Isolation and cultivation in vitro to the infective, metacyclic stage of Trypanosoma (Nannomonas) simiae from Glossina morsitans submorsitans

Acta Tropica, 46(1989)191-203 Elsevier

191

ATP 00023

Isolation and cultivation in vitro to the infective, metacyclic stage of Trypanosoma (Nannomonas) simiae from Glossina morsitans submorsitans P. Dukes 1, J. Faye 2, J.J. McNamara 1, W.F. Snow 2, P. Rawlings 2, R.H. D w i n g e r 2 a n d R. B r u n 3 1Tsetse Research Laboratory, ODA/University of Bristol, Bristol, U.K., 2International Trypanotolerance Centre, Banjul, The Gambia, and 3Swiss Tropical Institute, Basel, Switzerland (Received 1 July 1988; revised version received 4 November 1988; accepted 30 November 1988) Two separate trypanosome isolations were made from a single Nannomonas-infected Glossina morsitans submorsitans from The Gambia. Inoculation of a piglet with the infected hypopharynx produced an infection with Trypanosoma simiae. DNA was isolated from the bloodstream forms to prepare a probe specific for this species. Trypanosomes isolated from the fly midgut were frozen in liquid nitrogen and then cultivated in vitro. Amplification of this population and elimination of a yeast contaminant were achieved by two passages through laboratory G. m. morsitans. Further cultivation in vitro resulted in the production of epimastigotes and, later, metacyclic forms. Two pigs inoculated with cultivated metacyclic forms developed infections with atypical, relapsing parasitaemias and extended survival time. Neither the metacyclic forms, nor bloodstream forms derived from them, infected calves. The identity of various stages of the in vitro cultivated, procyclic-derived stock was confirmed morphologically and with the T. simiaespecific DNA probe. Key words: Trypanosoma simiae, in vitro cultivation; Trypanosoma simiae, infection; Pigs; Cattle

Introduction C u l t i v a t i o n in vitro o f t r y p a n o s o m e s o f v a r i o u s life-cycle stages c a n p r o v i d e v a l u a b l e m o d e l s o f infection a n d p a r a s i t e d e v e l o p m e n t , as well as m a t e r i a l for b i o c h e m i c a l , serological a n d d r u g studies. Insect stage t r y p a n o s o m e s o f Trypanosoma (Nannomonas) congolense a n d T. (Trypanozoon) brucei are readily c u l t i v a t e d in semi-defined m e d i a as p r o c y c l i c f o r m s (see Evans, 1978 a n d G r a y et al., 1987). O n l y recently has c u l t i v a t i o n o f T. congolense to the e p i m a s t i g o t e stage, with significant yields o f infective m e t a c y c l i c forms, b e c o m e p o s s i b l e ( G r a y et al., 1984). W e describe the i s o l a t i o n o f T. ( N . ) simiae f r o m a n a t u r a l l y infected Glossina morsitans submorsitans, a n d its c u l t i v a t i o n u n d e r c o n d i t i o n s similar to those devised b y G r a y et al. (1984). W e also e x a m i n e d the infectivity o f the c u l t i v a t e d m e t a c y c l i c f o r m s to pigs a n d cattle.

Correspondence address: P. Dukes, Tsetse Research Laboratory, ODA/University of Bristol, Langford, Bristol BSI8 7DU, U.K. 0001-706X/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

