METACYCLIC PROMASTIGOTES OF LEISHMANIA IN THE SALIVARY GLANDS OF EXPERIMENTALLY INFECTED PHLEBOTOMINE SANDFLIES

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METACYCLIC PROMASTIGOTES OF LEISHMANIA IN THE SALIVARY GLANDS OF EXPERIMENTALLY INFECTED PHLEBOTOMINE SANDFLIES

KILLICK-KENDRICK R.*, KILLICK-KENDRICK M.*, TANG Y.* & BASTIEN P.*

Summary:

Thirty one female Phlebotomus duboscqi experimentally infected with Leishmania tropica were found with metacyclic promastigotes in their salivary glands. The presence of parasites in the glands was correlated with heavy infections of metacyclic promastigotes in the stomodaeal valve and thoracic midgut of the fly and it is suggested that the parasites may have migrated through the gut wall into the glands which lie against the valve in the thorax of the fly. No evidence was found that gland invasion is an obligatory stage of the life-cycle of Leishmania, but reports of wild-caught flies with parasites in the glands, coupled with these laboratory

observations, raise the possibility that regurgitation of parasites or the migration of metacyclic promastigotes into the mouthparts may not be the only mechanisms of transmission by bite.

KEY WORDS : Leishmania tropica, Phlebotomus duboscqi, life-cycle, salivary glands, transmission.

For the past sixty years, there has b e e n m u c h discussion o n the p r e c i s e m e c h a n i s m by w h i c h a p h l e b o t o - m i n e sandfly deposits an infective d o s e o f leishmanial promastigotes into the skin o f a vertebrate host. Reports o f p a r a s i t e s in t h e p r o b o s c i s o f t h e fly ( A d l e r &

T h e o d o r , 1 9 2 9 , 1 9 3 0 , 1 9 3 5 ; Adler ET AL., 1 9 3 8 ) sug- g e s t e d that invasion o f the mouthparts w a s the cul- mination o f the d e v e l o p m e n t in the alimentary tract o f the vector, and that it w a s a prerequisite if the fly w e r e to b e a b l e to pass o n the infection (Killick-Kendrick, 1 9 7 9 ) . An alternative idea w a s b a s e d o n o b s e r v a t i o n s that the pharynx and o e s o p h a g u s o f infected flies w e r e s o m e t i m e s b l o c k e d by a mass o f parasites (Shortt ET AL., 1 9 2 6 ) . Influenced b y the discovery o f the trans- mission o f plague b y fleas with the alimentary canal b l o c k e d b y YERSINIA PESTIS ( s e e Parrot & D o n a t i e n , 1 9 5 2 ) , the notion a r o s e that a plug o f promastigotes in the foregut or anterior midgut o f the sandfly was regurgitated into the skin o f the invertebrate w h e n an infected insect t o o k a b l o o d meal (Shortt & Swaminath,

* Department of Biology, Imperial College at Silwood Park, Ascot, Berks SL5 7PY, UK.

Résumé : PROMASTIGOTES MÉTACYCLIQUES DE LEISHMANIA DANS LES GLANDES SALIVAIRES DE PHLÉBOTOMES INFECTÉS EN LABORATOIRE Des promastigotes métacycliques ont été observés dans les glandes salivaires de 3 I Phlebotomus duboscqi femelles infectées en laboratoire par Leishmania tropica. La présence de parasites dans ces glandes est corrélée à des infestations importantes de promastigotes métacycliques dans la valve stomodéale et la portion thoracique de l'intestin moyen du phlébotome, et il est suggéré que les parasites peuvent avoir migré à travers la paroi intestinale vers les glandes qui s'étendent le long de la valve thoracique du phlébotome. Aucune preuve n'a été apportée du caractère obligatoire d'une phase d'invasion des glandes dans le cycle des Leishmania; cependant, des observations de parasites dans les glandes de phlébotomes capturés dans la nature, couplées à ces observations de laboratoire, nous font émettre l'hypothèse que la régurgitation des parasites ou la migration de promastigotes métacycliques vers les pièces buccales ne sont peut- être pas les seuls mécanismes de transmission par piqûre.

MOTS CLES : Leishmania tropica, Phlebotomus duboscqi, cycle évolutif, glandes salivaires, transmission.

