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Axenic culture of African trypanosome bloodstream forms
80
Parasitology Today, vol. i 0, no. 2, I 994
T@GhlnBqu@,” Axenic Culture of African Trypanosome Bloodstream Forms H. Hirumi and K. Hirumi In this article,
Hiroyuk Hirumi and KCJZU~O Hirumi review recent technical developments of in vitro systems that support the growth of bloodstream firms of Afn’can trypanosomes in the absence of mammalian feeder layer cells, which were required in earlier methods. Salivarian trypanosomes cause African ttypanosomiases in humans, as well as in a variety of domestic and wild animals. Main species involved are Trypanosoma brucei gambiense and T. b. rhodesiense in humans, and T. b. brucei, T. congolense, T. simiae and T. vivax in cattle, sheep, goats, pigs and horses. Most trypanosomes are transmitted by tsetse (genus Glossina) and are extracellular parasites throughout their life cycle, both in the mammalian hosts and in the insect vectors. The availability of in vitro systems that support the continuous growth of all the developmental stages and the transformation from one stage to a subsequent stage is essential for conducting laboratory studies of the diseases in areas as varied as parasite
biology, biochemistry, genetics and immunology. Basic procedures that support the growth of three principal pathogens of African ttypanosomiases, namely, T. brucei, T. congolense and T. vivax, have been established since the first continuous cultivation of T. b. brucei bloodstream forms (BPS) was achieved in 1977 (Ref. I), although salivarian ttypanosomes had been considered to be the most difficult members of the order Kinetoplastida to culture, in vitro2. All the developmental stages (Box I) of the three species (at least those of stocks or clones tested to date) can now be cultured, in vitro (for reviews see Brun and ]enni3 and Gray and colleagues4). In the earlier methods, BSFs of the three species were propagated, in vitro, in the presence of mammalian feeder layer cells. These feeder layer systems are suitable for large-scale production of BSFs for studies in which a large number of the parasite materials is required regardless of the culture conditions used, but are unsuitable for studies in which ‘background noise’
caused by the feeder layer cells interferes with results. Recently, the number of studies that require ‘background noise’-free (axenic culture) systems has been increasing rapidly, particularly in the areas of: (I) growth-promoting and growth-inhibiting factors; (2) sensitivity to trypanocidal drugs; (3) mechanisms that underlie drug resistance; (4) the in vitro screening of new drugs; and (5) molecular mechanisms that regulate differentiation of the parasites. To facilitate such studies, attempts were made to eliminate the need of feeder layer cells, specifically for culturing BSFs of the three principal species.
Jwpanosoma
brucei
The first axenic cultivation of T. brucei BSFs was independently repot-ted by Baltz et a1.5 and Duszenko et aL6 In the Baltz system, BSFs of ten stocks of the T. brucei subgroup were successfully cultivated by using a modified Eagle’s minimum essential medium (MEM) supplemented with 2-mercaptoethanol,
Box
I. The Life Cycle of Trypanosoma brucei In tsetse Trypanosomes undergo a series of differentiations in mammalian hosts and tsetse (see Fig.). Parasites injected into the blood of a mammal by an infected tsetse become the long slender bloodstream forms (BSFs) (a), rapidly multiply by binary fission and establish the infection in the host. The BSFs are covered with variable surface glycoproteins (VSGs) and are infective for mammals. As the parasitaemia arises, the slender forms change to the intermediate forms (b) and subsequently to the stumpy forms (c). when a BSF population containing (a), (b) and (c) is ingested along with bloodmeals by a tsetse, they lose VSGs and transform In the mid-gut to the procyclic forms (d), which are non-infective For mammals, after which the parasites enter the proventriculus and later salivary glands, where they assume epimastigote forms (e). The procyclic and the epimastigote forms also multiply rapidly by binary fission in the vector. In the salivary gland, the epimastiptes further transform to the non-dividing metacyclic forms (f), which are covered again with VSGs and re-acquire the infectivity for mammals. The life cycles of T. congolense and T. vivox are basically the same as that of T. brucei, with some differences in development sites in the tsetse. Axenic culture systems that support the continuous growth of (a), as well as the transformation from non-dividing (f) to dividing (a), are highly useful tools for studying African trypanosomes under well-defined laboratory conditions. (ILRAD Annual Report 1990, after Vickerman19.)
Division
Mitochondrion
Parasitology
Today, vol. IO, no. 2, I994
81
Box 2. Procedure
for the axenic cultivation of Trypanosoma brucei bloodstream forms (I) Isolate bloodstream forms (BSFs) from infected mouse blood at the rising parasitaemia by suspending the blood (one part) in HMI-9 medium (nine parts) in 5 ml Wheaton V-vials and centrifuging at 200g for 5 min. (2) Transfer BSF-containing supernatants to 25cm2 T-type (T-25) flasks (5 ml per flask) and keep the flasks for 2-3 h at 37°C to settle remaining blood cells on the bottom of the flasks. (3) Pool the BSF-containing; medium from the flasks by gentle pipetting and adjust the concentration of BSFs to 6-8 X 10sBSFs ml-’ by adding an appropriate volume of fresh medium. (4) Place the adjusted trypanosome suspension in new T-25 flasks (4ml per flask), incubate the flasks of which caps are kept loose in a CO, incubator (2% CO, in air) for 3-I h at 37°C until phenol red in the medium indicates pH7.2f0.2 and then close the caps. (5) Readjust ‘daily seeding density’ to 7 X IO5 BSFs ml-’ by replacing 2.0 f0.5 ml of the medium every 24 h. (6) Make subcultures (if required) by transferring the trypanosome-containing culture fluids (1.5-2.0 ml per flask) to new T-25 flasks, readjust the BSF density to 7 X I05 BSFsml-1 by adding an appropriate volume of fresh medium and repeat the Steps (4) and (5) every 5-7 days. In some primary cultures Iof certain T. brucei populations, many BSFs die during the initial 5-7 days. In such a case, pool the culture fluids from flasks, remove cell debris by means of diethylaminoethyl cellulose (DE52) column chromatography under a sterile condition, collect B!SFs by centrifugation at 1600+5Og for IO min, resuspend BSFs in fresh medium (7 X IO5 BSFs ml-‘) and repeat Steps (4)-(6).