192 Materials and Methods

Isolation

As part of a study of Nannornonas infection rates in tsetse in The Gambia (Snow et al., 1989), two separate primary isolates were made from a single G.m. submorsitans caught in an area of savannah woodland about 16 km north of the village of Keneba (13020 ' N; 16°01' W), some 100 km from the coast. The fly was dissected in sterile physiological saline. The posterior midgut, salivary glands and mouthparts were examined for trypanosome infection. The anterior midgut was placed in a drop of saline on a glass slide until the other tissues had been examined. The first primary isolate (GMOS/GM/85/SAMIWOLO-008 [A-Midgut Isolate]World Health Organization (1986) recommended code; abbreviated to GMOS-008Alaboratory code) was made by transferring the fly's anterior midgut to 1.0 ml of Cunningham's medium (Cunningham, 1977) with 25% v/v foetal bovine serum (FBS), 100 I~g Gentamycin (Flow Laboratories) m1-1 and 10% v/v glycerol. For convenience, trypanosomes were kept overnight at 8 to 10°C. They were then cooled slowly in the neck of a liquid nitrogen vessel (3 to 4 h) before storage within the vessel. The stabilate was designated itc/trl 001. The second primary isolate (GMOS/GM/85/SAMIWOLO-008 [B-Proboscis Isolate]) was made by removing the infected hypopharynx and labrum from under the coverslip and inoculating them with 0.5 ml sterile saline subcutaneously into a piglet (85/11). The pig was monitored daily for infection (see below). A stabilate (ITC 011) of GMOS-008B, prepared on day 11, remained in The Gambia (Fig. 1). The piglets in these studies were purchased in a tsetse free area, to which they were returned within 2 or 3 days of inoculation. In vitro cultivation and in vivo amplification of the procyclic-derived stock GMOSO08A

After thawing, stabilate itc/trl 001 was diluted slowly over 2 min to 5 ml with Cunningham's medium containing 20% v/v FBS and 10 lag gentamycin m1-1 (medium CM) at 4°C. The gut tissue was lightly homogenized with a rubber-tipped glass rod. The suspension was centrifuged at 1000 x g for 10 min at 4°C, resuspended to 2 ml in CM and incubated at 25°C in two 1.0 ml wells of a 24-well plate (Nunc). To maintain adequate humidity, the remaining wells contained distilled water. After two days, a yeast infection was evident in the two culture wells. The infected suspensions were pooled, washed twice in physiological saline, resuspended in five-times washed horse red cells (approx. 66% v/v) in saline (5 ml total) and fed to laboratory reared G. morsitans morsitans through a silicone membrane. Two of eight flies dissected on day 43 post infection (PI) had mature Nannomonas infections. The anterior midgut populations were isolated into CM and used to re-infect more tsetse two days later. After 30 days, anterior midgut infections from two of seven flies with mature infections were pooled and cultivated in 2 ml (final volume) of medium CM mixed with an equal volume of SDM-79 medium (Brun and Schrnenberger, 1979) with a final concentration of 20% FBS and 10 lag gentamycin ml- 1 (medium SDM + CM), in one well of a 6-well culture plate. Three days later, the culture was expanded and transferred to a 2 5 cm 2 culture flask. Once established, the medium was changed about three times each week by removal and replacement of 4 ml of the total 5 ml

193 procyclics

~

metacyclics

~

Stabitate

GMOS-OOSA-'~{'~" B.__F__LY~l~GMOS-OOSB -- I 85/11 I "-~="ITC011

+

I Stabilate itc/trl 001 I Culture (CM) I 2 days

DNAprobe preparation in situ DNA hybridization

~GLETS 86/1 & 8612

procyclic8 (2 flies)

t

Cuiture (CM) I 2 days I

I

I

procyclics I Culture

(SDM*CM)

