MODELLING HUMAN FILARIASIS
CLINICAL STUDY OF THE OCULAR LESIONS
INDUCED BY MONANEMA MARTINI IN ITS MURID HOSTS
AIMARD L.*, WANJI S.*, V U O N G P.N.**, PETIT G.*, BAIN O.*
KEYWORDS : ocular lesions, ophthalmology, murids. filaria. Monanema. oncho- cerciasis.
T
he filaria Monanema martini with skin-dwelling microfi- lariae induces in its natural murid hosts lesions similar to t h o s e in h u m a n o n c h o c e r c i a s i s . T h i s w a s d e m o n s t r a t e d b y histo-pathological studies ( V u o n g et ai, 1 9 9 1 ) but it a p p e a - red useful to evaluate the m o d e l by a clinical study. An o p h - thalmological analysis (Aimard et ai, 1 9 9 3 ) w a s performed o n the t w o s p e c i e s o f hosts, inoculated by o n e , t w o or mul- tiple d o s e s o f larvae, with patent infections s i n c e at least o n e year. A total o f 140 e y e s w a s e x a m i n e d (anterior a n d poste- rior s e g m e n t s ) ; a system o f values w a s established for the different types a n d intensities o f lesions ; a file w a s prepared for e a c h e y e a n d an attempt at quantification w a s performed.T h e significant lesions w e r e different in the t w o host s p e c i e s . In Arvicanthis niloticus, in w h i c h motile microfilariae w e r e s e e n in the anterior segment, punctate keratitis w a s predomi- nant. In Lemniscomys striatus, the posterior s e g m e n t s h o w e d c o m p l e t e chorioretinal atrophy, similar to the ultimate stage o f o n c h o c e r c a l chorio-retinitis.
T h e p a t h o g e n i c m e c h a n i s m is p r o b a b l y not u n i q u e a n d it m a y vary a c c o r d i n g t o the s p e c i e s o r individuals. It is n o t e d for e x a m p l e that L. striatus h a s levels o f skin microfilariae m u c h h i g h e r than A. niloticus. M. martini r e p r e s e n t s in its natural hosts t w o c o m p l e m e n t a r y m o d e l s for the study o f p a t h o g e n e s i s a n d treatment o f h u m a n o n c h o c e r c i a s i s .
ACKNOWLEDGEMENTS
T
h i s i n v e s t i g a t i o n w a s s u p p o r t e d b y S c i e n c e a n d T e c h n o l o g y for D e v e l o p m e n t P r o g r a m m e o f E u r o p e a n C o m m u n i t y , Contract n ° T S E - 0 0 6 7 - F a n d T S 3 C T 9 - 0 0 3 7 , a n d b y U N D P / W o r l d B a n k / W H O p r o g r a m m e for R e s e a r c h a n d Training in T r o p i c a l D i s e a s e s ID n ° 9 0 0 0 5 3 5 a n d 9 1 0 3 3 7 .R E F E R E N C E S
AIMARD L . , W A N J I S . , V U O N G P . N . , P E T I T G . , BAIN O . : Ophthalmological study of the lesions induced by the filarial worm with dermal microfilariae, Monanema martini, in its murid hosts. Current Eye Research , 1993, 12, 885-891.
VUONG P.N., WANM S., SAKKA L., KLAGER S. and BAIN O . : The murid filaria Monanema martini : a model for onchocerciasis. Part I : Description of lesions. Ann. Parasit. Hum. Comp., 1991, 66, 109- 120.
* Laboratoire de Biologie Parasitaire. Protistologie, Helminthologie, CNRS-URA 114, Museum National d'Histoire Naturelle. 61 rue Buffon, F-75231 Paris Cedex 05, France.
** Département dAnatomie et de Cytologie Pathologiques, Hôpital Saint-Michel, 33 rue Olivier de Serres, F-75015 Paris, France.
Table I. - Scores of significant ocular lesions in L. striatus and A.
niloticus infected with Monanema martini.