hypoxanthine, sodium pyruvate, thymidine and 10% (v/v) of mammalian serum derived from various animals. However, the cultures we-e first maintained in the presence air mammalian feeder cells (macrophages) during an ‘initiation stage’ and also in a sub‘adaptation stage’. In the sequent Duszenko system, BSFs of a cloned population of T. brucei MTI I .4 were continuously cultured by using a modified MEM that contained L-cysteine which was added at regular intervals (twice a day) and in appropriate concentrations. To overcome -the problems encountered in these systems (ie. the requirement of feeder layer cells during the initial stages, the need to prepare a fresh medium for every use, and the need for frequent medium changes), an improved method for culturing T. brucei BSFs has been established7. In the improved method, BSFs of cloned populations of T. b. brucei
GUTat 3. I and T. b. gambiense IL2343 have been coritinuously cultured in a relatively simple manner (Box 2), using HMI-9 medium (Table I), which was modified from lscove’s modified Dulbecco’s modified MEM (Iscove’s modified DMEM) by supplementing with foetal bovine serum, Serum Plus and six additional BSF-supporting factors, in amounts indicated in Table I. This study confirmed the findings in the earlier work5.6, namely, that L-cysteine, hypoxanthine, sodium pyruvate and thymidine are essential to support the growth of T. brucei BSFs under axenic culture conditions. Furthermore, two improvements were made in this system: (I) complete elimination of the requirement of feeder cells; and (2) simplification of the medium preparation and changes. The need to prepare fresh medium for every use and frequent medium changes because of toxic effects of hydrogen peroxide pro-
duced by the autoxidation of L-cysteine (which was supplemented in the medium), has been minimized by adding bathocuproine sulphonate (a chelating agent) and by regulating the daily seeding density at a standardized level (7 X 10s BSFsml-I). HMI-9 medium containing L-cysteine can be used at least ten days after its preparation (the maximum shelf time has not been established) and one medium change is sufficient to produce 2-3 X I06BSFs ml-l every 24 h by readjusting the daily seeding density to 7 X IO5BSFsml-l. In certain biochemical and/or immunological experiments, the presence of serum protein in medium interferes with results. Thus, further attempts were made to develop a serum-free medium. To date, a completely serumfree medium is not yet available. However, HMI- I8 medium Fable I) containing a low concentraIion of serum protein has been developed7 by replacing foetal bovine serum in HMI-9 medium (IO%, v/v) with Serum Plus (increasing its amount from IO to 20%). Although the elimination of an addition of foetal bovine serum minimized variation in results that often have been attributed to variability of serum batches, the Serum Plus (lot no. I400 1004) still contains a low level of foetal bovine serum protein (I 3 kgml-I), in addition to specific growth-promoting factors, transport proteins, hormones, a high level of glucose (I 2.24mgml-I), and low amounts of hemoglobin ( IO kg ml-l) and endotoxin (5.8 ngml-I) with a pH of 7.32 and osmolarity of 378 mOsm (according to information released by the manufacturer; no further details are available). Thus, further improvement is still needed to establish a completely serum-independent culture medium. Nevertheless, 2-3 X I06BSFs ml-l can be produced by using HMI- I8 medium when BSF populations are maintained by readjusting the daily seeding density to 9 X I05BSFs ml-l.
Table I. Axenic culture media for bloodstream forms of African trypanosomesa Supplements Trypanosome species
Medium
Serum (%, v/v) YGS FBS
SP
BSF-S Factors (mM)b BAC CYS HYP
2ME
PYR
THY
T. brucei
HMI-9
IO
-
IO
0.05
I.5
I.0
0.20
I
0.16
T. brucei
HMI-I8
-
-
20
0.05
1.5
I .o
0.20
I
0.16
T. congolense
HMI-93
-
20
5
0.05
1.5
0.5
0.12
I
0.16
T. v&ox
HMI-I63
-
20
3
0.03
0.8
0.2
0.07
I
0.10
“Basalmedium was Iscove’smodified Dulbecco’s modified Eagle’sMEM (Iscove’sDMEM) (Flow Laboratories, Irvine, UK). bBSF-SFactors. bloodstream form-supporting factors; FBS, foetal bovine serum (heat inactivated, 56”C, 30 min) (Flow Laboratories; HyClone Laboratories, Logan, USA); YGS, young goat serum (heat inactivated) (prepared at ILRAD); SP, Serum Plus (Hazleton Biologics,presently JRH Biosciences, Lenaxa. USA); BAC, bathocuproine sulphonate (Sigma, St Louis, USA); CYS, L-cysteine (Sigma); HYP, hypoxanthine (Calbiochem, La Jolla, USA); 2ME, 2-mercaptoethanol (BDH Chemicals, Poole, UK); PYR, sodium pyruvate (Sigma);THY, thymidine (Sigma).
ParasitologyToday, vol. IO. no. 2, / 994
82
Trypanosoma
congolense
The development of an axenic culture system for T. congolense BSFs8was made by modifying HMI-9 medium used for the axenic cultivation of T. brucei BSFs. First, the ability of the six BSFsupporting factors supplemented in HMI-9 medium to support the transformation of in vitro-produced metacyclic forms8 (Box I Fig. f) of a cloned T. congolense IL3000 population to BSFs (Box I Fig. a) in vitro, was examined by testing eight culture media (not shown) containing various amounts of the factors and serum supplements in two basal media (MEM and Iscove’s DMEM). The results of this study revealed that: (I) the presence of the supporting factors is essential for the transformation; (2) Iscove’s DMEM is a better basal medium for transformation than MEM at 34°C; (3) MEM appeared to be superior for the propagation of epimastigotes (Box I Fig. e) at 27°C; and (4) the media supplemented with 10% (v/v) foetal bovine serum and 10% (v/v) Serum Plus support the transformation better than those supplemented with 20% foetal bovine serum. Second, the ability of each supporting factor to support the continuous growth of the metacyclic-derived BSFs of T. congolense IL3000 was examined by testing I I media (not shown) containing various combinations of the supporting factors at 34°C. The results of this study revealed that: (I) the presence of bathocuproine sulphonate, L-cysteine and thymidine is essential to support the continuous growth of the BSFs, although an addition of either thymidine alone or L-cysteine together with bathocuproine sulphonate does not support the growth; (2) an addition of hypoxanthine in the presence of these three ‘essential factors’ enhances the growth of BSFs to a certain extent; (3) a further addition of 2-mercaptoethanol and sodium pyruvate (HMI-9 medium) gives the best growth; and (4) additions of hypoxanthine, 2-mercaptoethanol and/or pyruvate in the absence of any of ‘the essential factors’ do not support the growth. HMI-9 medium was further tested for the axenic cultivation of BSFs of another five T. congolense populations (one clone: ILNat3. I and four stocks: lL2079, lL2466, IL3266 and CP-81) derived either from infected mouse blood or from metacyclic forms produced in vitro. However, HMI-9 medium unexpectedly did not support the growth of these additional populations”
A series of media were, therefore, modified from HMI-9 medium and tested for the cultivation of the other BSF populations. Among these, HMI-93 medium in which 10% foetal bovine serum in HMI-9 medium was replaced with 20% young goat serum and the amounts of Serum Plus, hypoxanthine and 2-mercaptoethanol were reduced (Table I), was established to be the optimum medium in terms of the initiation and the maintenance of not only the metacyclic-derived but also the mouse-derived BSFs of all populations testeds. The establishment of the optimum medium was carried out in Plastek M T-25 flasks (Tekmat, Ashland, USA) basically following the procedures used for the BSFs of T. brucei (Box 2). Furthermore, a simple method for initiating primary cultures with a small amount of tail blood of infected mice using the optimum medium was also standardized using 24-well culture plates (24 Well Tissue Culture Cluster, Costar, Cambridge, MA, USA)8. This method could also be applicable for in vitro cloning of BSFs. Both the metacyclic- and the mousederived BSFs of T. congolense rapidly (~4 h) attached to the bottom surface of flasks, as well as of wells, and continued to proliferates, unlike BSFs of T. brucei which proliferated in suspension. During 24 h of cultivation, BSFs formed attached populations and detached populations in the culture fluids. The detached BSFs usually increased in number when cultures became overpopulated. In such cultures, the colour of the medium changed to yellow (indicating a pH ~6.8) and the parasites became sluggish and died within I2 h if the medium was not changed. The attached forms, however, could be continuously maintained by subcultivating in new flasks. Growth rates of the attached populations were consistently higher in cultures that were initiated with a seeding density at 106BSFsml-I (or lower) in a small volume of medium (2ml per flask) than in those initiated with a higher seeding density in a larger volume (3-5 ml per flask). In a standardized method for the axenic cultivation of T. congolense BSFs, subcultures were initiated by: (I) discarding the culture fluids containing detached BSFs and resuspending the attached forms by gentle pipetting in fresh medium; and (2) transferring 2 ml of the BSF suspension containing lO6BSFsml-1 to a new T-25 flask. The pH of culture medium was, thereafter, adjusted to 7.2 + 0.2 by following the same procedure applied