Stabliete TRL 304 Stabilate

I

Stabilate

El~maNig~e Stabilate chromosome metacyclics

~

Mediumchange

~

~

r Day 13

(MEM-C) Day30

Day48

Day 55

preparation Day 64

1 Day 78

Stabilate

1 Day

111

Fig. 1. The pedigreeof T. simiae stock GMOS-008A and primaryisolate GMOS-008B, both isolated from a single G.m. submorsitans from Samiwolo, near Keneba in The Gambia. volume. The culture was divided between two flasks, and on day 30 the medium in one flask was replaced by the antibiotic-free MEM-based medium of Gray et al. (1981), with 10mM glutamine (Ross, 1987) (medium MEM-C). The culture was readily expanded by subpassage of epimastigotes (1 part) into new flasks (25 cm 2 and thence larger) with fresh medium (4 parts). Stabilates of the procyclic-derived stock (GMOS-008A) were made on seven occasions after isolation (Fig. 1). Metacyclic-like forms were harvested by centrifuging epimastigote culture supernatant (1000 x g, 10 min, 4°C) and resuspending trypanosomes in 2 ml of sterile PSG 6:4 (Lanham and Godfrey, 1970). The metacyclic forms were separated from the epimastigotes on a DEAE-cellulose column prepared in a 10 ml washed syringe barrel. If the column was overloaded so that epimastigotes appeared in the eluate, the eluted trypanosomes were concentrated to 1.0 ml by centrifugation and were applied to a second, similar column. The metacyclic forms were cryopreserved in freezing medium: MEM-C with FBS at 25% and 10% glycerol. Viability of stabilates, as measured by phase-contrast assessment of trypanosome morphology and motility after freezing, was 8 to 13%, irrespective of glycerol concentration (5, 7.5 or 10%) or FBS concentration (20, 25 or 90%) (R. Barker and P. Dukes, unpublished data).

Infection of piglets (a) With metacyclic forms from a naturally infected G. m. morsitans (Primary isolate GMOS-OO8B) Pig 85/11 was inoculated with a single trypanosome-infected proboscis (see Isolation above). The progress of infection was assessed by (1) examination of ear

194

vein blood samples by microhaemotocrit/dark ground (HCT/DG; Murray et al., 1977) or wet blood film (WF) microscopy; (2) rectal temperature; (3) packed cell volume (PCV) of ear vein blood after microhaematocrit centrifugation; and (4) trypanosome morphology in wet preparations and Giemsa stained blood films prepared on days 9 and 10 after inoculation. The pig was killed on day 11 and the trypanosomes harvested from 400 ml of heparinized cardiac blood (20 units ml- 1) on a DEAE-cellulose column using PSG 3:7 (Lanham and Godfrey, 1970). The trypanosomes were lysed and DNA prepared by standard methods (van der Ploeg et al., 1982).

(b) With cultivated metacyclic forms of stock GMOS-OOSA Piglets 86/1 and 86/2 were each inoculated subcutaneously in three sites with 0.2 ml of diluted, thawed stabilate (TRL 304) of metacyclic trypanosomes. Each piglet received a total of 5 × 10 3 parasites, 7 to 10% of which were estimated to be fully motile: the estimated infective dose was therefore about 5 x 102 trypanosomes per pig. The pigs were examined every one, two or three days for the first month postinoculation (PI); thereafter, they were bled once a week if persistently HCT/DG positive. Infections were assessed as for Pig 85/11.

(c) With metacyclic forms from pooled, naturally infected flies Data are also presented for two piglets fed on, whilst restrained, by groups of naturally infected G.m. submorsitans trapped in the Keneba area (Snow et al., 1989). The flies were dissected after feeding and examined for trypanosome infection. Pig infections were assessed as for Pig 85/11.

Infection of calves with GMOS-OO8A metacyclic and bloodstream forms Two male N'Dama cattle of two years of age, raised in a tsetse-free area, weighing between 85 and 105 kg, with no antibodies in the indirect fluorescent antibody (IFA) test to T. brucei, T. congolense or T. vivax, were inoculated intradermally at ten sites on the flank with thawed stabilate TRL 304 (10 x 0.1 ml at 7 to 10 × 102 fully motile trypanosomes ml- 1). Four similar cattle were inoculated with stock GMOS-008A as bloodstream forms from Pig 86/I: two were inoculated intradermally (10 x 0.1 ml at 1.6x 10 7 trypanosomes m1-1) and two intravenously (1.5 ml at 1.2x 10a trypanosomes ml-1). Two more cattle were inoculated with phosphate-buffered saline glucose (PBSG, Lanham and Godfrey, 1970) as controls. The following features were measured daily for 30 days and then twice weekly during the following month: (1) skin thickness (first 14 days only) (Dwinger et al., 1986); (2) PCV; and (3) rectal temperature. Serum samples were taken weekly for the IFA test using T. simiae antigen (Katende et al., 1987). Body weights were measured weekly.

