J P r o r o w d , 26(3). 1979. pp 428-432 C I979 by the Society of Protozoologists

Infectivity of Trypanosoma brucei Cultivated Tsetse Fly Salivary Glands*

at 28 C with

ISABEL CUNNINGHAM and A. M. TAYLOR Centre .for Tropicul Vererinury Medicine. Unirzersiry of Edinburgh. Easter Bush, Roslin. Midlothian, Scotland

SYNOPSIS. When transformed procyclic noninfective trypanosomes of several unrelated stocks of Trypanosoma brucei were cul- tivated in T-30 Falcon flasks at 28 C in a liquid medium containing head-salivary gland explants of Glossina morsitans rnorsitans some of the organisms developed into forms infective for mice. Infective trypanosomes were detected 7 to 14 days after the cultures were prepared and they persisted for varying periods of up to 88 days when the cultures were terminated. A few of the salivary glands became invaded with parasites about the time infective organisms appeared in the cultures. Using T . brucei TREU 929, it was shown that trypanosomes grown with between 27 and 50 explants were capable of producing infections consistently for prolonged periods. On the other hand, trypanosomes cultivated with 25 or fewer explants rarely infected mice. Infectivity titrations on trypanosome suspen- sions from cultures of stocks TREU 1275 and TREU 929 revealed that the maximum number of infective organisms was present 26 to 50 days after initiation of the cultures. Control cultures of trypanosomes grown in medium alone were generally not infective but 2 of the 6 stocks gave rise to a few sporadic infections. A few epimastigote-like and metacyclic-like trypanosomes were seen in stained preparations of infective inocula. Index Key Words: Trypanosornu brrrcei: Glossina head-salivary gland explants: infectivity: procyclic trypomastigotes; metacyclic forms

HEN Trypunosornn hriicei bloodstream trypanosomes W are placed in culture at 25-28 C they transform into pro- cyclic trypomastigotes morphologically similar to those found in a tsetse fly midgut, and are generally not infective to mam- malian hosts. The commonly used blood-enriched media and semi-defined or defined media (7, 16. 19) cannot support reliably the development of metacyclic trypanosomes.

Arthropod tissue culture has provided a useful tool for stud- ies of vector stages of various parasites. Trager (20) succeeded in cultivating salivarian trypanosomes in the presence of tsetse fly, Glossinn pulpulis, tissues. When his cultures of Trypano- soinu \irIa.r were placed overnight at 38 C some of the try- panosomes developed into metacyclic stages infective for sheep. However, infective stages of T . brrrcei did not occur in similar tsetse tissue culture systems employed by Trager (20) and subsequently by Nicoli & Vattier (15) and Cunningham (4).

Recently, Cunningham & Honigberg (6) demonstrated de- velopment into metacyclic stages among procyclic noninfective trypanosomes of a fly transmissible stock of T. briirei grown in a liquid medium (5) at 28 C, in association with tsetse fly head-salivary gland explants. In the present report we describe the application and extension of this method to several unre- lated stocks of T . brrrcei from geographically well separated localities in East and West Africa.

MATERIALS AND METHODS History of the T. brucei Stocks.-The terminology used in

this publication follows the proposals for nomenclature of sal- ivarian trypanosomes published in the Bulletin of the World Health Organization (2).

TREU (Trypanosome Research Edinburgh University) 1275 was obtained from one mouse passage of TREU 667 (EVE 10) which was isolated by the Edinburgh Veterinary Expedition (EVE) to the Busoga District of Uganda in 1966 (17). When stabilates were received at the Centre for Tropical Veterinary Medicine of Edinburgh University, 3 rats were infected: sta- bilates produced from these rats were designated TREU 667.

TREU 1320 was obtained by cyclical transmission of TREU 1275. Metacyclic trypanosomes extruded from an infected G. t?iorsitrins into a I-ml pool of defibrinated bovine blood + 10%

This investigation was supported by research grants from the Min- istry of Overseas Development, London.

