The polyamines and endophytes of gousiekte-causing and non-pathogenic Vangueria and Pavetta species

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.