in the Step 4 for the cultivation of T. brucei BSFs (Box 2). The shortest population doubling time achieved in the standardized system was nine hours. All the mouse-derived BSFs examined to date were obtained from mice inoculated with BSFs,which had undergone the complete cyclic development, in vitro. Thus, it may be necessary to examine whether populations that had not undergone the in vitro transfon-ration (such as stocks isolated directly from naturally infected hosts) can also be axenically cultured in this system. If such populations could not be adapted to the system, it would be advisable to pass the populations through the cyclic transformation, in vitro, and then to initiate the cultures with the in vitroproduced metacyclic forms
Trypanosoma
vivax
A recent attempt to culture BSFs of four T. vivax stocks under axenic culture conditions has been reported by Zweygatth and colleaguesg. In this system, BSFs were first adapted to in vitro conditions in the presence of mammalian feeder cells and then maintained axenically up to 60 days in 24-well culture plates using two media modified from MEM. However, the maximum density of BSFs achieved in the system was low (5 X IOsBSFsml-I) and the population doubling times were rather long (I 848 h). Furthermore, the modified media did not support the continuous growth of two cloned BSF populations derived from a West African T. vlvax stock (ILI 392) and an East African stock (IL367 I) (Hirumi, unpublished). Therefore, an in vitro system that supports the growth of BSFs of the East, and the West African T. vivax has been established by developing three in vitro systemslO. ?? System I was established in three steps: (I) production of metacyclic forms of T. vivax ILI 392 using Matrix Gel Green-A beads in the presence of feeder layer cells at 27°C (technical details were reviewed by Gray and colleagues4); (2) transformation of the in vitro-derived metacyclic forms to BSFs which continued to proliferate at 34°C in the presence of feeder layer cells; and (3) establishment of an axenic culture condition for the in vitrotransformed BSFs by testing a total of 69 media (not shown) modified from HMI-93 medium using Plastek M T-25 flasks at 34°C. Although most media did not support the growth of the BSFs, HMI-I 62 medium (not shown)
ParasftologyToday,vol. IO, no. 2, I994
was found to be the best medium for supporting growth at that time. Thus, the axenic cultivation of 7; vivax BSFs was standardized using this medium. In the standardized method, an average population density of J. vivax ILI 392 was 3.4 X I06BSFsml-1 and the population doubling time was 13.6 h. ?? System II was established in two steps: (I) production of metacyclic forms of J. vivax I-3671 BSFs obtained from the infected bovine blood without feeder layer cells at 27’C; and (2) transformation of the in vitro-produced metacyclic forms to BSF*j and continuous cultivation of the in vitrotransformed BSFs under the axenic condition using HMI- I62 medium. Although HMI-I 62 medium also supported the continuous. growth of T. vivax IL3671 BSFs, the maximum density of the BSFs achieved in this medium was lower than IO6ml~l (average 0.7 X I 06mlV) and the population doubling time was longer than I8 h. Therefore, an additional medium, HMII63 (Table I), was mc’dified from HMI- I62 medium. The subcultivation was carried out by transferring the culture fluids containing free swimming BSFs (0.5 X I06BSFsml-1, I ml per flask) and fresh medium (I .5 ml per flask) to new flasks. The subcultures were maintained by changing the medium ( I-I .5 ml per flask) every 24 h until the experiment was terminated on Day 70 of the BSF ‘culture. The maximum density achieved in HMI- I 63 medium was I .8 X 106ml-1 and the shortest population doubling time was 16.1 h. ?? System 111 was established in one step initiating the cultures with proboscides of Glossina morsitans centralis which were infected w’th T. vivax IL367 I, in the absence of feeder layer cells at 34°C initially usil?g HMI-I 62 medium which was later rleplaced with HMI-I 63 after the latter medium was developed. The maximum density and the population doubling time of T. vivax IL3671 in the system we’-e similar to those obtained in System II. Systems II and Ill are highly useful tools for the in vitro study of T. vivax, since most populations of bovine-infective J. vivax are not infect.ive for small experimental animals (with few exceptions, such as IL1392 which is infective for rodents). Application Recently, a simple method for detecting the sensitivity of T. congolense
83
BSFs in vitro to four ttypanocidal drugs, diminazene aceturate (DA), homidium chloride (HC), isometamidium chloride (IC) and quinapyramine sulphate (QS) which are commonly in use, has been established by using the axenic culture system for T. congolense BSFs in 24-well culture plates”. Most problems that had hampered the application of earlier in vitro systems to the assessment of drug sensitivity of trypanosome populations12 have been overcome in this method. Prior to the sensitivity tests, BSFs of four T. congolense stocks (lL2079, lL2466, IL3266 and lL3338), four clones (ILI 180, lL2642, IL3000 and lL3035) and I6 clones obtained in vitro from lL2079, lL2466, IL3000, IL3266 and lL3338, were propagated in T-25 flasks using HMI-93 medium. Among these populations ILI 180, lL2466, IL2642 are known to be sensitive to DA and/or IC as tested in mice and/or cattle, while IL3035 and IL3338 are highly resistant to DA, IC and/or HC in cattle. Test plates were prepared by placing IO0 ~1 of test solutions containing various amounts of the drugs in distilled water to each well. The plates were then freeze-dried and stored at room temperature. In the standardized system, levels of resistance for each drug were expressed in ten steps from ten to one, denoting the following concentrations: DA at 600, 500, 400, 300, 200, 100, 80, 60, 40 and 20ngml-1; HC, IC and QS at tenfold serial dilutions from IO Fg to I Ofgml-1. The test plates wrapped with aluminium foil could be kept at ledst six months at room temperature without any detectable deterioriation of the ttypanotides’ eficacy. The tests were carried out by first placing 500 ~1 aliquots of ttypanosome suspension in HMI-93 medium containing 4 X I OsBSFsml-1 in each well and then incubating at 34°C in a CO,incubator for five days without medium change. Effects of the drugs were examined by phase-contrast microscopy every 24 h. Growth inhibition of BSFs could be detected by Day 3 and attached ttypanosomes died during the next 2+48 h. On the contrary, BSFs which were tolerant to the given concentrations continued to proliferate reaching the maximum population density by Day 3-5, and died during the next 24 h due to overgrowth. The pH indicator, phenol red, in medium in wells which contained affected populations indicated pH 8.0-8.5 (pinkish), whereas that in wells in which BSFs reached the maximum density changed
