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

In vitro cultivation of Babesia occultans

Babesia occultans, the causative agent of a benign form of cattle babesiosis in South Africa, was continuously cultivated in microaerophilous stationary-phase culture. A modified medium, 199, supplemented with either 40% (v/v) bovine or 40% (v/v) horse serum, was used. Cultures were initiated in a humidified atmosphere containing 2% 0₂, 5% C0₂, and 93% N₂. The highest percentage of parasitized erythrocytes (PPE) reached 4,5% in horse-serum- and 2,4% in bovine-serum-supplemented medium. Parasite suspensions were cryopreserved and successfully resuscitated.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. Adobe Acrobat X Pro was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.mn201

Cold storage and cryopreservation of tick cell lines

<p>Abstract</p> <p>Background</p> <p>Tick cell lines are now available from fifteen ixodid and argasid species of medical and veterinary importance. However, some tick cell lines can be difficult to cryopreserve, and improved protocols for short- and long-term low temperature storage will greatly enhance their use as tools in tick and tick-borne pathogen research. In the present study, different protocols were evaluated for cold storage and cryopreservation of tick cell lines derived from <it>Rhipicephalus </it>(<it>Boophilus) decoloratus</it>, <it>Rhipicephalus </it>(<it>Boophilus) microplus, Ixodes ricinus </it>and <it>Ixodes scapularis</it>. For short-term cold storage, cells were kept under refrigeration at 6°C for 15, 30 and 45 days. For cryopreservation in liquid nitrogen, use of a sucrose-phosphate-glutamate freezing buffer (SPG) as cryoprotectant was compared with dimethylsulfoxide (DMSO) supplemented with sucrose. Cell viability was determined by the trypan blue exclusion test and cell morphology was evaluated in Giemsa-stained cytocentrifuge smears.</p> <p>Results</p> <p>Cold storage at 6°C for up to 30 days was successful in preserving <it>R</it>. (<it>B.) microplus</it>, <it>R</it>. (<it>B.) decoloratus, I. ricinus </it>and <it>I. scapularis </it>cell lines; lines from the latter three species could be easily re-cultivated after 45 days under refrigeration. While cell lines from all four tick species cryopreserved with 6% DMSO were successfully resuscitated, the <it>R</it>. (<it>B</it>.) <it>decoloratus </it>cells did not survive freezing in SPG and of the other three species, only the <it>R</it>. (<it>B</it>.) <it>microplus </it>cells resumed growth during the observation period.</p> <p>Conclusions</p> <p>This constitutes the first report on successful short-term refrigeration of cells derived from <it>R</it>. (<it>B.) decoloratus</it>, <it>R</it>. (<it>B.) microplus</it>, and <it>I. ricinus</it>, and use of SPG as an alternative to DMSO for cryopreservation, thus making an important contribution to more reliable and convenient tick cell culture maintenance.</p

Evaluation of a short-term in vitro growth-inhibition test to determine susceptibility of Trypanosoma vivax stocks to various trypanocides

Two Trypanosoma vivax stocks were initiated in culture with tsetse or culture-derived metacyclics. They were propagated axenically as bloodstream trypomastigotes at 35°C in 4% C0₂ in air. Populations of trypanosomes were incubated with various concentrations of antitrypanosomal compounds, namely diminazene aceturate, quinapyramine sulphate, DL-α-difluoromethylornithine, isometamidium chloride, suramin and mel Cy. Growth was monitored after 24 h of incubation and the growth inhibition was calculated. All six drugs tested showed little effect upon the growth of the parasite populations. These results indicate that a 24-h growth-inhibition test was not suitable for determining the drug susceptibility of T. vivax stocks in vitro. Neither did the results correlate with those obtained with susceptible or resistant stocks of T. b. brucei, T. b. evansi or T. simiae described in the literature, or with the results of these two T. vivax stocks tested in cattle.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. Adobe Acrobat XI Pro was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.mn201

Cultivation, cryopreservation and resuscitation of Theileria annulata transformed cells in serum-free media

Introduction: Tropical theileriosis is a protozoan disease caused by Theileria annulata that affects cattle in Northern Africa, the Middle East and Asia where vector ticks of the genus Hyalomma occur. Various measures are applied to control the disease, including vaccination with attenuated T. annulata schizonts. Cultivation of T. annulata schizonts is mainly conducted in media containing Fetal Bovine Serum (FBS), which has some disadvantages such as costs, batch- to-batch variation and ethical concerns. Methods: In this study, we conducted three experiments to evaluate the ability of (1) T. annulata strains grown in RPMI with 10% FBS (RPMI-FBS) to adapt and grow in serum-free media (i.e., HL-1, RPMI without FBS supplementation, ISF-1, and M199), (2) a T. annulata strain grown in ISF-1 and subsequently frozen in this medium to grow in ISF-1 again after long-term storage in liquid nitrogen, and (3) a T. annulata strain freshly isolated from infected bovine lymphocytes to growin ISF-1, also after cryopreservation. Cell numbers, schizont index, the viability and generation doubling time were calculated in all experiments. Results and discussion: In the first experiment, the Hessiene and Beja cell lines from Tunisia previously cultivated in RPMI-FBS and adapted to serum-free media continued to grow significantly better in RPMI-FBS compared to the serum-freemedia. In the second experiment, a Tunisian cell line (Hessiene) cryopreserved in ISF-1 with 5%[v/v] dimethylsulfoxide (DMSO) grewbetter after thawing in RPMI-FBS compared to ISF-1 with a highly significant difference in cell growth (p < 0.001), whereas the third experiment showed that the Ankara cell line had similar growth characteristics in both RPMI-FBS and ISF-1 before and after thawing, with a shorter generation doubling time in ISF-1 than in RPMI-FBS (p = 0.23). Our findings suggest that freshly isolated cells can be propagated, frozen and thawed in serum-free media such as ISF-1, but once cells are adapted to cultivation in the presence of FBS or resuscitated from frozen storage, propagation in serum-free media may not perform as well as cultivation in RPMI-FBS

