L-cysteine replaces microaerophilous culture conditions for the in vitro initiation of Theileria equi.

Fifty-one blood samples of carrier horses from Theileria equi-endemic localities in South Africa were used for two different methods of in vitro culture initiation of T. equi parasites. Cultures were initiated either in a oxygen-reduced gas mixture or in a 5% CO2-in-air atmosphere in combination with L-cysteine-supplemented culture medium. Out of the 51 blood samples, 43 and 42 cultures, respectively, became culture positive. A possible explanation for this observation is proposed.

Experimental transmission of field Anaplasma marginale and the A. centrale vaccine strain by Hyalomma excavatum, Rhipicephalus sanguineus and Rhipicephalus (Boophilus) annulatus ticks.

The cattle rickettsia Anaplasma marginale is distributed worldwide and is transmitted by about 20 tick species, but only Rhipicephalus simus, a strictly African tick species, has been shown to transmit the vaccine strain of A. centrale. The aim of the present study was to examine transmission of field strains of A. marginale and of the vaccine strain of A. centrale by three tick species -Hyalomma excavatum, Rhipicephalus sanguineus and Rhipicephalus (Boophilus) annulatus - to susceptible calves. Two genetically distinct Israeli field strains of A. marginale, tailed and non-tailed (AmIsT and AmIsNT, respectively), were efficiently transmitted by R. sanguineus, whereas H. excavatum transmitted only the tailed isolate, and R. (Boophilus) annulatus did not transmit A. marginale. None of the three tick species transmitted A. centrale. By means of msp1a primers in PCR assays, amplicons of similar sizes were obtained from either A. marginale-infected calves that were used for acquisition feeding, from R. sanguineus fed on the infected calves, or from calves to which anaplasmosis had been successfully transmitted by these ticks. Although an A. centrale-specific fragment was amplified from salivary glands of R. sanguineus, no transmission to susceptible cattle occurred during 3 months of observation, and anaplasmosis was not induced in splenectomized calves that were subinoculated with blood from calves on which R. sanguineus had fed.

In vitro isolation of Ehrlichia ruminantium from ovine blood into Ixodes scapularis (IDE8) cell cultures.

Four stocks of Ehrlichia ruminantium (Welgevonden, Ball3, Nonile and Blaauwkrans), the causative agent of heartwater in domestic ruminants, were isolated into Ixodes scapularis (IDE8) tick cells using the leukocyte fraction of the blood of infected sheep. Organisms of two of the E. ruminantium stocks (Welgevonden and Blaauwkrans) propagated in IDE8 cells were also successfully used to infect bovine endothelial cells. All stocks were successfully propagated in IDE8 cells using Dulbecco's modified Eagle's medium nutrient mixture Ham F-12 containing 10% foetal bovine serum (FBS). The technique should be included in any attempt to isolate uncharacterized E. ruminantium stocks.

Determination and quantification of the in vitro activity of Aloe marlothii (A. Berger) subsp. marlothii and Elephantorrhiza elephantina (Burch.) skeels acetone extracts against Ehrlichia ruminantium.

An Ehrlichia ruminantium culture system was utilized for the anti-rickettsial evaluation of two ethnoveterinary plants, Elephantorrhiza elephantina and Aloe marlothii. Well-established E. ruminantium cultures were incubated with the plant leaf acetone extracts and compared to oxytetracycline and untreated controls. Effectivity was established by comparing the percentage parasitised cells and the calculation of both EC50 and extrapolated EC90 in microg/ml. The plant extracts were also screened for antibacterial activity using bioautography. Elephantorrhiza elephantina and A. marlothii demonstrated anti-ehrlichial activity with an EC50 of 111.4 and 64.5 microg/ml and EC90 of 228.9 and 129.9 microg/ml, respectively. The corresponding EC50 and EC90 for oxytetracycline was 0.29 and 0.08 microg/ml. Both plants appeared to produce their inhibitory activity by a similar mechanism, unrelated to that of the tetracyclines. Both the plant acetone extracts demonstrated antibacterial activity against Escherichia coli and Staphylococcus aureus (ATCC strains).

In vitro cultivation of a south African isolate of an Anaplasma sp. in tick cell cultures.