Control : rodents not inoculated with infective filarial larvae (L3) ; Mono-inoc : inoculated with a single dose of 80 L3 ; Bi-inoc : ino- culated twice with 80 L3 at twelve months intervals ; Multi-inoc : inoculated with a mean sum of 340 L3 given in bi-monthly doses of 15-20 L3 ; n : number of L. s. or A. n. per group ; Mf/mm2 : mean microfilaria! densities in the ear-skin lobe during the patent phase (based on the microfilarial counts performed each three months until 18 months) ; Chor-ret atrophy Sc : mean score of chorioretinal atrophy (maximal theoretical value : 5) ; SPK-IPK Sc. : mean score of superficial punctate keratitis and interstitial punctate keratitis (maximal theoretical score : 3).
DRUG TRIALS W I T H MONANEMA MARTINI : EFFECT O N T H E ADULT W O R M S , T H E DERMAL MICRO- FILARIAE A N D T H E NATURAL M U R I D H O S T
WANJI S.*, V U O N G P.N.**, GANTIER J.C.***, B O U G N O U X M.E.****, B R E T O N B.*, BAIN O.*
T e c h n i c a l assistance : PLATEAUX S.*, DUMARQUEZ E.***
KEYWORDS : macrofilaricides. side-effects, histopathology. filariasis with dermal
microfilariae, onchocerciasis, murids.
SUMMARY :
The filaria Monanema martini, with skin-dwelling microfilariae, which induces onchocerca-like lesions, is well appropriate for drug trials.
These are performed together with a histopathological study of side- effects on the murid host.
* Laboratoire de Biologie Parasitaire, Prostilogie, Helminthologie, CNRS-URA 114, Museum national d'Histoire naturelle, 61 rue Buffon, F-75231 Paris Cedex 05.
** Laboratoire d'Anatomie et de Cytologie Pathologiques, 20 ave- nue de la Gare, 91570 Bièvres.
*** Laboratoire de Parasitologic, Faculté de Pharmacie, rue J . B . Clément, 92290 Chatenay-Malabry.
**** Laboratoire de Parasitologic et Microbiologie. Hôpital Ambroise Paré, 92100 Boulogne, France.
Parasite, 1994, /, 1S 33
Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/199401s1033b
MODELLING HUMAN FILARIASIS
Table I. - Efficiency of 12 drugs on Monanema martini and their side-effects on Lemniscomys striatus, forty days after the beginning of treatment.
Note that the three benzimidazoles are micro and macrofilaricides, and that ivermectin does not reduce the microfiladermia. Frequently drugs which do not reduce significantly the microfiladermia have a slight effect, demonstrated by the lesions induced in the cutaneous tis
sue due to a small proponion of microfilariae driven outside the lymphatic vessels.
TFMGP : Trifluoromethylguanidinopyrimidine ; TCMTD : Trichloromethylthiadiazole, ALBZ : albendazole ; sc : drug administration by sub
cutaneous route ; 0 : not treated control rodents. - mg/day x n day : mg of drug1 per kg of body weight per day, and number of days of drug administration. - Mf/mm2 : microfiladermia at the ear-lobe, on day 0 (left number) and day 40 (right number). - F/L3 : the number of filariae recovered divided by the number of infective larvae inoculated xlOO. - % Lu-He : percentage of filariae located in the lungs and heart. - Vise. les. ; more frequent and intense visceral lesions. - Cut. les. : more frequent and intense cutaneous lesions of the inflamma
tory process, with its different types : 2 : acute, 3 : sub-acute, 4 : granulomatous inflammation and 5 : lesions of scarring fibrosis (a + is added when the lesion is very intense ; nothing is written when the lesion is not frequent and weak) ; Ly : lymphagitis (type 3 associated to lymphatic vessels) ; PNE : eosinophilic polynuclear cells ; Thr : thrombosis of the pulmonary arteries ; inf : infarction ; nephritis* : obser
ved on a treated rodent dead on day 16. - % dead : percentage of rodents treated and dead.
1. Drug amount per day expressed in mmol.10-3 : Artemether, 84.2 ; TFMGP, 39.6 ; TCMTD, 226.8 ; UMF 289, 49.4 ; UMF 078, about 153.4 ; ALBZ, 154.3 ; CGP 6140, 269.5 ; CGI 18041, 168.6 ; CGP 20376, 32.6 ; levamisole, 29.1 ; suramin, 27.4 ; ivermectin, 5.7.