1 9 2 8 ; Napier, 1 9 4 6 ) . Adler and T h e o d o r ( 1 9 3 5 ) strongly o p p o s e d this idea o n the grounds that the b l o c k a g e w a s m o r e apparent than real. T h e y p o i n t e d out that t h e p h a r y n x w a s highly e x p a n d a b l e b y t h e l a r g e m u s c l e s that e n a b l e d it to w o r k as a p u m p , a n d pre- s e n t e d a formidable c a s e to support the idea that transmission w a s by small, highly active promastigotes in the mouthparts ( r e v i e w e d by Killick-Kendrick, 1979, 1 9 8 6 ) .

B y 1 9 8 5 , Adler's theory had r e c e i v e d strong support from reports o f promastigotes in mouthparts in 18 c o m - binations o f sandflies and LEISHMANIA s p e c i e s (Killick- Kendrick, 1 9 8 6 ) . D o u b t s w e r e then cast o n his ideas w h e n laboratory transmissions o f LEISHMANIA w e r e a c h i e v e d in the apparent a b s e n c e o f parasites in the proboscis (Lainson ET AL, 1 9 7 7 ; Pozio ET AL, 1985). After infected sandflies w e r e s h o w n to have difficulty in taking a b l o o d m e a l (Killick-Kendrick ET AL, 1 9 7 7 b ) , it w a s suggested that parasites in the b u c c a l cavity might interfere with the function o f internal receptors thereby changing the feeding b e h a v i o u r o f the fly and enhan- cing the c h a n c e s o f parasites from the foregut b e i n g d e p o s i t e d in the skin o f the vertebrate host (Killick- Kendrick & M o l y n e u x , 1 9 8 1 ) . H o w e v e r , a detailed

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Parasite, 1996, 3, 55-60 55

Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/1996031055

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KILLICK-KENDRICK R., KILLICK-KENDRICK M . TANG Y S BASTIEN P.

study s h o w e d that, unlike other Diptera, p h l e b o t o m i n e sandflies s e e n to have n o c h e m o r e c e p t o r s in t h e ciba- rium (Jefferies, 1 9 8 7 ) , a n d it appears that this is n o t the e x p l a n a t i o n o f multiple probing b y infected flies.

A n e w explanation o f the c h a n g e d feeding behaviour e m e r g e s from highly original observations b y Schlein a n d c o l l e a g u e s w h o s h o w e d t h a t p r o m a s t i g o t e s e n c a s e d in the peritrophic m e m b r a n e in the midgut o f a sandfly p r o d u c e chitinolytic e n z y m e s that not only b r e a k d o w n the m e m b r a n e a n d permit the parasites to e s c a p e , but also d a m a g e the chitin o f the s t o m o - daeal valve (the cardia o f s o m e authors) (Schlein ET AL, 1 9 9 1 ) . Schlein ET AL. ( 1 9 9 2 ) published e l e c t r o n m i c r o - graphs o f a d a m a g e d valve a n d illustrated h o w they thought this c o u l d lead to its malfunction resulting in the d e p o s i t i o n o f p r o m a s t i g o t e s from t h e anterior midgut a n d foregut into the skin o f a vertebrate as an infected fly attempted to feed. T h e action o f the chi

tinolytic e n z y m e s is inhibited b y h a e m o g l o b i n (Schlein

& J a c o b s e n , 1 9 9 4 ) , w h i c h e x p l a i n s t h e a b s e n c e o f signs o f d a m a g e in electronmicrographs o f the cuti

cular intima o f t h e valve o r hindgut o f several diffe

rent s p e c i e s o f flies infected with various parasites e x a mined before, o r shortly after, the digestion o f the infecting b l o o d meal. Schlein's ideas o n the mechanism of transmission o f LEISBMANIA b y the bite o f an infected sandfly are plausible a n d offer an e x p l a n a t i o n o f mul

tiple probing b y infected flies a n d t h e difficulty they have in obtaining a full b l o o d meal (Killick-Kendrick ET AL., 1 9 7 7 b , B e a c h ET AL., 1 9 8 5 ) .

T h e possibility a p p e a r s n e v e r to have b e e n c o n s i dered that a n o t h e r m o d e o f transmission could s o m e times b e b y promastigotes in the salivary glands b e i n g d e p o s i t e d in t h e skin o f the vertebrate host with the saliva. In the present p a p e r w e describe such infec

tions in experimentally infected sandflies a n d suggest that there is p e r h a p s m o r e than o n e m e c h a n i s m o f transmission b y bite.