Definitions The prepatent period was the time which elapsed between inoculation (day 0) and first detection of trypanosomes. The survival period was the time which elapsed between parasites being first detected and the death of the host.

Trypanosome morphology Blood smears were fixed for 2 min in absolute methanol, stained for 60 min in 5% Giemsa stain (Gurr's Improved R66, BDH Chemicals Ltd.) in phosphate buffered

I

b

o

a

k tl

Fig. 2. T. simiae GMOS-008A, originally isolated as procyclic forms from a single G.m. submorsitans midgut. Magnification: × 1780. Abbreviations: k: kinetoplast; p: typical, ragged posterior end; n: nucleus; u: undulating membrane. (a) Bloodstream forms from Pig 86/2; 9 days after inoculation with cultivated metacyclic forms. (b) Epimastigote forms cultivated in vitro; 10 days after changing to medium MEM-C. (c) A metacyclic trypomastigote cultivated in vitro; 10 days after changing to medium MEM-C.

a

196

saline (PBS) pH 7.2 (4.41 mM K H z P O 4, 29.8 mM Na2HPO4), and rinsed for 1 s only in PBS. Later smears were stained with 10% Giemsa stain (Pigs 86/1 and 2, as in Fig. 2a). Culture forms (Fig. 2b and c) were examined in situ, and as stained smears prepared as follows (modified from Gray et ai., 1987). Thin films were spread on detergent-cleansed (7X-Omatic, Flow Laboratories) glass slides and dried in an air flow at ambient temperature on a heating plate at 37 to 40°C. Smears were fixed in absolute methanol for 2 min and air dried. They were then immersed in 5 M hydrochloric acid at ambient temperature for 60 min and rinsed by immersion in two changes of PBS pH 6.8 (Gurr, BDH Chemicals Ltd.). The second rinse lasted at least 5 min. The smears were stained in 4% Giemsa stain in PBS pH 6.8 at 37°C, and were rinsed for 3 s in each of three changes of PBS.

Identification by DNA probes D N A probes specific for T. brucei, savannah T. congolense and T. simiae (Gibson et al., 1988) were used to check the identity of epimastigote and bloodstream stages of the procyclic-derived stock (GMOS-008A). The T. simiae probe was prepared from bloodstream forms of the metacyclic-derived primary isolate (GMOS-008B) harvested from Pig 85/11 (Fig. 1) (Gibson et al., 1988).

Epimastigotes 109 epimastigotes (with < 10% metacyclic forms) were harvested on day 64 from several culture flasks using a cell scraper (Costar) and centrifuged at 1000 × g for 10 min at 4°C. The trypanosomes were washed in Earle's buffered salts solution (Gibco) and prepared in agarose blocks for pulsed field gradient gel electrophoresis (van der Ploeg et al., 1984). The results were reported by Gibson et al. (1988).

Bloodstream forms 1.0 I~1 of ear vein blood from Piglet 86/2, infected with cultivated metacyclic forms, was diluted ten-fold in PSG 3:7 and spotted on nitrocellulose paper. The paper was stored dry at ambient temperature. Hybridization with D N A probes specific for T. brucei, three kinds of T. congolense and T. simiae was carried out as described by Gibson et al. (1988).

Results

Cultivation, decontamination and differentiation of the procyclic-derived stock (GMOS-OO8A) Two days after inoculation, a yeast contaminant was observed in the primary isolate culture. To try and eliminate the yeast infection, the pooled culture supernatants were fed to a group of teneral laboratory G.m. morsitans. Several flies were dissected 21 days later, but the trypanosomes were still contaminated with yeast. Consequently, procyclic forms from two additional flies, dissected 43 days PI, were incubated in medium CM for two days and then passaged through a second group of tsetse. Trypanosomes dissected and pooled from two of these flies 30 days PI grew readily in medium SDM + CM at 25°C and were free of yeast. Parallel cultures in medium CM alone deteriorated and died out from day 5 PI. Although in this study medium

