A chemically defined medium for the growth of Cowdria ruminantium

Chemically defined media, termed SFMC-23 and SFMC-36, were devised for the in vitro culture of Cowdria ruminantium, the causative agent of heartwater in domestic ruminants. Both media were based on Dulbecco's modified Eagle's medium nutrient mixture Ham F-12 (DME/F-12) containing various supplements. Medium SFMC-23 and SFMC-36 supported the long-term growth of the Welgevonden stock of C. ruminantium for a total of 55 and 28 passages, respectively, with regular passage intervals of 3 days. Using SFMC-23, split ratios varied from 5-10, depending on which host cell line was used. Other stocks of C. ruminantium (Sankat, Blaauwkrantz, Senegal) were successfully propagated for a test period of ten passages.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. Adobe Acrobat v.9 was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.Agricultural Research Council of South Africa. European Union (Cowdriosis Network) Grant no. IC18-CT95-0008 (DG12-SNRD).mn201

Continuous in vitro propagation of Cowdria ruminantium (Welgevonden stock) in a canine macrophage-monocyte cell line

The Welgevonden stock of Cowdria ruminantium, aetiologic agent of heartwater, was continuously propagated in DH82 cells, a continuous canine macrophage-monocyte cell line. Cultures of DH82 cells were readily infected provided that the culture medium was supplemented with cycloheximide. Cultures were split at regular 3-day intervals and infection rates ranged between 60% and 95%. Cultures were continuously propagated through more than 125 passages over a period of more than one year.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. Adobe Acrobat v.9 was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.Agricultural Research Council of South Africa and the European Union (Cowdriosis Network) Grant no. IC18-CT95-0008 (DG12-SNRD).mn201

Ehrlichia ruminantium variants which do not cause heartwater found in South Africa

In 1994 a batch of apparently healthy goats was selected for intended export to the USA from a heartwater-free and vector tick-free region of South Africa. The animals were tested serologically for heartwater, using either or both an IFA and an ELISA test, and 52% were found to be serologically positive. A PCR assay based on Ehrlichia ruminantium 16S gene sequences gave positive results for 54% of the animals, suggesting that apparently non-pathogenic E. ruminantium variants existed in this heartwater-free area. To identify and characterise the agents responsible for the positive serological and PCR results, ticks and animal blood samples were collected from two of the three farms involved in the original survey during two successive seasons of expected peak tick activity. Ticks were kept alive for a minimum of 3 weeks to allow digestion of any blood meal before being processed. Over the two seasons, 28% of the livestock and 15% of the ticks sampled were found to be carrying E. ruminantium. E. ruminantium 16S and pCS20 sequences were detected in all of the four tick species collected from the livestock (Rhipicephalus evertsi evertsi, Rhipicephalus evertsi mimeticus, Hyalomma truncatum, Hyalomma marginatum rufipes), suggesting that some of the species may act as vectors. Animals generally carried multiple E. ruminantium 16S genotypes, whereas ticks rarely carried more than one. Infection levels in both animals and ticks were too low to generate a marked response when a blood stabilate was sub-passaged in a clean sheep, preventing the subsequent establishment of any of the organisms in culture.This research was funded by the Red Meat Research and Development Trust (RMRDT) of South Africa. We thank Prof. Ivan Horak for advice and critical reading of the manuscript

The Kumm isolate of Ehrlichia ruminantium: in vitro isolation, propagation and characterization

An effective culture system for Ehrlichia (Cowdria) ruminantium comb. nov. was first established in 1985 and many stocks were subsequently isolated and propagated in vitro. A notable exception, however, was the Kumm isolate that resisted all attempts at in vitro culture until the successful experiment described here. In one experiment white blood cells were harvested from heparinized blood derived from a sheep infected with the Kumm isolate. The cells were added to DH 82 cells and incubated at 37degreesC. The high metabolic activity of the DH 82 cells necessitated that cell growth be retarded by the addition of cycloheximide. Colonies were first detected 19 days after culture initiation and, once the cultures were established, they could be passaged every 3 days. Bovine and sheep endothelial cells were readily infected with culture supernatant obtained from the infected DH 82 cells. In a further experiment, another sheep was infected, using a higher dose of the same batch of Kumm stabilate, and we attempted to infect several different cell lines: these were DH 82 cells, bovine aorta (BA 886) cells, sheep brain endothelial (SBE 189) cells and sheep fibroblastoid cells (E₂). Ten days after culture initiation, only the E₂ cells had become positive for E. ruminantium. Culture supernatant from the first cultured isolate (Kumm-1) was less virulent for mice than that of the second cultured isolate (Kumm-2) which killed all mice. Upon molecular characterization with E. ruminantium 16S probes, we found that Kumm-1 hybridized with a Senegal 16S genotype probe, whereas Kumm-2 hybridized only with an Omatjenne 16S genotype probe. The original stabilate used to infect the sheep hybridized with both probes. These results clearly indicate that two different stocks had been isolated in culture.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. Adobe Acrobat v.9 was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.Agricultural Research Council of South Africa. European Union (Cowdriosis Network) Grant no. IC18-CT95-0008 (DG12-SNRD)

In vitro infection by Ehrlichia ruminantium of baby hamster kidney (BHK), Chinese hamster ovary (CHO-K1) and Madin Darby bovine kidney (MDBK) cells

The Welgevonden stock of Ehrlichia ruminantium, aetiological agent of heartwater, was propagated in baby hamster kidney (BHK) cells, Chinese hamster ovary (CHO-K1) cells and Madin Darby bovine kidney (MDBK) cells. The cultures required supplementation of the medium with cycloheximide for reliable growth of E. ruminantium. Growth of the Welgevonden stock in BHK and CHO-K1 cells could lead to the development of suspension cultures suitable for the mass production of E. ruminantium for an inactivated elementary body vaccine.The articles have been scanned with a HP Scanjet 8300; 600dpi, saved in TIFF format. Adobe Acrobat v.9 was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.Agricultural Research Council of South Africa. The European Union (Cowdriosis Network) Grant no. IC18-CT95-0008 (DG12-SNRD)