MATERIALS AND METHODS

Sandflies

T

h e flies w e r e from a laboratory c o l o n y o f PHLE

BOTOMUS DUBOSCQI N e v e u - L e m a i r e set u p in J u n e , 1 9 8 8 , with the eggs o f females caught in CDC miniature light traps at Keur Moussa, Senegal. T h e m e t h o d s for the initiation a n d m a i n t e n a n c e o f the colony are given b y Killick-Kendrick & Killick-Kendrick ( 1 9 8 7 , 1 9 9 1 ) . T h e lines u s e d originated from the pro

g e n y o f individual f e m a l e s that w e r e i n b r e d a n d s e l e c t e d for their susceptibility to infections b y LEISH

MANIA TROPICA Wright. T h e observations reported in the present p a p e r w e r e m a d e o n t h e 13th to 24th g e n e rations after inbreeding w a s b e g u n .

Parasite

The parasite w a s isolated in NNN m e d i u m from a cuta

neous lesion o f a patient in Kabul, Afghanistan, in J u n e , 1988. It w a s typed at t h e Faculty o f M e d i c i n e , Mont- pellier, the Istituto Superiore di Sanita, R o m e , a n d the London S c h o o l o f H y g i e n e a n d Tropical M e d i c i n e a n d identified as L . TROPICA, z y m o d e m e M O N - 5 8 ( = LON- 2 0 ) . T h e strain, No M H O M / A I 7 8 8 / K K 2 7 ( = LEM 1422

= ISS 4 2 3 ) , w a s maintained in g o l d e n hamsters b y the p a s s a g e o f amastigotes ( B a s t i e n & Killick-Kendrick, 1 9 9 2 ) .

Infection o f sandflies

W a s h e d s u s p e n s i o n s o f amastigotes w e r e p r e p a r e d from infected hamster skin tissue. T h e parasites w e r e c o u n t e d b o t h in a h a e m o c y t o m e t e r a n d b y t h e m i c r o - b e a d m e t h o d o f Cenini ET AL. ( 1 9 8 9 ) . T h e proportion o f viable amastigotes w a s estimated with fluorescent illumination after staining with ethidium b r o m i d e a n d fluorescein diacetate (Cenini ET AL, 1 9 8 9 ) , a n d t h e sus

p e n s i o n w a s diluted with inactivated rabbit b l o o d t o give an estimated 3 x 1 0⁵ viable amastigotes per 1.0 ml.

T h e sandflies w e r e permitted to feed o n t h e suspen

sion through a hamster c h e e k - p o u c h attached to a glass feeder w a r m e d b y circulating w a t e r ( B a s t i e n , 1 9 9 0 ) . E a c h fly with a full b l o o d meal w a s estimated to have taken about 100 parasites.

M a i n t e n a n c e a n d examination o f infected flies O n the d a y after the meal, fed flies w e r e individually tubed, provided with a solution o f s u c r o s e ( 5 0 : 5 0 v / v ) and kept at 2 5 °C. T h e y w e r e e x a m i n e d at least twice a day until they h a d laid eggs w h e n they w e r e i m m e diately dissected a n d e x a m i n e d . T h e h e a d o f e a c h fly w a s cut off a n d an attempt w a s m a d e to dissect out b o t h salivary glands a n d mount them intact u n d e r a separate coverslip b e f o r e t h e gut w a s r e m o v e d . T h e y w e r e e x a m i n e d u n d e r p h a s e contrast o r interference illumination. Flies w e r e n o t c o n s i d e r e d t o h a v e nega

tive salivary glands unless both glands w e r e e x a m i n e d . The intensity o f infection in different parts o f the ali

mentary tract w a s subjectively a s s e s s e d a n d s c o r e d +++++, ++++, +++, ++ a n d +.

RESULTS

P

romastigotes w e r e s e e n in the salivary glands o f 31 sandflies 9-21 days after infection. In 22 o f t h e s e flies, o n l y o n e o f t h e t w o glands a p p e a r e d to b e infected. T h r e e flies h a d promastigotes in b o t h glands but, in six others, a gland w a s lost during dissection a n d only o n e w a s available for e x a mination. T h e n u m b e r s o f parasites s e e n w e r e g e n e rally low. O f 3 4 infected glands, 21 h a d 1-5 parasites,

56 Mémoire Parasite, 1996, .5, 55-60

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METACYCLIC; PROMASTIGOTES OF

Leishmania

IN THE SALIVARY GLANDS

Parasite, 1996, 3, 55-60

Mémoire 57

Fig.l, 430X; Figs 2 & 4, 1180X; Fig. 3, 800X. — Metacyclic promastigotes of Leishmania tropica in freshly dissected salivary glands of experimentally infected Phlebotomus duboscqi. Phase contrast illumination.