(v iv ) glycerol were cryopreserved. Trypanosomes comprising TREU 1275 and 1320 are pleomorphic and cause relapsing par- asitaemias and chronic infections in mice.

TREU 1397 (8118) was isolated from a pig at Nsukka, An- ambra State, Nigeria, in 1962. It has undergone several pas- sages in rodents in the course of adaptation and refreezing be- tween isolation and the latest cryopreservation in 1970. This stock is cyclically transmissible and produces heavy and lethal infections in rats and mice.

TREU 927 was obtained by inoculating the infected proboscis and salivary glands of a G. pallidipes caught at Kiboko, Kenya, in 1970. into a mouse. After a further 7 passages in mice it was cryopreserved and received at the CTVM. It was subsequently passaged twice in mice and frozen. The trypanosomes are pleo- morphic and cause fatal infections in mice.

TREU 1396 was isolated in 1970 from an ox which was in- fected by G. inorsitans caught in the Yankari Game Reserve, Bauchi State, Nigeria. It was passaged through 10 rats and one mouse and the infected blood was frozen in 1971. It is pleo- morphic and gives rise to heavy and fatal infections in rats and mice.

TREU 1399 is a cloned population prepared in 1970 from trypanosomes originally obtained from an ox at Raav, Benue State, Nigeria, in 1966. The original stock had been maintained by regular syringe passage in laboratory hosts before it was closed. TREU 1399 appears to be predominantly monomorphic; it produces non-relapsing fulminating infections in mice.

TREU 929 was derived from an infected Glossitin pallidipes caught at Lugala, Uganda, in 1969. The salivary glands of the tsetse fly were inoculated into a mouse and the infected blood from the 4th passage was frozen and sent to the CTVM. Sta- bilate 929 was prepared after one additional mouse passage. It is pleomorphic and kills mice.

Preparation and Maintenance of the Cii1tiires.-With all the stocks except TREU 1397, cultures of procyclic trypanosomes were established from 3-day or 4-day infections in mice. The cultures of TREU 1397 were initiated from a stabilate of in- fected mouse blood. Bloodstream trypanosomes were mixed with culture medium (5) to give a final concentration of 2-3 x lo6 trypanosomesiml. About 0.5 ml of the suspension was placed in a 25 cmz Falcon tissue culture flask and incubated at 28 C. As the flagellates multiplied and transformed into pro- cyclic forms culture medium was added up to a volume of 2 ml.

428

CULTIVATION OF INFECTIVE T. britcei 429

TABLE 1. Infectivity of variorrs stocks of Trypanosoma brucei cultivated with tsetse jly head-salivary gland explants.

Infections in mice Days in culture No. No. of

Days in before invaded infected culture No. of restoration salivary Period of Prepatent mice Time to without explants1 of glands infectivity No. infected/ period surviving death

Stock explants flask infectivity observed (days) No. inoculated (days) to day 20 (days)

TREU 14 1320 15

2 2 4 212 6-8 2 1 32* 919 6-8 9 23 26 9

30 50 9 4 23* 12/13 7-8 12 62 None 0 None 0 0119

5: 11 9

1 2 18* 414 7 0 11 2 18* 717 6-8 0 9-14

ii 11 8 19 28 8 38 None 0 None 0 0112

TREU 12 1397 14

TREU 9 45 9 5 21* loll1 5-7 9 16 927 14 50 7 6 18* 9/11 3-7 9

21 43 9 4 9* 516 5-7 5 39 None 30 None 7 3/12 10-16 3

TREU 22 25 10 3 43* 15/16 3-7 2 14-18 1396 21 30 11 4 43* 15/16 4-8 4 15-18

20 50 12 4 43 * 15/16 5-8 4 15-18 16 52 7 5 52* 19/19 4-7 2 11-16 75 None 34 None 23 7/20 6-11 7

TREU 19 37 12 2 23* 13/14 7-8 13 1399 36 52 12 2 10* 517 5-8 5

54 None 0 None 0 0115

TREU 35 15 10 1 14 6/22 6-12 6 929 40 15 28 0 4 2/17 6-12 2

40 25 28 0 1 1/17 16 1 26 27 8 3 81* 37/38 2-7 0 11-16 33 40 12 5 70* 27/29 3-7 0 9-15 13 50 14 5 88* 4 1/43 2-8 0 10-16

115 None 0 None 0 0145

* Experiments terminated. ] Pooled cultures.