to pH 6.5 or lower (yellowish) by Day 3-5. Calorimetry of the media on Day 5 was thus also used to distinguish levels of drug sensitivity for the ttypanosome populations. The calorimetric reaction of the culture media, similar to that observed in this study, was reported earlier for testing ttypanocidal activity in T. b. brucei BSFs using 96-well culture plates and an ELISA readeri3. In the in vitro assay described here’ 1,the effects of ttypanotidal drugs should be examined by means of phase-contrast microscopy every 24 h during the initial three days and, in general, the final detection of the end points could be made by colorimetty of the culture media by naked eye on Day 5. In this system, a few unaffected trypanosomes (even a single BSF) continue to multiply without medium change until the population reaches the maximum density. The ability to support growth of the trypanosomes without medium change for up to ten days is particularly advantageous in detecting maximum resistance concentrations of trypanocidal drugs, as well as in selecting a small number of drug-resistant parasites from large numbers of sensitive ttypanosomes. Thus, the in vitro system established would be a useful laboratory tool for: (I) the screening of new drugs: (2) the selection of experimentally induced drug-resistant populations for molecular biological studies; (3) cloning of drug resistant parasite: and (4) the identification of growth promoting, as well as inhibiting, factors. Another advantage of the in vitro assay is the requirement for only relatively simple laboratory equipment, such as a CO,-incubator, a phase-contrast microscope and a vacuum freeze dryer. There is, thus, no requirement for sophisticated equipment, such as a spectrophotometer, ELISA reader, liquid scintillation counter and a Coulter cell counter, used in earlier w0tW3-~*. Furthermore, the in vitro system would also be an excellent alternative to in vivo drug tests which require large numbers of experimental animals and are time-consuming. The results generated in this system using three test plates (triplicate) per drug per ttypanosome population and lasting five days, provided an equivalent amount of information about the drug sensitivity of a ttypanosome population as in viva tests which used at least 36 mice and required two months. In a preliminary study, the in vitro drug-sensitivity assay has been applied to several T. b. brucei and T. vivax
Parasitology
84
populations, using HMI-9 and HMI- I93 media, respectively. The results obtained to date indicate that the system would also be applicable to these species, although the yellowish colour of colorimetrtc reaction in T. b. brucei populations was somewhat less distinct (amber). Acknowledgements The authors wish ta thank A.R. Gray for his critical review of the manuscript, and JR. Wando for his technical assistance. This is ILfWD publication No. 1204. References I Hlrumi. H.. Doyle. 1.1.and Hirumi, K. (1977) j Science196,99%994 2 Evans, D.A. (I 978) in Methods m Cultjvatmg
Medical Insects and Arachnids edited by Richard P. Lone and Roger W. Crosskey, Chapman & Hali, 1993. f85.00 (xv + 723 pages) lS5N 0 4 I2 40000 6 Few young biologists choose medical entomology as a speciality. Not only is the subject generally poorly taught, but it also suffers from a Batesian image of Victorian times with practitioners who wear funny hats and prowl through forests with a net on a stick. The few who opt for medical entomology have no bandwagon to carry them to the heights of tropical medicine nor do they really need to study Swedish in case they are ever invited to Stockholm to receive a Nobel Prize. They will have to be enthusiasts like each of the authors of the chapters in this splendid new book with the unusual title Medical insects and Arachnids. It has a fine pedigree beginning during the Second World War with Smart’s Handbook for the Identification of Insects followed, in of Medical Importance 1973, by Insects ond Other Arthropods of Medical lrnportonce edited by K.G.V. Smith. Lane and Crosskey’s latest offering is completely rewritten and surpasses its predecessors. All medically important insects and arachnids are dealt with in an organized way which shows tight editing and an unusual degree of discipline and compliance by contributors. As expected in a book from the Natural History Museum, a large part of each chapter deals with classification and identifi-
Parasites in vitro 3
4
5 6 7 8 9 IO II 12
(Taylor,
A.E.R. and Baker,
I.R., eds). pp 55-88, Academic Press Btun, d. and Jenni, L. (I 987) in Methods for Poraslte CuItrvation (Taylor. A.E.R and Baker, I.R., eds), pp 94-l I?. Academic Press Gray, M.A.. Hlrumi, H. and Gardener, P.R. (1987) an Methods for Porostte Cultivation Taylor, A.&R and Baker, J.R., eds), pp I I 8-I 52, Academic Press Baltz,T.etol.(l985)EMBOJ.4, 1273-1277 Duszenko, M. et al. (1985) j. Exp. Med. 162, 1256-1263 Hirumi, H. and Hirumi. K. (I 989) J. Parosrtoi. 75,985-989 Hiruml, H. and Hltumi, K. (199 I) farasrtoiogy 102,223-236 Zweygarth, E., Gray, M.A. and Kamlnsky, R ( I99.lj Trap. Med. Porasrtol. 42, 4548 Hlrumi, H. et ol. (l99l)J. Protozooi Ues. I, I-12 Hlrumi, H.. Hirumi, K. and Peregrine, A.S. (I 993)J. Protozool. Res. 3. 52-62
,I~
I
n ,,nnn\ “_n~...-r_ h,.
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cation with no apologies offered. This is timely as we feel the first twinges of the birth pangs of a renaissance in taxonomy following many recent reports on the need to know more about the animals with which we share this planet. Whether traditionalists like it or not, molecular biology is going to make the same impact on medical entomology as it has on other biological disciplines. There may not be much evidence of this at the moment perhaps because (I) some entomologists are not moving with the times and (2) few molecular biologists know anything about ‘medical’ insects. How many of them know why the antennae of male and female mosquitoes are so different, how many larval instars does a flea have, or if male sandflies take bloodmeals? They should know that sibling species are not brothers and sisters. Above all, these are the workers who need this book - although every medical entomologist will find many things of interest between its covers. After classification, each chapter deals with biology, medical importance, control and, finally, collection, preservation and rearing. The new taxonomy will need living material and the next time this book appears under another title, rearing and colonization should not be relegated to a paragraph or two at the end of each chapter. Furthermore, preservation in the future will not be solely by pinning or pickling. It will be in cryobanks. Cryopresetved specimens can be mounted for classical taxonomy (far more easily than dried or pickled specimens), and are of immense value to molecular biologists.
2x205-2 13 Zinsstag, Parosrtol. 14 Brun, R.
Today, vol. IO, no. 2, I 994
IO 1,. Rerun, R and Gessler, M. (I 99 I) Res. 77, 33-38 and Kunz, C. (I 989) Acta Trap. 46,
361-368 15 Brun, R. and Rab, S. (1991) Porasltol. Res. 77, 341-345 16 Kaminsky, R. and Zweygarth. E. (1989) Antimlcrob. Agents Cbemother. 33, 88 l-885 17 Kaminsky, R, Chuma, F. and Zweygarth, E. (I 989) Exp. Porasitol. 69. 28 l-289 I8 Ross, C.A. and Taylor, A.M. (I 990) Purusitol. I Res. 76,326-33 I9 Vickerman, K. (I 97 I) in Ecology and PhysiaIogy of Parasrtes (Fallis, A.M., ed.), pp 58-91, Unlverslty of Toronto Press A-2 jlULU~~ Vf17111r nwmt ’’ uIIu ore at tionol Laboratory for Research on Animal DE ieases [ILRADJ, PO Box 30709, Hir*yuk the
H;->_; liullll
lnterna
h,r;“nh; I(n,
Ignoring this glimpse ‘into the future, it must be said that this is the best book available on the subject. It should be used not just by entomologists, but by all workers and teachers involved with vector-borne diseases. It is well written, authoritative and full of ‘gems’, the best of which concerns spiders. It is said that most bites of Latrodectus in Texas are inflicted on men using outdoor privies. (Why not women?) It is recommended that webs should be moved from under the seats so that ‘the spiders would not be stimulated to attack the delicate portion of the anatomy causing them to vibrate’ (p. 678). There is no reference to this story. In fact, for a reference book there are too few references. This book will undoubtedly stimulate more young biologists to become medical entomologists. Perhaps the future of the subject is brighter than we think and they might find it worthwhile to study Swedish. R. Killick-Kendrick Department of Biology lmperlal College at Silwood Park Ascot, UK SL5 7PY
Indexes We hope that you find the enclosed subject and author indexes useful. If you are interested in buying any back issues of Parosir~lo~ Joday, please contact Alison Fricker at our Cambridge offIce, and she will help you.