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KILLICK-KENDRICK R., KILLICK-KENDRICK M., TANG Y . & BASTIEN P.

five h a d 6 - 1 0 , t w o had 1 1 - 1 5 and six h a d > 2 0 . T h e r e w a s a strong correlation b e t w e e n infected glands and the intensity o f infection in the midgut, linked to the p r e s e n c e o f n u m e r o u s m e t a c y c l i c forms. T w e n t y four o f the flies with parasites in the glands h a d b o t h h e a vily infected s t o m o d a e a l valves ( s c o r e d as +++++ or ++++) and thoracic midguts ( + + + + ) . Eighteen o f the 24 had extraordinarily large numbers o f metacyclic promastigotes in t h e s e parts o f the alimentary tract.

Lightly infected glands initially s e e m e d often to b e negative. After long observation, the c o m m o n e s t first indication o f infection w a s an intermittently moving flagellum. It was n e c e s s a r y to wait until the parasite m o v e d revealing the body, and the parasite c o u l d then b e s e e n pushing b e t w e e n the cells o f the gland.

In glands with m o r e than a d o z e n parasites, the promastigotes w e r e visible without difficulty with p h a s e contrast or interference microscopy (but not with bright field). Cells o f o n e infected gland a p p e a r e d to h a v e b e e n d a m a g e d b y the parasites (Fig. 3 ) . All parasites in the glands l o o k e d like m e t a c y c l i c promastigotes.

T h e y w e r e small with a b o d y length o f o n l y 5-10 um a n d a flagellum length at least twice that o f the b o d y (Figs 1-4). N o n e w a s in division.

It is not p o s s i b l e to estimate the true p r e v a l e n c e o f gland infections b e c a u s e it is certain that s o m e glands r e c o r d e d as negative w e r e infected, but the parasites w e r e i m p o s s i b l e to s e e . Nevertheless, n o parasites w e r e s e e n in the glands o f m a n y h u n d r e d s o f our experimentally infected flies and the overall prevalence w a s s e e m i n g l y low.

DISCUSSION

O

ur observations are insufficient e v i d e n c e by t h e m s e l v e s to c o n c l u d e that invasion o f the salivary glands o f sandflies by promastigotes is a normal part o f the life c y c l e o f LEISHMANIA. Fur

thermore, our w o r k w a s with a LEISHMANIA s p e c i e s in a fly w h i c h is n o t its natural v e c t o r . T h e r e a r e , h o w e v e r , t w o reports from Brazil o f promastigotes in the salivary glands o f wild-caught sandflies. Arias ET AL.

( 1 9 7 8 ) found three female flies with infected glands a m o n g c a t c h e s m a d e in A m a z o n a s State. T h e identity o f t w o o f the flies is not clear, but the third w a s a m e m b e r o f the SHANNONI group w h i c h c o u l d not b e identified further b e c a u s e this g r o u p c o n t a i n s s p e c i e s with isomorphic females. T h e parasite from the midgut o f this fly failed to infect a hamster, a n d its identity remains u n k n o w n . In the s e c o n d report, Naiff ET AL.

( 1 9 9 1 ) found promastigotes in the salivary glands o f o n e o f t w o naturally infected s p e c i m e n s o f LU. SQUA- MIVENTRIS (Lutz & Neiva) caught at B a l b i n a , A m a z o n a s State. T h e midgut a n d Malpighian tubules o f the fly

w e r e heavily infected, but a hamster i n o c u l a t e d with the parasites did not develop a lesion. Eighteen months later, the strain w a s r e c o v e r e d b y culturing from the site o f inoculation a n d w a s t y p e d b y Grimaldi ET AL.

( 1 9 9 1 ) as LE. NAIFFI Lainson & S h a w ; it is r e a s o n a b l e to s u p p o s e that the promastigotes in the salivary glands w e r e the s a m e s p e c i e s .