When the population reached -2 x 107/ml, the trypanosomes free from blood cells were transferred into flasks containing 3.5 ml of medium to yield an initial concentration of 3 x 106/ml; the parasites were thereafter passaged 3 times each week.

The tsetse fly salivary gland-trypanosome culture system was prepared by the method described by Cunningham & Honig- berg (6). Samples of established noninfective procyclic try- panosomes to give an initial concentration of 3 x 106/ml were added to 25 cmz Falcon flasks containing 3.5 ml culture medium and head-salivary gland explants freshly dissected from pharate G . rnorsitans immediately before emergence. The flasks con- tained between 15 and 52 explants, depending on the experi- ment. The cultures were incubated at 28 C and maintained by removing the trypanosome suspension, except for 0.3-0.5 ml, and replacing it every 2 days with a volume of fresh medium equal to that removed. The tsetse organ-trypanosome cultures were maintained with medium containing 100 IU/ml penicillin and 100 pgiml streptomycin for the first 5-7 days after they were prepared. Thereafter medium without antibiotics was used.

Control cultures of parasites grown in flasks with 3.5 ml me- dium without antibiotics and in the absence of tsetse fly head- salivary gland explants were handled in the same manner as the experimental preparations.

Inoculations of Mice.-Three times weekly trypanosomes were taken from the cultures and injected into mice. Approxi- mately 3 ml trypanosome-containing medium, from individual flasks was centrifuged at 1000 g for 10 min. All but 0.5 ml of the supernatant fluid was discarded. The trypanosomes were resuspended in the medium left in the tube and inoculated in- traperitoneally into a mouse. With stocks TREU 1320 and TREU 1397 populations of trypanosomes pooled from 2 or 3 flasks were inoculated into mice.

In all the experiments random bred albino CFl , mice 6-9 weeks old, were used and their tail blood examined daily for the presence of trypanosomes. The mice were kept under ob- servation for 20 days postinoculation.

Infectivity Titrution.-The number of infective organisms in cultures of stocks TREU 1275 and TREU 929 grown with 45 and 40 explants respectively was estimated by the infectivity titration method of Lumsden et al. (13). Trypanosome suspen- sions grown for 2 days after the addition of fresh medium to the flasks were diluted serially tenfold with culture medium, and 0.1 ml aliquots were inoculated into groups of 6 mice. Ti- trations were carried out weekly for 7 weeks with TREU 1275 and 9 weeks with TREU 929. The numbers of organisms in the original suspensions were counted in a Neubauer hemacy- tometer.

430 CULTIVATION OF I N F E C T I V E T. hrircei

TREU 929

8,

5 1 1

Number of

per ml 0 - 0 trypanosomes

Numbers of

I, 1D63 per /i

1

Days in c u l t u r e

8

7

6

5

4

3

2

Log

1

TREU 1275

Number of \-. t r y p a n o s o m e s *---*-*- --

per ml.

2 it20 30 4 0 50 60 7b

Days in c u l t u r e

Figs. 1, 2. [Estimations of the number of trypanosomes in, and the infectivity of, samples of T . brucei stocks cultivated with tsetse fly head-salivary gland explants.] 1 . TREU 929 cultivated with 40 ex- plants. 2. TREU 1275 cultivated with 45 explants.