This paper describes the first successful in vitro cultivation of a South African isolate of an Anaplasma sp., initially thought to be Anaplasma marginale, in the continuous tick cell line IDE8. Blood from a bovine naturally infected with A. marginale kept on the farm Kaalplaas (28 degrees 08' E, 25 degrees 38' S) was collected, frozen, thawed and used as inoculum on confluent IDE8 cell cultures. Twenty days after culture initiation small intracellular colonies were detected in a Cytospin smear prepared from culture supernatant. Cultures were passaged on Day 34. Attempts to infect IRE/CTVM18 cell cultures with the Kaalplaas isolate derived from IDE8 cultures failed, whereas a reference stock of A. marginale from Israel infected IRE/CTVM18 tick cell cultures. Attempts to infect various mammalian cell lines (BA 886, SBE 189, Vero, L 929, MDBK) and bovine erythrocytes, kept under various atmospheric conditions, with tick cell-derived Anaplasma sp. or the Israeli strain of A. marginale failed. Molecular characterization revealed that the blood inoculum used to initiate the culture contained both A. marginale and Anaplasma sp. (Omatienne) whereas the organisms from established cultures were only Anaplasma sp. (Omatjenne).

Identification of anti-babesial activity for four ethnoveterinary plants in vitro.

A commonly available Babesia caballi culture system was utilized for anti-babesial screening of four commonly used ethnoveterinary plants, Rhoiscissus tridentata, Elephantorrhiza elephantina, Aloe marlothii and Urginea sanguinea, in vitro. Well-established B. caballi cultures were initially incubated with either imidocarb diproprionate and diminazene aceturate to validate the model, where after the studies were performed on the four plants. Effectivity was established as the degree of inhibition using a colour change method as well as by evaluating percentage parasitized cells on thin culture smears and calculating the degree of residual infectivity. The model was effective in demonstrating the in vitro efficacy of the well known anti-babesial drugs imidocarb and diminazene indicating an EC50 value of 0.08 and 0.3 microg/ml, respectively. Only the E. elephantina rhizomes acetone extracts were effective at a concentration of 100 microg/ml. It was also shown that the colour change method of evaluation was not very sensitive for determining activity of crude plant extracts.

In vitro infection of nonendothelial cells by Ehrlichia ruminantium.

The Welgevonden stock of Ehrlichia ruminantium was propagated in eight nonendothelial cell cultures derived from different animal species, both ruminants and nonruminants. The origins of the cells were: bovine fetal testis (BFT), cat ovary (COC), donkey fibroblasts (DFC), sheep fibroblasts (E(2)), horse testis (HTC), lamb fetal testis (LFT), mouse connective tissue (L), and African green monkey kidney (Vero). Four cell culture types (BFT, E(2), LFT and Vero) required supplementation of the medium with cycloheximide for suitable growth of E. ruminantium, whereas the other four (COC, DFC, HTC, and L) did not. Three other stocks of E. ruminantium, Senegal, Ball 3, and Gardel, were also propagated, either in LFT cultures only or in both E(2) and LFT cell cultures. The Welgevonden stock was successfully initiated using E(2) and LFT cell cultures.

Amino acid content of cell cultures infected with Cowdria ruminantium propagated in a protein-free medium.

The in vitro culture of Cowdria ruminantium, the causative agent of heartwater in domestic ruminants, was first achieved in 1985. Culture media were usually supplemented with serum and tryptose phosphate broth, both undefined components, contributing to great variability. Recently, we reported about the propagation of stocks of C. ruminantium in a protein-free culture medium referred to as SFMC-23, which is chemically fully defined. To clarify whether the amino acid composition in SFMC-23 is adequate for the in vitro propagation of Cowdria, the Welgevonden stock was propagated in SFMC-23 medium. After a 3-day culture period, samples were taken from uninfected and infected bovine endothelial cell cultures. They were analyzed for free amino acids by the Pico Taq reversed-phase HPLC precolumn derivatization method. Eighteen different amino acids were examined. A considerable decrease in concentration was observed with proline (29%) and glutamine (62%). Further dramatic changes were observed with amino acids which accumulated in the culture medium: aspartic acid, serine, asparagine, tryptophane, glycine, and alanine. The concentration of alanine increased by approximately 660%. The concentrations of all other amino acids analyzed remained within a 25% range, either increasing or decreasing. These results suggest that only glutamine may run short during in vitro cultivation. It seems more likely that accumulation of various amino acids may impact negatively on long-term Cowdria propagation.