T
o bring filariasis u n d e r c o n t r o l a s e a r c h o n macrofilaric i d e s is b e i n g strongly stimulated (Macrofil P r o g r a m m e , W H O ) . T h e aim is to d e f i n e a treatment with a c o r r e c t e q u i librium b e t w e e n its efficiency a n d its side-effects. Drug acti
v i t i e s m u s t b e s c r e e n e d w i t h v e r y s m a l l a m o u n t o f products, a n d a h i s t o p a t h o l o g i c a l analysis o f t h e i n f e c t e d treated hosts is n e c e s s a r y . D u e to t h e s e t w o essential requi
r e m e n t s , small rodent m o d e l s in w h i c h parasitological c y c l e is c o m p l e t e d are n e e d e d .
Monanema martini with d e r m a l m i c r o f i l a r i a e b e l o n g s t o that k i n d o f m o d e l ; it is also t h e u n i q u e l a b o r a t o r y filaria w h i c h i n d u c e s an Onchocerca like p a t h o l o g y , as d e m o n s trated b y h i s t o p a t h o l o g i c a l ( V u o n g et al., 1 9 9 1 ) a n d o p h t h a l m o l o g i c ^ studies (Aimard et a!.. 1 9 9 3 ) . T h e b i o l o g y o f M. martini -rate o f d e v e l o p m e n t o f L3, distribution o f fila
riae, e v o l u t i o n o f microfiladermia- has b e e n previously pre
c i s e d (Wanji et a!., 1 9 9 0 ; Wanji, 1 9 9 2 ) .
M E T H O D S
f h e f i l a r i a is m a i n t a i n e d in its n a t u r a l m u r i d h o s t , Lemniscomys striatus, 3 9 g o f m e a n w e i g h t ; t h e infec
tive d o s e o f l a r v a e is 8 0 L 3 , i n o c u l a t e d s u b c u t a n e o u s l y . M i c r o f i l a d e r m i a is m e a s u r e d o n t h e left e a r - l o b e a n d e x p r e s s e d in m f / m m2. D i s s e c t i o n o f t h e r o d e n t s ( i n t e s t i n e , m e s e n t e r y , l u m b a r a n d r e n a l l y m p h n o d e s , heart, l u n g s ) a n d m o r p h o l o g i c a l study o n fresh w o r m s are p e r f o r m e d . E a c h rodent h e a d is fixed in formalin for e y e a n d c u t a n e o u s (right e a r ) lesions ; s o m e animals are fixed in toto for the histological study o f internal o r g a n s . Localization (intra or e x t r a l y m p h a t i c ) a n d density o f parasites are n o t e d ; lesions are identified (inflammatory reaction -types 1 to 5- a n d r e a c tive l e s i o n s ) a n d their intensity is n o t e d ; a s c o r e o f the cuta
n e o u s lesions is calculated. F o r e a c h rodent, all t h e results are registered o n a file.
R E S U L T S
1. M. martini has a very l o w susceptibility t o ivermectin. It h a s b e e n s h o w n b y in vitro study (microfilariae are i m m o b i l i z e d with 5 0 u g / m l , after 12 h o u r s ) a n d in vivo s t u d y ( B o u g n o u x , 1 9 8 7 ) . T h e distribution o f t h e d a i g in L. striatus is n o r m a l (analysis o f M e r c k , S h a r p a n d D o h m e , 1 9 8 7 ) . T h i s unusual b e h a v i o u r p r e s e n t e d b y M. martini is o f interest t o
3 .
Parasite, 1994, 7, I S
u n d e r s t a n d the g e n e t i c m e c h a n i s m s o f susceptibility to iver- m e c t i n a n d a c q u i r e d refractiveness.