Parasitology Today, vol. i 0, no. 2, I 994
T@GhlnBqu@,” Axenic Culture of African Trypanosome Bloodstream Forms H. Hirumi and K. Hirumi In this article,
Hiroyuk Hirumi and KCJZU~O Hirumi review recent technical developments of in vitro systems that support the growth of bloodstream firms of Afn’can trypanosomes in the absence of mammalian feeder layer cells, which were required in earlier methods. Salivarian trypanosomes cause African ttypanosomiases in humans, as well as in a variety of domestic and wild animals. Main species involved are Trypanosoma brucei gambiense and T. b. rhodesiense in humans, and T. b. brucei, T. congolense, T. simiae and T. vivax in cattle, sheep, goats, pigs and horses. Most trypanosomes are transmitted by tsetse (genus Glossina) and are extracellular parasites throughout their life cycle, both in the mammalian hosts and in the insect vectors. The availability of in vitro systems that support the continuous growth of all the developmental stages and the transformation from one stage to a subsequent stage is essential for conducting laboratory studies of the diseases in areas as varied as parasite
biology, biochemistry, genetics and immunology. Basic procedures that support the growth of three principal pathogens of African ttypanosomiases, namely, T. brucei, T. congolense and T. vivax, have been established since the first continuous cultivation of T. b. brucei bloodstream forms (BPS) was achieved in 1977 (Ref. I), although salivarian ttypanosomes had been considered to be the most difficult members of the order Kinetoplastida to culture, in vitro2. All the developmental stages (Box I) of the three species (at least those of stocks or clones tested to date) can now be cultured, in vitro (for reviews see Brun and ]enni3 and Gray and colleagues4). In the earlier methods, BSFs of the three species were propagated, in vitro, in the presence of mammalian feeder layer cells. These feeder layer systems are suitable for large-scale production of BSFs for studies in which a large number of the parasite materials is required regardless of the culture conditions used, but are unsuitable for studies in which ‘background noise’
caused by the feeder layer cells interferes with results. Recently, the number of studies that require ‘background noise’-free (axenic culture) systems has been increasing rapidly, particularly in the areas of: (I) growth-promoting and growth-inhibiting factors; (2) sensitivity to trypanocidal drugs; (3) mechanisms that underlie drug resistance; (4) the in vitro screening of new drugs; and (5) molecular mechanisms that regulate differentiation of the parasites. To facilitate such studies, attempts were made to eliminate the need of feeder layer cells, specifically for culturing BSFs of the three principal species.
Jwpanosoma
brucei
The first axenic cultivation of T. brucei BSFs was independently repot-ted by Baltz et a1.5 and Duszenko et aL6 In the Baltz system, BSFs of ten stocks of the T. brucei subgroup were successfully cultivated by using a modified Eagle’s minimum essential medium (MEM) supplemented with 2-mercaptoethanol,
Box
I. The Life Cycle of Trypanosoma brucei In tsetse Trypanosomes undergo a series of differentiations in mammalian hosts and tsetse (see Fig.). Parasites injected into the blood of a mammal by an infected tsetse become the long slender bloodstream forms (BSFs) (a), rapidly multiply by binary fission and establish the infection in the host. The BSFs are covered with variable surface glycoproteins (VSGs) and are infective for mammals. As the parasitaemia arises, the slender forms change to the intermediate forms (b) and subsequently to the stumpy forms (c). when a BSF population containing (a), (b) and (c) is ingested along with bloodmeals by a tsetse, they lose VSGs and transform In the mid-gut to the procyclic forms (d), which are non-infective For mammals, after which the parasites enter the proventriculus and later salivary glands, where they assume epimastigote forms (e). The procyclic and the epimastigote forms also multiply rapidly by binary fission in the vector. In the salivary gland, the epimastiptes further transform to the non-dividing metacyclic forms (f), which are covered again with VSGs and re-acquire the infectivity for mammals. The life cycles of T. congolense and T. vivox are basically the same as that of T. brucei, with some differences in development sites in the tsetse. Axenic culture systems that support the continuous growth of (a), as well as the transformation from non-dividing (f) to dividing (a), are highly useful tools for studying African trypanosomes under well-defined laboratory conditions. (ILRAD Annual Report 1990, after Vickerman19.)
Division
Mitochondrion
Parasitology
Today, vol. IO, no. 2, I994
81
Box 2. Procedure
for the axenic cultivation of Trypanosoma brucei bloodstream forms (I) Isolate bloodstream forms (BSFs) from infected mouse blood at the rising parasitaemia by suspending the blood (one part) in HMI-9 medium (nine parts) in 5 ml Wheaton V-vials and centrifuging at 200g for 5 min. (2) Transfer BSF-containing supernatants to 25cm2 T-type (T-25) flasks (5 ml per flask) and keep the flasks for 2-3 h at 37°C to settle remaining blood cells on the bottom of the flasks. (3) Pool the BSF-containing; medium from the flasks by gentle pipetting and adjust the concentration of BSFs to 6-8 X 10sBSFs ml-’ by adding an appropriate volume of fresh medium. (4) Place the adjusted trypanosome suspension in new T-25 flasks (4ml per flask), incubate the flasks of which caps are kept loose in a CO, incubator (2% CO, in air) for 3-I h at 37°C until phenol red in the medium indicates pH7.2f0.2 and then close the caps. (5) Readjust ‘daily seeding density’ to 7 X IO5 BSFs ml-’ by replacing 2.0 f0.5 ml of the medium every 24 h. (6) Make subcultures (if required) by transferring the trypanosome-containing culture fluids (1.5-2.0 ml per flask) to new T-25 flasks, readjust the BSF density to 7 X I05 BSFsml-1 by adding an appropriate volume of fresh medium and repeat the Steps (4) and (5) every 5-7 days. In some primary cultures Iof certain T. brucei populations, many BSFs die during the initial 5-7 days. In such a case, pool the culture fluids from flasks, remove cell debris by means of diethylaminoethyl cellulose (DE52) column chromatography under a sterile condition, collect B!SFs by centrifugation at 1600+5Og for IO min, resuspend BSFs in fresh medium (7 X IO5 BSFs ml-‘) and repeat Steps (4)-(6).