Serum-free media for the in vitro cultivation of Cowdria ruminantium.

The in vitro culture of Cowdria ruminantium, the causative agent of heartwater in domestic ruminants, was first achieved in 1985; since then, most groups working with this culture system have used media which were supplemented with serum and, in addition, most of them contained tryptose phosphate broth. These undefined products vary from batch to batch and often fail to support the growth of C. ruminantium. We are therefore working towards the development of a completely chemically defined medium for Cowdria culture. We attempted the propagation of the Welgevonden stock of C. ruminantium in bovine endothelial cell cultures in a variety of serum-free culture media. Four synthetic media gave unsatisfactory results, these were: SFRE-199, Iscove's modified Dulbecco's medium, Dulbecco's modified Eagle's medium, and Leibovitz L-15. These media were all supplemented with a proprietary solution A (components solution A of the HL-1 medium kit, containing transferrin, testosterone, sodium selenite, ethanolamine, saturated and unsaturated fatty acids, and stabilizing proteins). Three other serum-free media did support the growth of C. ruminantium: a modified HL-1 medium, Dulbecco's modified Eagle's medium nutrient mixture Ham F-12 (DME/F-12), and RPMI 1640. The chemical composition of DME/F-12 and RPMI 1640 are published, but not that of the HL-1 medium. Each of these media was supplemented with proprietary solution A. Various supplements were investigated as alternative to the incompletely specified solution A; bovine lipoproteins and bovine transferrin were identified as essential supplements which effectively replaced compound solution A. C. ruminantium was propagated in the three growth-supportive media for at least 10 passages.

Purification of Cowdria ruminantium organisms for use in genome analysis by pulsed-field gel electrophoresis.

Cowdria ruminantium is an obligate intracellular rickettsial pathogen which is responsible for a tick-borne disease of domestic and wild ruminants called heartwater or cowdriosis. Although several genes have been cloned and partially sequenced, the genome size, gross structure, and organization of the C. ruminantium genome is unknown. Genome analysis of the organism has been hindered because it is difficult to obtain C. ruminantium DNA free from contaminating host cell DNA, and this probably accounts for the lack of genome size data for this organism. In this study we investigated several methods for purifying C. ruminantium from bovine cellular contaminants and organisms of a relatively high purity were obtained. These were used to prepare Cowdria DNA which was analyzed by pulsed-field gel electrophoresis (PFGE) and which revealed a genome approximately 1900 kbp in length plus an additional extra-chromosomal fragment migrating with an apparent size of 815 kbp. This is the first time that the genome size of C. ruminantium has been determined and the first demonstration of an extrachromosomal element.

Evaluation of DL-alpha-difluoromethylornithine against susceptible and drug-resistant Trypanosoma brucei brucei.

The antitrypanosomal activity of the ornithine decarboxylase inhibitor DL-alpha-difluoromethylornithine (DFMO, eflornithine) was tested in ten stocks and one clone of the hemoflagellate Trypanosoma brucei brucei in an in vitro system. They showed varying levels of susceptibility to DFMO, their IC50 (the concentration which inhibited growth by 50%) values ranging from 81-691 microM. Differences in DFMO susceptibility were also demonstrated in mice. Combinations of melarsonyl potassium (mel W; trimelarsan) and DFMO showed an additive effect in vitro in a mel W-susceptible and a mel W-resistant stock, but an antagonistic effect in a mel W- and DFMO-susceptible clone. Combinations of suramin and DFMO showed an antagonistic effect in vitro in a suramin-susceptible clone, but a potentiation in a suramin-resistant stock.

The effect of verapamil alone and in combination with trypanocides on multidrug-resistant Trypanosoma brucei brucei.

Following previous studies of verapamil reversal of multidrug resistance in cancer cells and chloroquine resistance in malaria, the effect of the calcium channel blocker verapamil was investigated on multidrug-resistant and susceptible Trypanosoma brucei brucei. Resistance of cloned parasites to diminazene aceturate (Berenil) and isometamidium chloride (Samorin) was expressed in a cell-free in vitro culture system. Verapamil showed antitrypanosomal activity against both, multidrug-resistant and susceptible trypanosomes at concentrations above 1 micrograms/ml. Verapamil did not reverse multidrug resistance when used at concentrations of 0.1 or 1.0 micrograms/ml in combination with diminazene aceturate or isometamidium chloride. Results obtained in vitro correlate with observations in mice. It is suggested that multidrug resistance in African trypanosomes is due to mechanisms other than those occurring in cancer cells, malaria or South-American trypanosomiasis.

