Survival of a Recombinant Rumen Bacterium in the Rumen of Sheep

Abstract

Survival of the recombinant rumen bacterium Butyrivibrio fibrisolvens NO4, expressing an exogenous xylanase and erythromycin resistance, was assessed in vitro and in vivo. Selective enumeration of the recombinant strain from indigenous rumen bacteria was performed using an erythromycin-supplemented anaerobic medium.When incubated in vitro with mixed rumen bacteria and protozoa obtained from sheep, the numbers of the recombinant strain stabilised at 103/ml (48h). When incubated with mixed microbes from the goat rumen, strain NO4 disappeared 48h after inoculation. The presence of protozoa in the culture tended to decrease the numbers of strain NO4 ten-fold compared to bacteria alone. In vivo, numbers declined rapidly after inoculation, and the recombinant strain was no longer detectable in the sheep rumen after 144h.Animal Science Journal 72 (4) Recent developments in genetic engineering are applicable to rumen microbes, allowing improvements to their metabolic capabilities, e.g. fibrolysis, protein synthesis and detoxification6). One such application is close to be at the animal production stage3). Studies tracking Prevotellaruminicola2) and a Ruminococcus albus transconjugant8), inoculated into the rumen, suggested that rapid elimination or poor establishment occurs with bacteria that have been maintained in culture for a considerable time. However, Gregget al.3) observed that inoculated wild and recombinant Butyrivibrio fibrisolvens in the rumen of sheep were detectable by PCR even at 105-7 dilutions. Identification of factors important for survival and establishment of specific bacterial strains (including recombinants) in the rumen is required. The present paper describes the survival of the recombinant rumen anaerobic bacterium Butyrivibrio fibrisolvens NO4 in simple batch culture and the sheep rumen. Materials and MethodsButyrivibrio fibrisolvens NO45) expressing a Eubacterium ruminantium xylanase gene (xynA) and a was used. NO4 was anaerobically grown until mid logarithmic phase (approximately, OD600=0.5) in a reduced CCA medium1), and used directly for in vitro inoculation.A rumen cannulated sheep and goat (weighing 45 and 21kg) were used as rumen fluid donors for in vitro experiments. The animals were fed a cubed alfalfa hay and concentrate diet (400g:100g (sheep) and 200g:50g (goat) respectively) twice daily.Rumen fluid was collected prior to morning feeding, strained through 2 layers of surgical gauze and centrifuged of the rumen fluid was incubated with an acetate buffer at 37 were washed twice with an anaerobic dilution solution9), resuspended in McDougal's artificial saliva and combined if necessary (bacteria (B) and bacteria plus protozoa (BP)). Both B and BP were mixed with 2g ground alfalfa hay, for the simple batch culture (100ml in volume) experiments. Samples were taken at 0, 2, 4, 6, 8, 12, 24 and 48h after inoculation of strain NO4 to enumerate total bacteria , strain NO4 and protozoa.The donor sheep was used as the recipient for strain NO4 inoculation in vivo (managed according to M...

Factors that limit maintenance of recombinant rumen bacterium in sheep rumen

“…The culturable recombinant was selectively enumerated with erythromycin‐supplemented medium (Kobayashi et al. 2001).…”

“…This allowed tracking studies of the recombinant with a culturing enumeration. In the preceding report (Kobayashi et al. 2001), we demonstrated that protozoal predation and washout of the recombinant from the rumen were factors limiting the survival of the recombinant in vivo .…”

“…The tested environment, however, never eliminated the recombinant, allowing survival of the recombinant at a lower level in the rumen. Disappearance of the recombinant in vivo is probably led by the cell‐free antibacterial substances and early mentioned factors such as washout (Kobayashi et al. 2001).…”

“…1998; Kobayashi et al. 2000, 2001). Our preceding report (Kobayashi et al 2001) indicated that the recombinant Butyrivibrio fibrisolvens was poorly survived in the rumen of sheep, probably due to its less competitive ability against indigenous microbes and/or washout from the rumen.…”

“…Our preceding report (Kobayashi et al 2001) indicated that the recombinant Butyrivibrio fibrisolvens was poorly survived in the rumen of sheep, probably due to its less competitive ability against indigenous microbes and/or washout from the rumen. In particular, rapid elimination of the culturable recombinant after inoculation was demonstrated (Kobayashi et al. 2001), partly due to additional but unidentified limiting factors present in the rumen.…”