevertheless, neither transforming activity of the DNA recovered from the alimentary tract of grass grubs larvae nor competence of bacterial cells recovered from inoculated larvae could be shown.

Biological risk assessment of food containing recombinant DNA has exposed knowledge gaps related to the general fate of DNA in the gastrointestinal tract (GIT). Here, a series of experiments is presented that were designed to determine if genetic transformation of the naturally competent bacterium Acinetobacter baylyi BD413 occurs in the GIT of mice and rats, with feed-introduced bacterial DNA containing a kanamycin resistance gene (nptII). Strain BD413 was found in various gut locations in germ-free mice at 103-105 CFU per gram GIT content 24–48 h after administration. However, subsequent DNA exposure of the colonized mice did not result in detectable bacterial transformants, with a detection limit of 1 transformant per 103-105 bacteria. Further attempts to increase the likelihood of detection by introducing weak positive selection with kanamycin of putative transformants arising in vivo during a 4-week-long feeding experiment (where the mice received DNA and the recipient cells regularly) did not yield transformants either. Moreover, the in vitro exposure of actively growing A. baylyi cells to gut contents from the stomach, small intestine, cecum or colon contents of rats (with a normal microbiota) fed either purified DNA (50 µg) or bacterial cell lysates did not produce bacterial transformants. The presence of gut content of germfree mice was also highly inhibitory to transformation of A. baylyi, indicating that microbially-produced nucleases are not responsible for the sharp 500- to 1 000 000-fold reduction of transformation frequencies seen. Finally, a range of isolates from the genera Enterococcus, Streptococcus and Bifidobacterium spp. was examined for competence expression in vitro, without yielding any transformants. In conclusion, model choice and methodological constraints severely limit the sample size and, hence, transfer frequencies that can be measured experimentally in the GIT. Our observations suggest the contents of the GIT shield or adsorb DNA, preventing detectable exposure of feed-derived DNA fragments to competent bacteria.

An improved Theileria parva DNA detection assay based on the polymerase chain reaction (PCR) using primers derived from the 104 kDa antigen (p104) gene was developed to detect parasite DNA in blood spots on filter paper. The specificity of the assay was validated using DNA from a wide range of cattle-derived and buffalo-derived stocks of T. parva. DNA of T. annulata, T. buffeli, T. lestoquardi, T. mutans and T. taurotragi was not amplified using the p104 primers. The detection threshold of the assay was ∼1–2 parasites/μl of infected blood. PCR amplification using the p104 primers was applied to sequential samples from groups of cattle experimentally infected with either the T. parva Marikebuni stock that induces a long-term carrier state or the Muguga stock, which does not induce a carrier state. The study extended for up to 487 days post-infection and PCR data from defined time points were compared with parasitological microscopy and serological data, together with xenodiagnosis by experimental application of ticks. Microscopy first detected piroplasms between days 13 and 16 after infection whereas all cattle became PCR +ve between days 9 and 13. Animals infected with the Muguga stock of T. parva had parasite DNA in the peripheral blood, which could be detected by PCR, for between 33 and 129 days post-infection in different animals. By contrast parasite DNA in the blood of cattle infected with the Marikebuni stock could be detected consistently from day 9 up to 487 days, when the study terminated. The data suggest that the nature and persistence of the carrier state may differ markedly between different T. parva parasite stocks.