197

SDM + CM was superior to CM alone, subsequent Nannomonas isolates have been grown satisfactorily in CM alone. We (JJM and PD) routinely grow primary isolates from tsetse midguts in three media in parallel: (1) medium SDM + CM, (2) a mixture of media CM and MEM-C (1:1) (medium CM + MEM-C), and (3) CM alone. Stocks are usually grown in medium CM + MEM-C from the first subpassage. On day 30, small stellate clusters of inflexible, elongated trypanosomes were observed adhering to the flask surface in the manner of T. congolense epimastigotes described by Gray et al. (1984). The S D M + C M was immediately replaced by epimastigote culture medium MEM-C and incubated at 28°C. Within ten days, the epimastigote clusters had expanded into large, dense adhering sheets. Giemsa staining confirmed the epimastigote morphology (Fig. 2b) and revealed the presence of 1 to 4% trypomastigote, metacyclic-like forms (Fig. 2c).

Identification by DNA probes Gibson et al. (1988) showed that size fractionated large DNA molecules prepared from the procyclic-derived epimastigotes were recognized by the T. simiae-specific probe prepared from GMOS-008B, but not by probes for savannah T. congolense or T. brucei. Bloodstream forms derived from cultivated metacyclics and blotted on nitrocellulose paper hybridized strongly to the DNA probe specific for T. simiae (GMOS-008B), but not to probes for T. congolense or T. brucei.

Bloodstream form morphology The morphology of bloodstream form trypanosomes of the two stocks, derived separately from metacyclic (GMOS-008B) and procyclic (GMOS-008A) forms, were examined using the criteria of Stephen (1966) and Hoare (1972) (Table 1). All except a few specimens were without a free flagellum. The short free flagella occasionally observed were attributable to sample preparation, the undulating membrane lying immediately above or below the body of the trypanosome. The kinetoplast was conspicuous, frequently marginal, and often appearing to protrude laterally. These characteristics identified the population as Nannomonas. The mean overall length, high parasitaemia, high frequency of dividing forms and high percentage of trypanosomes with a well-developed undulating membrane were typical of T. simiae, differentiating the specimens from T. congolense. Pleomorphism was not readily evident.

Infections in pigs (a) Two pigs infected with cultivated metacyclic forms of stock GMOS-OO8A The parasitaemias, rectal temperatures and PCVs of the two pigs inoculated with cultivated metacyclic trypanosomes of stock GMOS-008A are shown in Figs. 3a and 3b. The high parasitaemias were within the range expected of T. simiae, and greater than those characteristic of T. congolense. However, the prepatent periods of 7 and 9 days were longer than Stephen (1966) considers typical of T. simiae (4 to 6 days) and within the range expected of T. congolense (6 to 15 days; Hoare, 1972). Several waves of parasitaemia were observed for both animals, with increasing periods of remission between relapses. Each relapse was accompanied by a corresponding increase in

112(7) 22(1) Means 12.2 to 17.6 ~tm Means 15.3 to 19.7 lam

17.7 (2.5) 16.9 (1.4)

17.3 (1.7) 18.1 (2.0)

Mean overall length ~tmb (SD)

Generally absent Absent in the Frequent majority

0.0 to 0.31 0.0

0.15 0.01

Range of mean free flagellum lengthsc (p.m)

aNumber of slides examined, each prepared on a separate day. bMean of all the trypanosomes examined, slides l to n. ORange of the mean values obtained for slides 1 to n.

'Typical T. simiae'

'Typical T. congolense'

85/2 85/3

GMOS 005 GMOS 006

Total number of trypanosomes examined (No. of slides)a

40(2) 40(1)

Pig number

GMOS 008B 85/11 GMOS 008A 86/1

Primary isolate number

8.3 to 30.0 9.1

15.2 & 22.7 27.5

% in division Range

Generally inconspicuous Conspicuous: one, two or more waves

65 to 95 70

70 & 70 62.5

% with /> 2 undulating membrane loops Range

Frequent

Variable

45 to 50 40

50 & 55 45

% with a rounded posterior Range

Frequent

Infrequent

25 to 90 55

40 & 50 43

% with a vacuole Range

Data taken from Stephen (1966) & Hoare (1972)

From hypopharynx From midgut

Notes

Morphological characteristics of 7". simiae bloodstream forms: Primary isolate GMOS-008B and stock GMOS-008A, isolated from a single fly, compared with data for two other T. simiae primary isolates obtained by feeding different batches of G.m. submorsitans from Samiwolo, near Keneba, on piglets (full primary isolate codes: GMOS/GAM/85/SAMIWOLO002 etc.)