hypoxanthine, sodium pyruvate, thymidine and 10% (v/v) of mammalian serum derived from various animals. However, the cultures we-e first maintained in the presence air mammalian feeder cells (macrophages) during an ‘initiation stage’ and also in a sub‘adaptation stage’. In the sequent Duszenko system, BSFs of a cloned population of T. brucei MTI I .4 were continuously cultured by using a modified MEM that contained L-cysteine which was added at regular intervals (twice a day) and in appropriate concentrations. To overcome -the problems encountered in these systems (ie. the requirement of feeder layer cells during the initial stages, the need to prepare a fresh medium for every use, and the need for frequent medium changes), an improved method for culturing T. brucei BSFs has been established7. In the improved method, BSFs of cloned populations of T. b. brucei
GUTat 3. I and T. b. gambiense IL2343 have been coritinuously cultured in a relatively simple manner (Box 2), using HMI-9 medium (Table I), which was modified from lscove’s modified Dulbecco’s modified MEM (Iscove’s modified DMEM) by supplementing with foetal bovine serum, Serum Plus and six additional BSF-supporting factors, in amounts indicated in Table I. This study confirmed the findings in the earlier work5.6, namely, that L-cysteine, hypoxanthine, sodium pyruvate and thymidine are essential to support the growth of T. brucei BSFs under axenic culture conditions. Furthermore, two improvements were made in this system: (I) complete elimination of the requirement of feeder cells; and (2) simplification of the medium preparation and changes. The need to prepare fresh medium for every use and frequent medium changes because of toxic effects of hydrogen peroxide pro-
duced by the autoxidation of L-cysteine (which was supplemented in the medium), has been minimized by adding bathocuproine sulphonate (a chelating agent) and by regulating the daily seeding density at a standardized level (7 X 10s BSFsml-I). HMI-9 medium containing L-cysteine can be used at least ten days after its preparation (the maximum shelf time has not been established) and one medium change is sufficient to produce 2-3 X I06BSFs ml-l every 24 h by readjusting the daily seeding density to 7 X IO5BSFsml-l. In certain biochemical and/or immunological experiments, the presence of serum protein in medium interferes with results. Thus, further attempts were made to develop a serum-free medium. To date, a completely serumfree medium is not yet available. However, HMI- I8 medium Fable I) containing a low concentraIion of serum protein has been developed7 by replacing foetal bovine serum in HMI-9 medium (IO%, v/v) with Serum Plus (increasing its amount from IO to 20%). Although the elimination of an addition of foetal bovine serum minimized variation in results that often have been attributed to variability of serum batches, the Serum Plus (lot no. I400 1004) still contains a low level of foetal bovine serum protein (I 3 kgml-I), in addition to specific growth-promoting factors, transport proteins, hormones, a high level of glucose (I 2.24mgml-I), and low amounts of hemoglobin ( IO kg ml-l) and endotoxin (5.8 ngml-I) with a pH of 7.32 and osmolarity of 378 mOsm (according to information released by the manufacturer; no further details are available). Thus, further improvement is still needed to establish a completely serum-independent culture medium. Nevertheless, 2-3 X I06BSFs ml-l can be produced by using HMI- I8 medium when BSF populations are maintained by readjusting the daily seeding density to 9 X I05BSFs ml-l.
Table I. Axenic culture media for bloodstream forms of African trypanosomesa Supplements Trypanosome species
Medium
Serum (%, v/v) YGS FBS
SP
BSF-S Factors (mM)b BAC CYS HYP
2ME
PYR
THY
T. brucei
HMI-9
IO
-
IO
0.05
I.5
I.0
0.20
I
0.16
T. brucei
HMI-I8
-
-
20
0.05
1.5
I .o
0.20
I
0.16
T. congolense
HMI-93
-
20
5
0.05
1.5
0.5
0.12
I
0.16
T. v&ox
HMI-I63
-
20
3
0.03
0.8
0.2
0.07
I
0.10
“Basalmedium was Iscove’smodified Dulbecco’s modified Eagle’sMEM (Iscove’sDMEM) (Flow Laboratories, Irvine, UK). bBSF-SFactors. bloodstream form-supporting factors; FBS, foetal bovine serum (heat inactivated, 56”C, 30 min) (Flow Laboratories; HyClone Laboratories, Logan, USA); YGS, young goat serum (heat inactivated) (prepared at ILRAD); SP, Serum Plus (Hazleton Biologics,presently JRH Biosciences, Lenaxa. USA); BAC, bathocuproine sulphonate (Sigma, St Louis, USA); CYS, L-cysteine (Sigma); HYP, hypoxanthine (Calbiochem, La Jolla, USA); 2ME, 2-mercaptoethanol (BDH Chemicals, Poole, UK); PYR, sodium pyruvate (Sigma);THY, thymidine (Sigma).
ParasitologyToday, vol. IO. no. 2, / 994
82
Trypanosoma
congolense
The development of an axenic culture system for T. congolense BSFs8was made by modifying HMI-9 medium used for the axenic cultivation of T. brucei BSFs. First, the ability of the six BSFsupporting factors supplemented in HMI-9 medium to support the transformation of in vitro-produced metacyclic forms8 (Box I Fig. f) of a cloned T. congolense IL3000 population to BSFs (Box I Fig. a) in vitro, was examined by testing eight culture media (not shown) containing various amounts of the factors and serum supplements in two basal media (MEM and Iscove’s DMEM). The results of this study revealed that: (I) the presence of the supporting factors is essential for the transformation; (2) Iscove’s DMEM is a better basal medium for transformation than MEM at 34°C; (3) MEM appeared to be superior for the propagation of epimastigotes (Box I Fig. e) at 27°C; and (4) the media supplemented with 10% (v/v) foetal bovine serum and 10% (v/v) Serum Plus support the transformation better than those supplemented with 20% foetal bovine serum. Second, the ability of each supporting factor to support the continuous growth of the metacyclic-derived BSFs of T. congolense IL3000 was examined by testing I I media (not shown) containing various combinations of the supporting factors at 34°C. The results of this study revealed that: (I) the presence of bathocuproine sulphonate, L-cysteine and thymidine is essential to support the continuous growth of the BSFs, although an addition of either thymidine alone or L-cysteine together with bathocuproine sulphonate does not support the growth; (2) an addition of hypoxanthine in the presence of these three ‘essential factors’ enhances the growth of BSFs to a certain extent; (3) a further addition of 2-mercaptoethanol and sodium pyruvate (HMI-9 medium) gives the best growth; and (4) additions of hypoxanthine, 2-mercaptoethanol and/or pyruvate in the absence of any of ‘the essential factors’ do not support the growth. HMI-9 medium was further tested for the axenic cultivation of BSFs of another five T. congolense populations (one clone: ILNat3. I and four stocks: lL2079, lL2466, IL3266 and CP-81) derived either from infected mouse blood or from metacyclic forms produced in vitro. However, HMI-9 medium unexpectedly did not support the growth of these additional populations”
A series of media were, therefore, modified from HMI-9 medium and tested for the cultivation of the other BSF populations. Among these, HMI-93 medium in which 10% foetal bovine serum in HMI-9 medium was replaced with 20% young goat serum and the amounts of Serum Plus, hypoxanthine and 2-mercaptoethanol were reduced (Table I), was established to be the optimum medium in terms of the initiation and the maintenance of not only the metacyclic-derived but also the mouse-derived BSFs of all populations testeds. The establishment of the optimum medium was carried out in Plastek M T-25 flasks (Tekmat, Ashland, USA) basically following the procedures used for the BSFs of T. brucei (Box 2). Furthermore, a simple method for initiating primary cultures with a small amount of tail blood of infected mice using the optimum medium was also standardized using 24-well culture plates (24 Well Tissue Culture Cluster, Costar, Cambridge, MA, USA)8. This method could also be applicable for in vitro cloning of BSFs. Both the metacyclic- and the mousederived BSFs of T. congolense rapidly (~4 h) attached to the bottom surface of flasks, as well as of wells, and continued to proliferates, unlike BSFs of T. brucei which proliferated in suspension. During 24 h of cultivation, BSFs formed attached populations and detached populations in the culture fluids. The detached BSFs usually increased in number when cultures became overpopulated. In such cultures, the colour of the medium changed to yellow (indicating a pH ~6.8) and the parasites became sluggish and died within I2 h if the medium was not changed. The attached forms, however, could be continuously maintained by subcultivating in new flasks. Growth rates of the attached populations were consistently higher in cultures that were initiated with a seeding density at 106BSFsml-I (or lower) in a small volume of medium (2ml per flask) than in those initiated with a higher seeding density in a larger volume (3-5 ml per flask). In a standardized method for the axenic cultivation of T. congolense BSFs, subcultures were initiated by: (I) discarding the culture fluids containing detached BSFs and resuspending the attached forms by gentle pipetting in fresh medium; and (2) transferring 2 ml of the BSF suspension containing lO6BSFsml-1 to a new T-25 flask. The pH of culture medium was, thereafter, adjusted to 7.2 + 0.2 by following the same procedure applied