RESULTS The details of the restoration of infectivity of the various

stocks of T. brucei grown in the presence of tsetse fly head- salivary gland explants and the characteristics of the infections produced in mice are shown in Table 1 . Trypanosome suspen- sions of stocks TREU 1320 and TREU 1397 pooled from 2 and

3 flasks containing between 21 and 34 explants gave rise to infections in mice 9 and 8 days after the cultures had been prepared. These cultures remained infective for varying periods of up to 32 days when they were discarded. Parasites from an individual flask of TREU 1320 cultivated in association with 50 explants produced infective forms for 23 days when mouse in- oculations were terminated. The prepatent periods in mice in- fected from cultures of these 2 stocks grown with tsetse tissues ranged from 6-8 days. Mice infected with TREU 1320 had re- lapsing parasitemias from the onset of infection. Those infected from similar cultures of TREU 1397 died 9 to 14 days postin- oculation. Animals inoculated with trypanosomes from control cultures of these stocks maintained in the absence of tsetse fly head-salivary gland explants never became infected.

With all the other stocks mice were injected with trypano- some suspensions from individual culture flasks. As indicated in Table 1 . cultures of procyclic trypanosomes of TREU 927, 1396, and 1399. grown in the presence of between 25 and 52 explants, began to develop into infective forms 7 to 12 days after they had been prepared. The infectivity of these trypano- some populations persisted for protracted periods of up to 52 days when inoculations of mice were discontinued. The pre- patent periods of these infections ranged from 3 to 8 days and caused fatalities in most of the mice infected with TREU 1396. The control cultures of TREU 1399 were not infective, but a few mice inoculated with TREU 927 and TREU 1396 grown without explants developed parasitemias. The infections in these animals had longer prepatent periods and fewer trypano- somes were seen in the blood than were found in mice inocu- lated with infective cultures containing explants.

Stock TREU 929 was used (a) to obtain data on the number of explants necessary to induce the development of infective organisms in a culture flask with 3.5 ml medium and (b) to ascertain the period during which the cultures could continu- ously produce infective trypanosomes. Trypanosomes grown with 25 or less explants infected mice for only limited periods (Table 1). The cultures containing uninvaded salivary glands gave rise to a few sporadic infections 28 days after they had been prepared. On the other hand, culture flasks which con- tained 27 or more explants and included 3 to 5 invaded glands produced infective trypanosomes continuously for up to 88 days when tests were terminated. All the mice infected from this stock grown in the presence of 27 or more explants devel- oped lethal infections 2 to 8 days post inoculation. The prepa- tent periods in the few mice infected from cultures with 25 and 15 explants ranged from 6-16 days and were followed by very low parasitemias.

Counts were made periodically of trypanosome suspensions during the experiments, and there was little variation in the number of organisms in the flasks with and without explants. The numbers of trypanosomes in samples of suspensions with- drawn from the cultures for inoculation into mice ranged from 3.5 to 4.2 x 107/ml.

The numbers of infective trypanosomes in cultures of TREU 929 and TREU 1275 grown with 40 and 45 explants respective- ly , estimated during 9 and 7 weeks, are shown in Figs. 1 and 2. The total number of organisms during maintenance of the cultures remained at between 3.5 and 4.0 x 107/ml. Only a small proportion of this population was infective and the max- imum number of infective forms (ID6J of TREU 929 was pres- ent between 26 and 40 days after initiation of the culture and decreased significantly after 54 days (Fig. 1 ) . The number of infective trypanosomes (ID,) in the culture of TREU 1275 was greatest at -35 days after its preparation and did not differ significantly during the 7-week period of the test.

One notable feature observed throughout these investigations

CULTIVATION OF INFECTIVE T. briicei 43 1

Figs. 3-7.

Fig. 8.

[Trypanosoma brucei in noninfective (Fig. 3) and infective cultures. The scale in Fig. 4 applies to all figures. Giemsa stain. x 1560.1 3. Procyclic forms. 4, 5. Epimastigote-like forms. 6, 7. Metacyclic-like forms.

Bloodstream forms of T. brucei from mice infected with culture material. For scale, see Fig. 4. Giemsa stain. x 1560.

was the invasion of a few of the salivary glands by trypano- somes at about the time that infective organisms began to ap- pear in the cultures. The parasites appeared as a shimmering mass within the glands for as long as the cultures were main- tained.