2. M. martini is n o r m a l l y s u s c e p t i b l e to suramin, l e v a m i s o l e a n d a l b e n d a z o l e .
3. Eight drugs p r o v i d e d b y W H O h a v e b e e n tested : C G P 2 0 3 7 6 , C G P 6 1 4 0 , C G I 1 8 0 4 1 , A r t e m e t h e r , T r i c h l o r o m e t h y l t h i a d i a z o l e , T r i f l u o r o m e t h y l g u a n i d i - n o p y r i - m i d i n e , a n d t h e t w o b e n z i m i d a z o l e s , U M F 0 7 8 a n d U M F 2 8 9 . F o u r principal data w e r e s h o w n :
a. T h e macrofilaricidal activity is first d e m o n s t r a t e d b y the a b n o r m a l i t i e s o f t h e e m b r y o g e n e s i s in t h e uteri o f t h e f e m a l e w o r m s ; divided e g g s are the m o s t fragile stages ; uterine microfilariae persist ( C G P 6 1 4 0 ) o r are d e s t r o y e d (UMF 0 7 8 a n d 2 8 9 , e t c . ) .
b. A migration o f the adult w o r m is generally observed. M.
martini filariae are normally in the lymphatic vessels o f the i n t e s t i n e wall. W h e n treated, t h e y are n o m o r e a b l e to maintain themselves in situ and they are drawn passively towards the pulmonary b l o o d system via the thoracic duct and right heart ventricule. This migration induces lymphan- gitis a n d s o m e t i m e s t h r o m b o s i s f o l l o w e d b y i n f a r c t i o n ( a l b e n d a z o l e , CGI 1 8 0 4 1 ) . Similar events may o c c u r in all lymphatic filariae (Brugia, Wuchereria, and ? non nodular Onchocerca volvulus).
c. W h e n a macrofilaricide is a l s o microfilaricide, it m a y i n d u c e a Mazzotti r e a c t i o n , as with D E C : microfilariae are driven from the l y m p h a t i c v e s s e l s , in w h i c h t h e y nor- mally inhabit, a n d they are d e s t r o y e d in the perivascular c o n n e c t i v e tissue b y a multitude o f s y n c h r o n o u s inflam- matory' r e a c t i o n s ( a l b e n d a z o l e , UMF 0 7 8 , U M F 2 8 9 ) . d. A p r o p o r t i o n o f microfilariae and adult filariae d o not migrate a n d are d e s t r o y e d in situ in the l y m p h a t i c v e s - sels, inducing lymphangitis.
O n t h e b a s i s o f t w o trials p e r f o r m e d , U M F 2 8 9 s e e m s t o link high macrofilaricide efficacy a n d m o r e rapid r e c o v e r y o f the side-effects than t h e t w o o t h e r b e n z i m i d a z o l e s .
ACKNOWLEDGEMENTS
T h i s i n v e s t i g a t i o n w a s s u p p o r t e d b y S c i e n c e a n d T e c h n o l o g y for D e v e l o p m e n t P r o g r a m m e o f E u r o p e a n C o m m u n i t y , Contract n ° T S E - 0 0 6 7 - F a n d T S 3 C T 9 - 0 0 3 7 , a n d by U N D P / W o r l d B a n k / W H O p r o g r a m m e for R e s e a r c h and Training in T r o p i c a l D i s e a s e s I D n ° 9 0 0 0 5 3 5 and 9 1 0 3 3 7 .
R E F E R E N C E S
AIMARD L . , W A N J I S . , V I ' O N G P . N . , P E T I T G . , BAIN O . Ophthalmologic^ study of the lesions induced by the filarial worm with dermal microfilariae, Monanema martini, in its murid hosts. Current Eye Research , 1993, / 2 , 8 8 5 - 8 9 1 .
BOUGNOUX M.E. : La filaire à microfilaires dermiques, Monanema martini, modèle expérimental pour l'onchocercose ; complé- ments biologiques et essais thérapeutiques. DEA Parasitologic (Parasitologic : écologie, pathologie), Acad. Montpellier, Univ.
Se. Techn. Languedoc, 30 septembre 1987, 32 p.
VUONG P.N., WANJI S., SAKKA L., KLAGER S. and BAIN O . : The murid filaria Monanema martini : a model for onchocerciasis. Part I : Description of lesions. Ann. Parasit. Hum. Comp., 1991, 66, 109- 120.