Trypanosoma brucei evansi: dyskinetoplasia and loss of infectivity after long-term in vitro cultivation.

Bloodstream forms of a stock of Trypanosoma brucei evansi were propagated in vitro for more than 14 months. After that period, all organisms were dyskinetoplastic and had lost their infectivity for mice. An increase in diminazene aceturate resistance in vitro was observed whereas the susceptibility to isometamidium chloride, quinapyramine sulphate and suramin was unaltered. Trypanosomes derived from the long term culture had a surface coat.

Time-dose-response of Trypanosoma brucei brucei to diminazene aceturate (Berenil) and in vitro simulation of drug-concentration-time profiles in cattle plasma.

Bloodstream form Trypanosoma brucei brucei of axenically growing populations were incubated in the presence of 10.0, 1.0 or 0.1 micrograms/ml diminazene aceturate (Berenil) at 37 degrees C for various periods and, subsequently, either inoculated into mice or further propagated in vitro in drug-free medium. Growth was monitored for 10 days. The ability of trypanosomes of drug-sensitive CP 2137 (clone 1) to grow in vitro was irreversibly damaged after short incubation (< 1 min) with 10.0 micrograms/ml or after 15 min with 1.0 micrograms/ml diminazene aceturate. In contrast, drug-resistant CP 2469 (clone 1) trypanosomes tolerated incubation with 10 micrograms/ml of drug for up to 6 h and 1.0 micrograms/ml of drug for up to 24 h. Differences in drug susceptibility were also detected regarding infectivity to mice and changes in trypanosome cell volume. The results demonstrated that less than 1 min exposure to diminazene aceturate at concentrations as seen in bovine plasma at the initial peak after diminazene aceturate treatment is enough to irreversibly damage drug-sensitive trypanosomes. However, these concentrations were not sufficient to completely eliminate drug-resistant trypanosomes after exposure for 1-6 h; trypanosomes continued to grow for 48 h before the majority of them died and only a few organisms survived to revive the cultures. When drug-sensitive trypanosomes were exposed in vitro for 24 h to diminazene aceturate at the level of concentrations found in cattle after treatment with 3.5 mg/kg, most of the trypanosomes died and none of the surviving parasites could be propagated in vitro in the absence of drug for more than 2 days. However, a small population of drug-resistant trypanosomes was not irreversibly damaged and a few surviving trypanosomes were able to establish growing cultures. The addition of feeder layer cells did not change the outcome of these experiments.

A simple and rapid method to initiate Trypanosoma brucei brucei and T. brucei evansi bloodstream form cultures.

Initiation of cultures of bloodstream forms of trypanosomes from the Trypanozoon subgenus is an established laboratory procedure. The trypanosomes are usually separated from the blood cells of the donor animal by centrifugation on a density gradient (Hirumi et al., 1977) or by differential centrifugation (Hill et al., 1978; Brun et al., 1981; Zweygarth et al., 1982; Baltz et al., 1985). Isolation on an anion exchange column (Lanham and Godfrey, 1970) has been little used for this purpose. We describe a simple and rapid method for the initiation of trypanosome bloodstream form cultures from infected host blood, avoiding centrifugation and anion exchange column procedures. Two T.b. brucei stocks, CP 271 and CP 547, and two T.b. evansi stocks, CP 1134 and CP 2087, were used. Stock CP 271 was isolated in 1980 from a goat in Matuga/Kenya, stock CP 547 was isolated in 1985 from a naturally infected bovine in Jilib/Somalia. The two T.b. evansi stocks were both isolated from camels, stock CP 1134 in 1979 in Kulal/Kenya, stock CP 2087 in 1980 in the Sudan.

Trypanosoma simiae: in vitro studies on drug susceptibility.