Although the majority of organisms observed in Giernsa- stained smears of infective inocula were procyclic trypomasti- gotes (Fig. 3), morphologically similar to those found in the midgut or proventriculus of a tsetse fly, a few epimastigote-like forms (Figs. 4, 5 ) and occasional shorter trypomastigotes (Figs.

432 CULTIVATION OF INFECTIVE T . brircei

6, 7) with a terminal kinetoplast were also noted. Noninfective cultures contained only procyclic trypomastigotes (Fig. 3). Tsetse fly midgut-like and proventricular-like forms were also most abundant in stained preparations of trypanosomes extrud- ed from fragments of invaded salivary glands.

The parasitemias in the mice inoculated with material from cultures which produced regular infections consisted of pleo- morphic trypanosomes (Fig. 8), and the course of infections resembled that of the parent stock except in the case of T R E U 1399. The "culture-transmitted" trypanosomes of this predom- inantly monomorphic and highly virulent stock caused parasit- emias of pleomorphic but less virulent forms.

DISCUSSION It is evident from this investigation that noninfective pro-

cyclic trypanosomes of several different stocks of T. brucei cultivated at 28 C in the presence of tsetse fly head-salivary gland explants by a method similar to that of Cunningham & Honigberg (6) developed into stages infective to mice. Resto- ration of infectivity was demonstrated with various pleomorph- ic and one cloned predominantly monomorphic stock.

In the Cunningham & Honigberg (6) culture system, infec- tions in mice were produced consistently only from large in- ocula of parasites pooled from several flasks containing up to 24 explants. The present studies indicate that a n increased num- ber of explants added to the culture flasks improved the yield of infective trypanosomes generated in the cultures-mice in- oculated with trypanosome suspensions from individual flasks became infected. However, infectivity titrations of 2 typical cultures revealed that the numbers of infective organisms rep- resented only a small proportion of the total populations in the flasks.

The results obtained with TREU 929 showed that there ap- peared to be a relationship between the number of invaded salivary glands and consistent infectivity of the cultures; how- ever, other factors emanating from the explants might be re- sponsible for the development of infective stages.

The reason for the occasional infections in mice injected with stocks TREU 927 and TREU 1396 grown in the absence of tsetse tissues is not known. Other workers have reported sim- ilar sporadic infections in mice inoculated with culture forms of T. brucei and T . rhodesiense grown at 25-28 C o n blood- agar base media with various additives (1, 12, 14) and in semi- synthetic media (3).

Among the workers who have cultivated salivarian trypano- somes in the presence of Glossina tissues or cells (4, 15, 18, 20, 21), only Trager (20) was able to produce metacyclic stages. His success was achieved with T. vivax and could not be ex- tended to similar cultures of Trypanosoma congolense and T . brucei. The tsetse organ cultures of Trager (20, 21) and the tsetse cell line used by Steiger e t al. (18) supported the devel- opment of T. vivax and T. congolense respectively to the epi- mastigote stage. The culture system reported here, and by Cun- ningham & Honigberg ( 6 ) , reproduces to some extent the life cycle of the trypanosomes as it occurs in the vector. The scar- city of epimastigote and metacyclic trypanosomes in the infec- tive cultures and in the invaded salivary glands might be the result of abnormal conditions in the explants maintained in vitro for prolonged periods. The success and reproducibility of the method now described offers a n opportunity for further inves- tigation of factors which control the completion of the life cycle of T. brucei in the insect vector. If these trypanosome stocks could also be cultivated as bloodstream forms in mammalian tissue culture systems (8-11), it would be possible to study the entire life cycle of the parasites and various aspects of their

physiology, including processes of antigenic variation, in the absence of their hosts.

ACKNOWLEDGEMENTS It is a pleasure to thank Professor Sir Alexander Robertson

and Professor D. W. Brocklesby for their encouragement dur- ing these studies.

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