Abstract:
Fadogia homblei, Pavetta harborii, P. schumanniana, Vangueria latifolia, V. pygmaea and V.
thamnus belong to the family Rubiaceae and induce the sickness gousiekte in ruminants
within southern Africa. Gousiekte is a plant induced cardiomyopathy with a latent period of
3-8 weeks before death of the ruminant occurs (Ellis et al., 2010a; Van der Walt et al., 1990;
Fourie et al., 1989; Prozesky et al., 1988). Gousiekte was discovered in 1908, 87 years later
the causative compound, pavettamine, was isolated; a further 15 years saw pavettamine
elucidated in 2010 (Bode et al., 2010; Fourie et al., 1995). The extraction method to isolate
pavettamine is very cumbersome as is the diagnosis of gousiekte due to many factors (Fourie
et al., 1995). Bacterial endophytes have been confirmed present in F. homblei, V. latifolia, V.
pygmaea and V. thamnus, however, to date no bacterial endophyte isolated from a gousiekteinducing
plant produces pavettamine (Van Elst et al., 2011; Van Wyk et al., 1990). There is a
theory that the bacterial endophytes present within the gousiekte inducing plants play a key
role in synthesis of pavettamine (Van Wyk et al., 1990). Pavettamine was not isolated from
any of the plants focused on in this study (Pavetta gardeniifolia, P. schumanniana, V.
infausta, V. macrocalyx, V. pygmaea or V. thamnus). Cytotoxicity screening on H9c2 cells
(derived from rat cardiac cells) gave unexpected results with P. schumanniana as the most
toxic followed by P. gardeniifolia, V. pygmaea, V. thamnus, V. macrocalyx and lastly V.
infausta. Using transmission electron microscopy bacterial endophytes were located in P.
schumanniana, V. infausta, V. macrocalyx, V. madagascariensis, V. pygmaea and V. thamnus
revealing that not only gousiekte-inducing plants contain bacterial endophytes, however, the
bacterial endophytes present in the gousiekte-inducing plants ( P. schumanniana, V. pygmaea
and V thamnus) all appeared morphologically similar. Seasonal colonisation fluctuations of
bacterial endophytes was observed within V. pygmaea using transmission electron
microscopy linking the season (summer) which has the highest quantity of bacterial colonies to the season when gousiekte cases are reported most frequently. Twelve culturable bacterial
endophytes were isolated from V. pygmaea, seven from V. thamnus and a single bacterium
was isolated from both plants. The bacteria were subjected to cytotoxicity screening on H9c2
cells (susceptible to pavettamine) which resulted in two toxic bacteria. One isolated from V.
pygmaea and the other was isolated from both V. pygmaea and V. thamnus. The DNA of the
toxic bacterium isolated from both V. pygmaea and V. thamnus was sequenced and found to
belong to the family Bacillus. The isolation of a toxic bacterium from gousiekte-inducing
plants supports the theory that bacterial endophytes play a role in pavettamine synthesis.