Two Trypanosoma simiae stocks were initiated in culture with tsetse-derived metacyclics. They were propagated axenically as trypomastigote forms at 35 degrees C in 4% CO2 in air. Populations of trypanosomes were incubated with various concentrations of antitrypanosomal compounds. Growth was monitored after 24 h of incubation and the growth inhibition was calculated. Diminazene aceturate, quinapyramine sulphate, DL-alpha-difluoromethylornithine, and Ro 15-0216 showed activity against the stocks. Suramin and Mel Cy showed little effect upon the growth of the parasite populations. Isometamidium chloride gave questionable results in the 24 h growth inhibition test, but the results of a long-term viability assay indicated some degree of drug resistance (or drug tolerance). The results obtained herein correlate with observations obtained from in vivo studies in pigs. It is thus concluded that in many cases the cryptic nature of T. simiae rather than drug resistance is responsible for the failure of chemotherapy of simiae-trypanosomiasis in pigs.

In vitro development of metacyclic Trypanosoma simiae derived from bloodstream trypomastigotes.

A stock of Trypanosoma simiae was transformed into procyclic forms at 26 C in a semi-defined maintenance medium. After transformation, the trypanosomes were maintained in a modified Eagle's MEM medium. On day 35 of cultivation, epimastigotes attached to the bottom of the culture flask. From day 44 onwards, metacyclic-like trypanosomes were observed. Subcutaneous injections into pigs of trypanosome suspensions obtained from cultures on day 10 were not infective, whereas culture-derived metacyclics (days 44, 63 and 69) were highly pathogenic.

Characterization of the pCS20 region of different Ehrlichia ruminantium isolates.

Heartwater is a serious tick-borne disease of ruminants caused by the rickettsial organism Ehrlichia (Cowdria) ruminantium. A diagnostic test, targeting the pCS20 genomic region and using PCR amplification and probe hybridization, detects E. ruminantium infection in ticks and animals. However, only the pCS20 sequence of the Crystal Springs E. ruminantium isolate is available and the existence of sequence variation amongst different E. ruminantium isolates has not been determined. Primers were designed from the published pCS20 sequence to obtain sequences of the pCS20 region of various E. ruminantium isolates. These primers were unable to amplify the pCS20 region from genomic Welgevonden DNA and genome walking was used to characterize the pCS20 region. This technique showed that the published pCS20 sequence is from a chimeric clone. Sequences of the pCS20 region of 14 different E. ruminantium isolates were determined after amplification with newly designed primers. Sequencing data indicated that West African E. ruminantium isolates are highly conserved, whereas more variation occurs amongst the southern African isolates. These results facilitated the design of a short pCS20 probe and a large PCR target that improved the sensitivity of the E. ruminantium detection assay.

Low molecular weight proteins of Cowdria ruminantium (Welgevonden isolate) induce bovine CD4+-enriched T-cells to proliferate and produce interferon-gamma.

An important objective in vaccination strategies is to activate lymphocytes with particular effector functions. Cellular immunity and the type I cytokine IFN-gamma have been implicated in protective immunity to heartwater. Furthermore, low molecular weight proteins of Cowdria ruminantium have been shown to induce peripheral blood mononuclear cells to proliferate. To determine which lymphocyte subset responds when stimulated with fractionated C. ruminantium proteins, specific short-term lymphocyte cultures were established from cattle immunized with the Welgevonden isolate. Four cattle were immunized, two by infection and treatment and two with inactivated organisms. Cell surface phenotypic analysis of the cultures indicated that CD4+ lymphocytes were enriched over time. This coincided with increased antigen-specific proliferation and IFN-gamma production. Proteins of molecular weights 13-18kDa induced the CD4+-enriched T-cell cultures, derived from each of the animals, to proliferate and produce IFN-gamma. Although the two groups of cattle were immunized differently, their lymphocytes responded similarly. These results extend previous findings by identifying the responder cells as being predominantly IFN-gamma producing CD4+ lymphocytes. This cytokine has been implicated in immunity to the parasite. The low molecular weight proteins that induced CD4+ lymphocytes to proliferate and produce IFN-gamma are therefore likely to be important in protection against heartwater and may have a role in vaccine development.

Trypanosoma simiae in the white rhinoceros (Ceratotherium simum) and the dromedary camel (Camelus dromedarius).

Trypanosoma simiae was identified as the cause of a disease outbreak in dromedary camels (Camelus dromedarius) introduced to Tsavo East National Park, confirming the susceptibility of camels to this pathogen. T. simiae was also isolated from a new host, the white rhinoceros (Ceratotherium simum) through xenodiagnosis with a susceptible tsetse species (Glossina morsitans centralis). A white rhinoceros showed some evidence of anaemia and lymphopaenia when harbouring T. simiae, but did not suffer any long-term health effects.