TABLE 1

199 Q

P

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2'0 . . . .

_PCV %

3'0 0

days (post infection)

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•, ,

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D o 0

,

, ~,

1~0 . . . . . 20 . . . . .

pcv% 41

39 30

0

days (post infection)

Fig. 3. The relapsing parasitaemias observed in cultivated metacyclic-stage T. simiae GMOS-008A inoculated into two pigs (a) Pig 86/1; (b) Pig 86/2. I , parasitaemia (p, logaotrypanosomes ml-X); D, temperature (°C); *, packed cell volume (PCV %); T, indicates the day parasitaemia was first observed. Low parasitaemias detected but not measured were arbitrarily given the value p = 5.0.

rectal temperature. The packed cell volume fell for both pigs, but appeared partially to recover in the case of Pig 86/1, which also appeared to control its parasitaemia better than did Pig 86/2. Variation of individual PCV values from the trend was attributable to occasional clotting of blood in the microhaematocrit capillaries. (b) The pig infected with metacyclie forms of primary isolate GMOS-OO8B Fig. 4 shows the course of infection of the primary isolate GMOS-008B in Pig 85/11, inoculated with a single, naturally infected proboscis. The 6 day prepatent period was shorter than those observed for the cultivated metacyclics (GMOS-008A), being within the range characteristic of T. simiae. However, because the experiment was terminated early (to harvest trypanosome DNA), we do not know whether the infection would have followed the prolonged course shown by the cultivated metacyclics (Fig. 3) or a more normal, rapidly fatal infection (Fig. 5). ( c ) Pooled-feed inoculations Fig. 5 shows the parasitaemias obtained after feeding groups of naturally infected flies on two pigs. The infection in Pig 85/3 was typical of T. simiae: three Nannomonas

200

/,

P 9.0 8.0 7.0 6.0 5.0

.

.

.

.

.

.

.

.

.

.

.

.

.

4~ 40 40"

r

0

i

r

i

r

~

u

i

20

~

0

10

18

days (post infection)

Fig. 4. Typically short prepatent period and rapid rise in parasitaemia of T. simiae in Pig 85/11, inoculated with a single hypopharynx (GMOS-008B). K: killed before natural death due to infection.

P

D

8.0

7.0 6.0 5.0 4.0 i

o

J

i

n

10

J

20

days (post infection)

Fig. 5. The parasitaemias observed in two wild-fly infected pigs: In, Pig 85/2 exposed to one Nannomonas infected fly; O, Pig 85/3 exposed to three Nannomonas infected flies; D, natural death due to infection.

infected flies were found in the group of flies offered feeds on this animal. The infection in Pig 85/2 was similar to that observed in the pigs inoculated with cultivated metacyclic forms: only one Nannomonas infected fly fed on this animal.

Discussion

Our results show that the host-restricted species, T. simiae can now be collected and characterized considerably more easily than hitherto. Procyclic forms can be isolated from tsetse midguts and grown to high yields in vitro, followed by differentiation to the infective, metacyclic stage. However, our original aim was simply to isolate and cultivate 7". simiae as procyclic forms from frozen tsetse midguts because of the restrictions governing the importation of infected pig blood into the U.K. The production of epimastigotes was fortuitous, particularly as it occurred without collagen and in the presence of gentamycin, thus differing from key conditions of the epimastigote/metacyclic system of Gray et al. (1984). Although Gray et al. (1984) consider collagen to be critical for epimastigote attachment and multiplication, our experience and that of Ross (cited by Gray et al., 1987) suggests that collagen is not always essential. The formation of epimastigotes in the presence of gentamycin was