T h e r e are t w o routes by w h i c h p r o m a s t i g o t e s c o u l d m o v e from the midgut to the salivary glands. Firstly, t h e y m a y p e n e t r a t e through the gut wall into t h e glands w h i c h lie p r e s s e d against the t h o r a c i c midgut (Jobling, 1 9 8 7 ) . Most flies with parasites in the glands had heavily infected s t o m o d a e a l valves a n d t h o r a c i c midguts and, from e l e c t r o n m i c r o g r a p h s , promastigotes are k n o w n to burrow into the cells o f the anterior midgut ( M o l y n e u x ET AL., 1 9 7 5 ; Killick-Kendrick ET AL, 1977'A). Furthermore, Adler & T h e o d o r ( 1 9 2 9 ) found parasites throughout the h a e m o c o e l o m i c cavity o f t w o sandflies that had b e e n i m b e d d e d a n d s e c t i o n e d ; curiously, they attached n o i m p o r t a n c e t o their o b s e r vation. T h e s e c o n d , less likely, route is by the salivary duct w h i c h o p e n s towards t h e anterior tip o f the mouthparts (Jobling, 1 9 8 7 ) . This w o u l d b e likely only if the p r o b o s c i s w e r e infected.

M o r p h o l o g i c a l l y , t h e p r o m a s t i g o t e s in the salivary glands r e s e m b l e d parasites r e c o v e r e d from the pro

b o s c i s o f infected flies (Adler & T h e o d o r , 1 9 3 1 , 1 9 3 5 ; Killick-Kendrick, 1 9 8 6 ) . T h e i r size a n d s h a p e w e r e the s a m e as m e t a c y c l i c p r o m a s t i g o t e s , t h e form p r e - adapted to life in the vertebrate host ( s e e review b y Killick-Kendrick, 1 9 9 0 ) , a n d it s e e m s p r o b a b l e t h e y would b e infective and b e deposited in the skin during the act o f biting. Such a m e c h a n i s m o f transmission c o u l d b e advantageous in the evolution o f LEISHMANIA spp. s i n c e there is n o w m u c h e v i d e n c e that sandfly saliva plays an important role enabling m e t a c y c l i c pro

mastigotes to b e c o m e established in skin at the site o f the bite (Titus & Ribeiro, 1 9 8 8 ; S a m u e l s o n ET AL, 1 9 9 1 ; T h e o d o s ET AL, 1 9 9 1 ) .

Salivary glands are seldom e x a m i n e d and further obser

vations o n b o t h natural and e x p e r i m e n t a l infections must b e m a d e b e f o r e the r e l e v a n c e o f promastigotes in the glands to transmission can b e judged. Light infec

tions are almost i m p o s s i b l e to detect w h e n glands are e x a m i n e d with bright field illumination, and it is n e c e s sary to use a 1 0 0 X oil-immersion o b j e c t i v e with either p h a s e contrast o r interference illumination. Even with this e q u i p m e n t , the glands h a v e often to b e o b s e r v e d for a long time b e f o r e it is certain they are infected.

A further difficulty in s e e i n g the promastigotes is that t h e y are o b s c u r e d b y d e g e r a t e cells a n d g r a n u l e s w h i c h a p p e a r with progressive c h a n g e s o f the glands following a b l o o d m e a l (Adler & T h e o d o r , 1 9 2 6 ) . A review o f publications o n transmission by bite leads to the c o n c l u s i o n that a s e a r c h for a single m e c h a n i s m

58 Parasite, 1996, 3, 55-60

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may b e in vain. T h e r e is strong e v i d e n c e that parasites may b e regurgitated following d a m a g e to the s t o m o - daeal valve b y chitinolytic e n z y m e s p r o d u c e d b y t h e parasite ( S c h l e i n et al, 1 9 9 1 , 1 9 9 2 ) . It a l s o s e e m s highly p r o b a b l e that transmission c o u l d o c c u r b y the transfer o f parasites from heavily infected mouthparts into the skin in the act o f biting (Killick-Kendrick, 1 9 7 9 ) . Possibly invasion o f t h e salivary glands a n d t h e deposition o f m e t a c y c l i c promastigotes into the skin with the saliva is a third w a y .

ACKNOWLEDGEMENTS

W

e are grateful to t h e M e d i c a l R e s e a r c h C o u n c i l , L o n d o n , a n d t h e L e v e r h u l m e Trust for financial support. T h e Afghan strain o f L. tropica w a s isolated w h i l e R.K-K. w a s a Short T e r m Consultant for the W H O Eastern Mediter- r a n e a n Regional Office.

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Reçu le 20 juin 1995 Accepté le 20 septembre 1995

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METACYCLIC PROMASTIGOTES OF LEISHMANIA IN THE SALIVARY GLANDS OF EXPERIMENTALLY INFECTED PHLEBOTOMINE SANDFLIES