in the Step 4 for the cultivation of T. brucei BSFs (Box 2). The shortest population doubling time achieved in the standardized system was nine hours. All the mouse-derived BSFs examined to date were obtained from mice inoculated with BSFs,which had undergone the complete cyclic development, in vitro. Thus, it may be necessary to examine whether populations that had not undergone the in vitro transfon-ration (such as stocks isolated directly from naturally infected hosts) can also be axenically cultured in this system. If such populations could not be adapted to the system, it would be advisable to pass the populations through the cyclic transformation, in vitro, and then to initiate the cultures with the in vitroproduced metacyclic forms
Trypanosoma
vivax
A recent attempt to culture BSFs of four T. vivax stocks under axenic culture conditions has been reported by Zweygatth and colleaguesg. In this system, BSFs were first adapted to in vitro conditions in the presence of mammalian feeder cells and then maintained axenically up to 60 days in 24-well culture plates using two media modified from MEM. However, the maximum density of BSFs achieved in the system was low (5 X IOsBSFsml-I) and the population doubling times were rather long (I 848 h). Furthermore, the modified media did not support the continuous growth of two cloned BSF populations derived from a West African T. vlvax stock (ILI 392) and an East African stock (IL367 I) (Hirumi, unpublished). Therefore, an in vitro system that supports the growth of BSFs of the East, and the West African T. vivax has been established by developing three in vitro systemslO. ?? System I was established in three steps: (I) production of metacyclic forms of T. vivax ILI 392 using Matrix Gel Green-A beads in the presence of feeder layer cells at 27°C (technical details were reviewed by Gray and colleagues4); (2) transformation of the in vitro-derived metacyclic forms to BSFs which continued to proliferate at 34°C in the presence of feeder layer cells; and (3) establishment of an axenic culture condition for the in vitrotransformed BSFs by testing a total of 69 media (not shown) modified from HMI-93 medium using Plastek M T-25 flasks at 34°C. Although most media did not support the growth of the BSFs, HMI-I 62 medium (not shown)
ParasftologyToday,vol. IO, no. 2, I994
was found to be the best medium for supporting growth at that time. Thus, the axenic cultivation of 7; vivax BSFs was standardized using this medium. In the standardized method, an average population density of J. vivax ILI 392 was 3.4 X I06BSFsml-1 and the population doubling time was 13.6 h. ?? System II was established in two steps: (I) production of metacyclic forms of J. vivax I-3671 BSFs obtained from the infected bovine blood without feeder layer cells at 27’C; and (2) transformation of the in vitro-produced metacyclic forms to BSF*j and continuous cultivation of the in vitrotransformed BSFs under the axenic condition using HMI- I62 medium. Although HMI-I 62 medium also supported the continuous. growth of T. vivax IL3671 BSFs, the maximum density of the BSFs achieved in this medium was lower than IO6ml~l (average 0.7 X I 06mlV) and the population doubling time was longer than I8 h. Therefore, an additional medium, HMII63 (Table I), was mc’dified from HMI- I62 medium. The subcultivation was carried out by transferring the culture fluids containing free swimming BSFs (0.5 X I06BSFsml-1, I ml per flask) and fresh medium (I .5 ml per flask) to new flasks. The subcultures were maintained by changing the medium ( I-I .5 ml per flask) every 24 h until the experiment was terminated on Day 70 of the BSF ‘culture. The maximum density achieved in HMI- I 63 medium was I .8 X 106ml-1 and the shortest population doubling time was 16.1 h. ?? System 111 was established in one step initiating the cultures with proboscides of Glossina morsitans centralis which were infected w’th T. vivax IL367 I, in the absence of feeder layer cells at 34°C initially usil?g HMI-I 62 medium which was later rleplaced with HMI-I 63 after the latter medium was developed. The maximum density and the population doubling time of T. vivax IL3671 in the system we’-e similar to those obtained in System II. Systems II and Ill are highly useful tools for the in vitro study of T. vivax, since most populations of bovine-infective J. vivax are not infect.ive for small experimental animals (with few exceptions, such as IL1392 which is infective for rodents). Application Recently, a simple method for detecting the sensitivity of T. congolense
83
BSFs in vitro to four ttypanocidal drugs, diminazene aceturate (DA), homidium chloride (HC), isometamidium chloride (IC) and quinapyramine sulphate (QS) which are commonly in use, has been established by using the axenic culture system for T. congolense BSFs in 24-well culture plates”. Most problems that had hampered the application of earlier in vitro systems to the assessment of drug sensitivity of trypanosome populations12 have been overcome in this method. Prior to the sensitivity tests, BSFs of four T. congolense stocks (lL2079, lL2466, IL3266 and lL3338), four clones (ILI 180, lL2642, IL3000 and lL3035) and I6 clones obtained in vitro from lL2079, lL2466, IL3000, IL3266 and lL3338, were propagated in T-25 flasks using HMI-93 medium. Among these populations ILI 180, lL2466, IL2642 are known to be sensitive to DA and/or IC as tested in mice and/or cattle, while IL3035 and IL3338 are highly resistant to DA, IC and/or HC in cattle. Test plates were prepared by placing IO0 ~1 of test solutions containing various amounts of the drugs in distilled water to each well. The plates were then freeze-dried and stored at room temperature. In the standardized system, levels of resistance for each drug were expressed in ten steps from ten to one, denoting the following concentrations: DA at 600, 500, 400, 300, 200, 100, 80, 60, 40 and 20ngml-1; HC, IC and QS at tenfold serial dilutions from IO Fg to I Ofgml-1. The test plates wrapped with aluminium foil could be kept at ledst six months at room temperature without any detectable deterioriation of the ttypanotides’ eficacy. The tests were carried out by first placing 500 ~1 aliquots of ttypanosome suspension in HMI-93 medium containing 4 X I OsBSFsml-1 in each well and then incubating at 34°C in a CO,incubator for five days without medium change. Effects of the drugs were examined by phase-contrast microscopy every 24 h. Growth inhibition of BSFs could be detected by Day 3 and attached ttypanosomes died during the next 2+48 h. On the contrary, BSFs which were tolerant to the given concentrations continued to proliferate reaching the maximum population density by Day 3-5, and died during the next 24 h due to overgrowth. The pH indicator, phenol red, in medium in wells which contained affected populations indicated pH 8.0-8.5 (pinkish), whereas that in wells in which BSFs reached the maximum density changed
