BackgroundSouth African plant species of the genera Fadogia, Pavetta and Vangueria (all belonging to Rubiaceae) are known to cause gousiekte (literally ‘quick disease’), a fatal cardiotoxicosis of ruminants characterised by acute heart failure four to eight weeks after ingestion. Noteworthy is that all these plants harbour endophytes in their leaves: nodulating bacteria in specialized nodules in Pavetta and non-nodulating bacteria in the intercellular spaces between mesophyll cells in Fadogia and Vangueria.Principal FindingsIsolation and analyses of these endophytes reveal the presence of Burkholderia bacteria in all the plant species implicated in gousiekte. Although the nodulating and non-nodulating bacteria belong to the same genus, they are phylogenetically not closely related and even fall in different bacterial clades. Pavetta harborii and Pavetta schumanniana have their own specific endophyte – Candidatus Burkholderia harborii and Candidatus Burkholderia schumanniana – while the non-nodulating bacteria found in the other gousiekte-inducing plants show high similarity to Burkholderia caledonica. In this group, the bacteria are host specific at population level. Investigation of gousiekte-inducing plants from other African countries resulted in the discovery of the same endophytes. Several other plants of the genera Afrocanthium, Canthium, Keetia, Psydrax, Pygmaeothamnus and Pyrostria were tested and were found to lack bacterial endophytes.ConclusionsThe discovery and identification of Burkholderia bacteria in gousiekte-inducing plants open new perspectives and opportunities for research not only into the cause of this economically important disease, but also into the evolution and functional significance of bacterial endosymbiosis in Rubiaceae. Other South African Rubiaceae that grow in the same area as the gousiekte-inducing plants were found to lack bacterial endophytes which suggests a link between bacteria and gousiekte. The same bacteria are consistently found in gousiekte-inducing plants from different regions indicating that these plants will also be toxic to ruminants in other African countries.

Pig manure may contain antibiotic residues, antibiotic-resistant bacteria or pathogens, which may reach the environment upon fertilization. During this study, 69 antibiotic residues belonging to 12 classes were quantified in 89 pig slurry samples. These samples were also studied for the presence of Salmonella and for E. coli resistant to meropenem, colistin, ciprofloxacin, or cefotaxim. The obtained isolates were further tested for antibacterial susceptibility. No antibiotic residues were detected in four samples, whereas in the other samples, up to 12 antibiotics were found. The most frequently detected antibiotic residues were doxycycline, sulfadiazine, and lincomycin. Doxycycline was found in the highest concentration with a mean of 1476 µg/kg manure (range: 18–13632 µg/kg). Tylosin and oxytetracycline were found with mean concentrations of 784 µg/kg (range: 17–5599 µg/kg) and 482 µg/kg (range: 11–3865 µg/kg), respectively. Lincomycin, had a mean concentration of 177 µg/kg manure (range: 9–3154 µg/kg). All other 18 antibiotic residues were found with mean concentrations of less than 100 µg/kg manure. Fifty-one slurry samples harbored Salmonella; 35% of the Salmonella isolates were sensitive to a panel of 14 antibiotics, whereas the other 65% were resistant up to five antibiotics. For E. coli, 52 manure samples contained E. coli isolates which were resistant to ciprofloxacin and 22 resistant to cefotaxime. All ciprofloxacin and cefotaxime-resistant isolates were multi-resistant, with resistance up to nine and eight antibiotics, respectively. This research indicates that pig slurry used for fertilization often contains antibiotic residues and antibiotic-resistant bacteria, including pathogens.

Antibiotic resistant bacteria and antibiotic residues can enter the environment when using animal manure as fertilizer. Twenty-five mixed beef cattle farmyard manure samples and 9 mixed fattening calf slurry samples from different farms across Belgium were investigated for the presence of 69 antibiotic residues, antibiotic resistant Escherichia coli and Salmonella spp. Doxycycline, oxytetracycline, ciprofloxacin, enrofloxacin, flumequine and lincomycin were detected in all fattening calf slurry samples with mean concentrations of 2776, 4078, 48, 31, 536 and 36 µg/kg manure, respectively. Sulfadiazine was detected at a mean concentration of 10,895 µg/kg. Further, antibiotic residues were found in only 4 of the 25 beef cattle farmyard manure samples. Oxytetracycline was detected twice below 500 µg/kg. Paromomycin, ciprofloxacin and enrofloxacin were detected in a concentration below 100 µg/kg. Of E. coli isolates, 88% and 23% from fattening calf slurry and beef cattle farmyard manure, respectively, were resistant to at least one of the antibiotics tested. Multi-drug resistance was observed at a maximum of 10 and 7 antibiotics, respectively. The occurrence of antibiotic resistant E. coli and antibiotic residues is shown to be higher in fattening calf slurry than in beef cattle farmyard manure used for agricultural field fertilization.

Plant poisoning of livestock is responsible for considerable economic losses in southern Africa.Six plant species of the Rubiaceae family are known to cause gousiekte, a cardiac syndrome of ruminants induced by ingestion of the toxic compound pavettamine. Progress in understanding the aetiology of this disease is largely hampered by the variable toxicity of the plants and the -2 -absence of a quantification method for pavettamine. The pavettamine concentration in leaf samples of Fadogia homblei, a known gousiekte causing plant, was analyzed by massspectrometry. In the most apical leaf pair, the highest concentration of pavettamine was detected. Distal leaves contained progressively less pavettamine. Besides a significant amount of free pavettamine, most pavettamine was found to occur in a conjugated form. To which molecules the pavettamine is conjugated remains unknown as is the function of conjugated pavettamine in the development of gousiekte. All know gousiekte-causing plants contain symbiotic bacteria in their leaves; it was hypothesized that these bacteria might be involved in the production of pavettamine. However, analysis of in vitro cultures of the Fadogia homblei endosymbiont revealed no production of pavettamine. Pavettamine is therefore not produced by the bacteria alone. It is either the product of the interaction with the plant or solely produced by the host.

Gousiekte, a cardiac syndrome of ruminants in southern Africa, is caused by the ingestion of plants containing the polyamine pavettamine. All the six known gousiekte-causing plants are members of the Rubiaceae or coffee family and house endosymbiotic Burkholderia bacteria in their leaves. It was therefore hypothesized that these bacteria could be involved in the production of the toxin. The pavettamine level in the leaves of 82 taxa from 14 genera was determined. Included in the analyses were various nodulated and non-nodulated members of the Rubiaceae. This led to the discovery of other pavettamine producing Rubiaceae, namely Psychotria kirkii and Ps. viridiflora. Our analysis showed that many plant species containing bacterial nodules in their leaves do not produce pavettamine. It is consequently unlikely that the endosymbiont alone can be accredited for the synthesis of the toxin. Until now the inconsistent toxicity of the gousiekte-causing plants have hindered studies that aimed at a better understanding of the disease. In vitro dedifferentiated plant cell cultures are a useful tool for the study of molecular processes. Plant callus cultures were obtained from pavettamine-positive species. Mass spectrometric analysis shows that these calli do not produce pavettamine but can produce common plant polyamines.