WANJI S., CABARET J . . GANTIER J . - C , BONNAND N . , BAIN O . : The fate
MODELLING HUMAN FILARIASIS
P A R E N T E R A L A D M I N I S T R A T I O N O F C Y T O K I N E S A N D D R U G S I N R O D E N T FILARIASIS V I A OSMOTIC P U M P S
RAPP J . * , DALLAH C.N.**, S C H U L Z - K E Y H.*
KEYWORDS : Interferon-γ, osmotic pump, lemniscomys striatus. Monanema mar
tini.
SUMMARY
Changes in the cytokine production profile are supposed to be respon
sible for the different courses of parasitic infections. Therefore, a big effort is made to understand the roles of cytokines produced by diffe
rent cells during parasitic infections. In the following text we describe first experiments with Interferon-γ done in a natural model for filariasis, the filariae Monanema martini in Lemniscomys striatus. For the applica
tion of this cytokine to the animals we implanted osmotic pumps, which deliver IFN-γ continuously.
I N T R O D U C T I O N
C y t o k i n e s play an important role in the regulation o f the i m m u n e r e s p o n s e c a u s e d b y parasitic infections. T w o sets o f c y t o k i n e s are r e s p o n s i b l e for the activation o f diffe
r e n t e f f e c t o r m e c h a n i s m s ( M o s m a n n et al., 1 9 8 9 ) . I n t e r l e u k i n - 2 ( I L - 2 ) a n d interferon-v (IFN-y), p r o d u c e d b y T h l - l y m p h o c y t e s , are important for the activation o f m a c r o p h a g e s , c y t o t o x i c T - l y m p h o c y t e s a n d natural killer c e l l s , w h e r e a s I L - 4 a n d I L - 5 , p r o d u c e d b y T h 2 - l y m p h o c y t e s , i n d u c e an Ig E r e s p o n s e a n d l e a d to the g e n e r a t i o n o f e o s i - nophilia. T h e type o f c y t o k i n e s p r o d u c e d d e p e n d s o n the host, the parasite, the site a n d t h e p h a s e o f infection. W h i l e T h l r e s p o n s e s a r e u s u a l l y d i r e c t e d a g a i n s t i n t r a c e l l u l a r parasites, the r e s p o n s e s o f T h 2 often act against intestinal helminth infections. T h e s e patterns, h o w e v e r , are not uni
form. Filarial infections often c a u s e T h 2 r e s p o n s e s , but fila
r i a e p o s s e s s d i f f e r e n t e v a s i o n s t r a t e g i e s , e s p e c i a l l y t h e d o w n r e g u l a t i o n o f the i m m u n e d e f e n s e resulting in c h r o n i c infections ( B e h n k e , 1 9 8 7 ) . I m m u n o l o g i c a l hyperreactivity is a s s o c i a t e d with s e v e r e p a t h o l o g y , i.e. s o w d a in the c a s e o f o n c h o c e r c i a s i s .
T h e d i c h o t o m y o f the T h l a n d T h 2 cell population is b a s e d o n multiple, subtle a n d d y n a m i c equilibria. For studies o n t h e s e m e c h a n i s m s a suitable e x p e r i m e n t a l filarial m o d e l is n e e d e d as w e l l as n e w t e c h n i q u e s for t h e c o n s t a n t a n d c o n t i n u o u s application o f c y t o k i n e s a n d o t h e r i m m u n o l o g i cal s u b s t a n c e s into the host. M e t h o d s to m o n i t o r c h a n g e s in c y t o k i n e profiles and cell reactivity are also required.
Monanema martini in t h e s t r i p e d m o u s e , Lemniscomys striatus, is at p r e s e n t t h e b e s t n a t u r a l r o d e n t m o d e l for
* Institut fur Tropenmedizin, University of Tubingen, Germany.
** Dept. o f Pharmacology/Therapeutics, University o f Nigeria, Enugu.
Parasite, 1994, 1, I S 35
of the filaria Monanema martini in two rodent hosts : recovery rate, migration and localization. Ann. Parasit. Hum. Comp., 1990, 6 5 , 80-88.
WANJI S. : Un nouveau modèle pour la thérapeutique de l'oncho- c e r c o s e : la filaire des muridés à microfilaires dermiques, Monanema martini. T h è s e de Doctorat de l'Université de Montpellier, 18 décembre 1992, 199p.