to pH 6.5 or lower (yellowish) by Day 3-5. Calorimetry of the media on Day 5 was thus also used to distinguish levels of drug sensitivity for the ttypanosome populations. The calorimetric reaction of the culture media, similar to that observed in this study, was reported earlier for testing ttypanocidal activity in T. b. brucei BSFs using 96-well culture plates and an ELISA readeri3. In the in vitro assay described here’ 1,the effects of ttypanotidal drugs should be examined by means of phase-contrast microscopy every 24 h during the initial three days and, in general, the final detection of the end points could be made by colorimetty of the culture media by naked eye on Day 5. In this system, a few unaffected trypanosomes (even a single BSF) continue to multiply without medium change until the population reaches the maximum density. The ability to support growth of the trypanosomes without medium change for up to ten days is particularly advantageous in detecting maximum resistance concentrations of trypanocidal drugs, as well as in selecting a small number of drug-resistant parasites from large numbers of sensitive ttypanosomes. Thus, the in vitro system established would be a useful laboratory tool for: (I) the screening of new drugs: (2) the selection of experimentally induced drug-resistant populations for molecular biological studies; (3) cloning of drug resistant parasite: and (4) the identification of growth promoting, as well as inhibiting, factors. Another advantage of the in vitro assay is the requirement for only relatively simple laboratory equipment, such as a CO,-incubator, a phase-contrast microscope and a vacuum freeze dryer. There is, thus, no requirement for sophisticated equipment, such as a spectrophotometer, ELISA reader, liquid scintillation counter and a Coulter cell counter, used in earlier w0tW3-~*. Furthermore, the in vitro system would also be an excellent alternative to in vivo drug tests which require large numbers of experimental animals and are time-consuming. The results generated in this system using three test plates (triplicate) per drug per ttypanosome population and lasting five days, provided an equivalent amount of information about the drug sensitivity of a ttypanosome population as in viva tests which used at least 36 mice and required two months. In a preliminary study, the in vitro drug-sensitivity assay has been applied to several T. b. brucei and T. vivax
Parasitology
84
populations, using HMI-9 and HMI- I93 media, respectively. The results obtained to date indicate that the system would also be applicable to these species, although the yellowish colour of colorimetrtc reaction in T. b. brucei populations was somewhat less distinct (amber). Acknowledgements The authors wish ta thank A.R. Gray for his critical review of the manuscript, and JR. Wando for his technical assistance. This is ILfWD publication No. 1204. References I Hlrumi. H.. Doyle. 1.1.and Hirumi, K. (1977) j Science196,99%994 2 Evans, D.A. (I 978) in Methods m Cultjvatmg
Medical Insects and Arachnids edited by Richard P. Lone and Roger W. Crosskey, Chapman & Hali, 1993. f85.00 (xv + 723 pages) lS5N 0 4 I2 40000 6 Few young biologists choose medical entomology as a speciality. Not only is the subject generally poorly taught, but it also suffers from a Batesian image of Victorian times with practitioners who wear funny hats and prowl through forests with a net on a stick. The few who opt for medical entomology have no bandwagon to carry them to the heights of tropical medicine nor do they really need to study Swedish in case they are ever invited to Stockholm to receive a Nobel Prize. They will have to be enthusiasts like each of the authors of the chapters in this splendid new book with the unusual title Medical insects and Arachnids. It has a fine pedigree beginning during the Second World War with Smart’s Handbook for the Identification of Insects followed, in of Medical Importance 1973, by Insects ond Other Arthropods of Medical lrnportonce edited by K.G.V. Smith. Lane and Crosskey’s latest offering is completely rewritten and surpasses its predecessors. All medically important insects and arachnids are dealt with in an organized way which shows tight editing and an unusual degree of discipline and compliance by contributors. As expected in a book from the Natural History Museum, a large part of each chapter deals with classification and identifi-
Parasites in vitro 3
4
5 6 7 8 9 IO II 12
(Taylor,
A.E.R. and Baker,
I.R., eds). pp 55-88, Academic Press Btun, d. and Jenni, L. (I 987) in Methods for Poraslte CuItrvation (Taylor. A.E.R and Baker, I.R., eds), pp 94-l I?. Academic Press Gray, M.A.. Hlrumi, H. and Gardener, P.R. (1987) an Methods for Porostte Cultivation Taylor, A.&R and Baker, J.R., eds), pp I I 8-I 52, Academic Press Baltz,T.etol.(l985)EMBOJ.4, 1273-1277 Duszenko, M. et al. (1985) j. Exp. Med. 162, 1256-1263 Hirumi, H. and Hirumi. K. (I 989) J. Parosrtoi. 75,985-989 Hiruml, H. and Hltumi, K. (199 I) farasrtoiogy 102,223-236 Zweygarth, E., Gray, M.A. and Kamlnsky, R ( I99.lj Trap. Med. Porasrtol. 42, 4548 Hlrumi, H. et ol. (l99l)J. Protozooi Ues. I, I-12 Hlrumi, H.. Hirumi, K. and Peregrine, A.S. (I 993)J. Protozool. Res. 3. 52-62
,I~
I
n ,,nnn\ “_n~...-r_ h,.
i_‘-
cation with no apologies offered. This is timely as we feel the first twinges of the birth pangs of a renaissance in taxonomy following many recent reports on the need to know more about the animals with which we share this planet. Whether traditionalists like it or not, molecular biology is going to make the same impact on medical entomology as it has on other biological disciplines. There may not be much evidence of this at the moment perhaps because (I) some entomologists are not moving with the times and (2) few molecular biologists know anything about ‘medical’ insects. How many of them know why the antennae of male and female mosquitoes are so different, how many larval instars does a flea have, or if male sandflies take bloodmeals? They should know that sibling species are not brothers and sisters. Above all, these are the workers who need this book - although every medical entomologist will find many things of interest between its covers. After classification, each chapter deals with biology, medical importance, control and, finally, collection, preservation and rearing. The new taxonomy will need living material and the next time this book appears under another title, rearing and colonization should not be relegated to a paragraph or two at the end of each chapter. Furthermore, preservation in the future will not be solely by pinning or pickling. It will be in cryobanks. Cryopresetved specimens can be mounted for classical taxonomy (far more easily than dried or pickled specimens), and are of immense value to molecular biologists.
2x205-2 13 Zinsstag, Parosrtol. 14 Brun, R.
Today, vol. IO, no. 2, I 994
IO 1,. Rerun, R and Gessler, M. (I 99 I) Res. 77, 33-38 and Kunz, C. (I 989) Acta Trap. 46,
361-368 15 Brun, R. and Rab, S. (1991) Porasltol. Res. 77, 341-345 16 Kaminsky, R. and Zweygarth. E. (1989) Antimlcrob. Agents Cbemother. 33, 88 l-885 17 Kaminsky, R, Chuma, F. and Zweygarth, E. (I 989) Exp. Porasitol. 69. 28 l-289 I8 Ross, C.A. and Taylor, A.M. (I 990) Purusitol. I Res. 76,326-33 I9 Vickerman, K. (I 97 I) in Ecology and PhysiaIogy of Parasrtes (Fallis, A.M., ed.), pp 58-91, Unlverslty of Toronto Press A-2 jlULU~~ Vf17111r nwmt ’’ uIIu ore at tionol Laboratory for Research on Animal DE ieases [ILRADJ, PO Box 30709, Hir*yuk the
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Ignoring this glimpse ‘into the future, it must be said that this is the best book available on the subject. It should be used not just by entomologists, but by all workers and teachers involved with vector-borne diseases. It is well written, authoritative and full of ‘gems’, the best of which concerns spiders. It is said that most bites of Latrodectus in Texas are inflicted on men using outdoor privies. (Why not women?) It is recommended that webs should be moved from under the seats so that ‘the spiders would not be stimulated to attack the delicate portion of the anatomy causing them to vibrate’ (p. 678). There is no reference to this story. In fact, for a reference book there are too few references. This book will undoubtedly stimulate more young biologists to become medical entomologists. Perhaps the future of the subject is brighter than we think and they might find it worthwhile to study Swedish. R. Killick-Kendrick Department of Biology lmperlal College at Silwood Park Ascot, UK SL5 7PY
Indexes We hope that you find the enclosed subject and author indexes useful. If you are interested in buying any back issues of Parosir~lo~ Joday, please contact Alison Fricker at our Cambridge offIce, and she will help you.