De novo assembly and annotation of the Amblyomma hebraeum tick midgut transcriptome response to Ehrlichia ruminantium infection

The South African bont tick Amblyomma hebraeum is a hematophagous vector for the heartwater disease pathogen Ehrlichia ruminantium in southern Africa. During feeding, the tick’s enterocytes express proteins that perform vital functions in blood digestion, including proteins that may be involved in E. ruminantium acquisition, colonization or immunity. To delineate the molecular mechanism of midgut response to E. ruminantium infection, we performed comparative analyses of midgut transcriptomes of E. ruminantium infected engorged A. hebraeum nymphs, and infected adult male and female ticks with their corresponding matched uninfected controls, before and during feeding. A total of 102,036 unigenes were annotated in public databases and their expression levels analyzed for engorged nymphs as well as unfed and partly-fed adult ticks. There were 2,025 differentially expressed genes (DEGs) in midguts, of which 1,225 unigenes were up-regulated and 800 unigenes were down-regulated in the midguts of infected ticks. Annotation of DEGs revealed an increase in metabolic and cellular processes among E. ruminantium infected ticks. Notably, among the infected ticks, there was up-regulation in the expression of genes involved in tick immunity, histone proteins and oxidative stress responses. We also observed up-regulation of glycoproteins that E. ruminantium could potentially use as docking sites for host cell entry. Insights uncovered in this study offer a platform for further investigations into the molecular interaction between E. ruminantium and A. hebraeum

In vitro cultivation of Babesia equi: detection of carrier animals and isolation of parasites

By means of an in vitro culture technique, 75 samples of horse blood were examined for Babesia equi, a causative agent of equine piroplasmosis. At the time of culture initiation, 15 samples were microscopically positive for B. equi, and this was subsequently confirmed by culture diagnosis. Sixty samples showed no parasites in Giemsa-stained thin blood smears. However, after the culturing process, parasites were found in blood smears of 36 of these samples. The sensitivity of the in vitro culture method was such that 2,5 µl (1/40 of the usual volume used for the above mentioned samples} of packed erythrocytes obtained from a carrier horse still yielded positive results after cultivation. Cultures were initiated from blood samples stored for up to 120 h at 8º C in vacuum tubes containing EDTA as anticoagulant. These results show that the in vitro culture method is highly sensitive. It can be used to identify B. equi carrier horses, to evaluate the effects of chemotherapeutic intervention, and to isolate field strains of B. equi or further characterization.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. Adobe Acrobat X Pro was used to OCR the text and also for the merging and conversion to the final presentation PDF-format

In vitro isolation of equine piroplasms derived from Cape Mountain zebra (Equus zebra zebra) in South Africa

Twenty blood samples of zebras ( Equus zebra zebra) from the Karoo National Park and the Bontebok National Park in South Africa, all seropositive for Theileria equi, were subjected to in vitro culture to identify carrier animals and to isolate the parasites. Sixteen animals had a detectable parasitaemia in Giemsa-stained blood smears examined before culture initiation, the remaining four animals were identified as T. equi carriers by in vitro culture. Cultures were initiated either in an oxygen-reduced gas mixture or in a 5% CO2-in-air atmosphere. Out of the 20 blood samples, 12 cultures of T. equi and two cultures of T. equi mixed with Babesia caballi were established. None of the four animals seropositive for B. caballi could be identified as carrier animals, whereas two seronegative samples became culture-positive for B. caballiThe 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.mn201

In vitro cultivation of a Babesia sp. from cattle in South Africa

A South African Babesia sp. of cattle which is as yet unclassified, was continuously cultivated in micro-aerophilous stationary-phase culture. The parasites were resuscitated from a blood stabilate stored in liquid nitrogen. A modified HL-1 medium supplemented with either horse or bovine serum was used. Cultures were initiated in a humidified atmosphere containing 2% 0₂ , 5% C0₂ and 93% N₂ at 37°C. Parasites were detected on Giemsa-stained smears after 2 d in culture. On day 4, the cultures were split at a ratio of 1:2 (v/v) and transferred into a humidified atmosphere of 5% C0₂ in air. Starting from day 6, subcultures were made daily at a ratio of 1:4 (v/v). The percentage of parasitized erythrocytes ranged from 2-5%. Addition of purine bases (hypoxanthine, adenine, adenosine or guanosine) was essential for the continuous propagation of the parasites when bovine, but not horse serum, was used for medium supplementation.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. Adobe Acrobat XI Pro was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.mn201