201 unexpected, given the sensitivity of T. congolense epimastigotes to antibiotics (Gray et al., 1984); and continued use of antibiotic would possibly have inhibited further epimastigote formation and differentiation. We have also demonstrated the usefulness of early passage of fly-derived primary isolates through laboratory-reared tsetse. This allowed amplification of the material available, providing more than one chance to adapt the material to cultivation in vitro. We (PD & JJM) now routinely passage most frozen primary isolates through laboratory flies. Subpassage in tsetse also allowed a yeast infection to be eliminated. However, to decontaminate cultures thus may not always be possible, as bacterial infections in particular are likely to kill tsetse. The species-specific DNA probes of Gibson et al. (1988) confirmed that we were at all stages dealing with T. simiae, and not over-growth by a T. congolense subpopulation undetected in the primary isolate. DNA based identification was particulary useful because the clinical manifestations of the cultivated-metacyclic induced infections in the two piglets differed from those considered typical of T. simiae, and because T. simiae and T. congolense bloodstream forms are difficult to distinguish on morphological criteria. It was not clear whether the apparently reduced virulence was a permanent feature involving a change in the genome, which arose during cultivation because of preferential growth of trypanosomes with attenuated virulence. Other possible influences on virulence, for example inoculum size and the temporary effects of freezing and handling, also need investigation. Changes in the virulence of T. simiae have been reported by other authors, for instance, Hoare (1972) considers that prepatent and survival periods in pigs infected with T. simiae by fly bite are generally longer than in mechanically infected pigs. However, Bruce et al. (1913) suggest that cyclical transmission enhances virulence. In the experiments of Peel and Chardome (1954), no consistent effect of either subpassage or the mode of transmission is evident. T. simiae is probably restricted in its range of natural hosts to warthogs and other wild Suidae (Hoare, 1972). Some natural T. simiae infections of cattle have been reported (Culwick and Fairbairn, 1947; Wilson, 1958; Killick-Kendrick and Godfrey, 1963). However, experimental inoculation of cattle with bloodstream forms has failed to produce detectable infections (see KiUick-Kendrick and Godfrey, 1963, and Stephen, 1966). In addition, Roberts (1971) was unable to infect zebu cattle with cyclically transmitted T. simiae. Our failure to infect N'Dama calves with cultivated T. simiae metacyclic forms, or with bloodstream forms derived from them, supports the contention that T. simiae rarely, if ever, infects cattle. However, we worked with only one stock. Although our T. simiae DNA probe recognized all the parasitologically proven T. simiae stocks isolated by us in The Gambia and a stock from East Africa (Gibson et al., 1988; McNamara et al., 1989), there may be more than one kind of T. simiae: Hoare (1972) cites a description by Chardome and Peel of a stock from Rwanda-Burundi with longer than typical prepatent and survival periods in pigs, and with a parasitaemia that relapsed; McNamara et al. (1989) found that many of their trypanosomes in infected tsetse-midgut blots from The Gambia were not recognized by any of their battery of DNA probes specific for Trypanozoon or the four known kinds of Nannomonas, suggesting that other kinds of Nannomonas may exist. If there are other strains of T. simiae distinguishable by DNA probes, they may differ also in their infectivity and virulence. Nonetheless, we have no evidence that the

202

fleeting low-grade Nannomonas parasitaemias sometimes detected in Gambian cattle might be transient T. simiae infections. In conclusion, we have shown that continuous and significant yields of T. simiae metacyclic forms can be produced in vitro. This opens up the possibility of studying the effects on infectivity and virulence of metacyclic infective dose; cultivation and freezing; and strain variation. A further advance would be to achieve cultivation of this severely host-restricted species in vitro as bloodstream forms.

Acknowledgements We thank Mr. Alistair Grieve (ITC) for advice and practical assistance and the ITC Entomological Team and Mr. R. Barker (TRL) for technical assistance. We are grateful to Dr. Wendy Gibson for the DNA hybridization and Dr. D.G. Godfrey for critical examination of the manuscript. This work was supported by funds from the Overseas Development Administration and the Medical Research Council of the U.K.

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