Operational application of various identification technics of vectors and parasites of Onchocerciasis in West Africa: the 20th international congress of Florence, Italy (August 24-31, 1996)

WORTD HEALTH ORGANIZATION ONCHOCERCIACIS CONTROL PROGRAMME IN WEST AFRICA ORGANISATION MONDIALE DE LA SANTE PROGRAMME DE LUTTE CONTRE L'ONCHOCERCOSE EN AFRIQUE DE L I OUEST TIre 2 O EtL F l-orernce , I nt e r r3-El f. i o rra- l-Errtorno aogy -I ta-l- ie ( Augrrst Corf.gtress of 24-3L,L996> Opera.t iorra.l- Aa>a>l-ica.tiorr of vErr iorrs id.errtif ic€rt iorl- techrrics of vectors a.rrd. E>ErrElsites of Orrchocercia.sis rrr Trfest Africa By Drs. AKPOBOUA t.K.B, BISSAN Y TOE L HOUGARD J.M iLq .: L)-N \- 1. Introduction. Onchocerciasis,or river bI dness results from infection witOnchocerca volvuIus. The para ite is endemic to West Africaboth rain forest and savan na bioclimes. ReIiable identificatio nosibling species of Srmu l-iun damnosom complex and Onchocercvolvulus parasite is essential for an understandin gofthepidemiology and transmis sion of human onchocerciasis.fn west Africa, the onchocercal blindness rate can reach as high as158 in the savana, with 1008 infectivity (Duke ,1990). The majorpatterns of epidemiologica1 variation are relat ed to the taxonomyof the parasite and the vector ( Post and Boakye, 7992), and so notall sibling species are equally importantVector taxonomy is based upon the analysis of the polytenechr omosomes from Iarva I silk gland, but there is a clearrequirement for the iden tification of adu lt female, because it isthis stage that actuall y transmits the parasite. This requirementhas long been a priori ty of the World Health Or ganaization (WHO,1,978) and a subjedt o f intensive study.Beside the cytotologicalidentification of 1a rvae of -q. dannosun s. I. , morphologicaltechniques have been found to be the most successful in terms ofspecificity, convenie nce and cost. Specific DNA probes developpedby Post and F1ook,19 92; Toe et aI. Lgg4; Tang et al.1995 ) haveprovided a tool for an accurate identification of the adults ofS. damnosum complex and a wide range of operationnal use of t heentomological parameters ln West Africa, onchocerc ias istransmission is broadl y divided into two different types. In theforest zones, high loads of the filarial wo rm undistingishable fromO. volulus, may be car ried by forest vect ors of ^9. dannosum s.1complex but severe ocu1ar symptoms are rare. In the savanna zonesthe savanna species o f the complex gener a1ly have lower parasit eloads but even compara tively moderade leve 1s of transmission can beassociated with blindness (buke, I96ga and b, Philippon ,L977 ) . Thepresence of at 1east two strains of the parasite is believed toaccount for the differences in pathogenicity ( Duke,19g1 , Dadzie etdl. , 1989 ) . Because of the diff erences in severity of O. volvulus_induced ocular disease in the rain forest and savanna, the OCp hasconcentrated its efforts on savanna regions (Le Berre et aI.1990 ,1 .\./" I I tL ) h n t a e Entomologic ( Duke et al . j_966 immunologic ( Lobos and Weiss ,1995 )and genetic (Flockhart et aI 1986; Ertmann K.D et a1. 7gg7 ) datahave accumulated which su9gest that distinct populations of theparasite exist in the rain forest and savanna. In additio nepidemiological studies have shown that distinct patterns ofonchocerca-induced ocular disease exist in different regions ofWest Africa. The development of onchocerca DNA probes (zimmerman et a1-1993;Ertmann et aI.1990 ) has been particurarly useful in separatinginfective stage of onchocerca ichengi (an animal parasite) and o.volvuLus which are transmitted by t-he 'same members of s.damnosuncomplex ' The operational implications for the control tperations inOCP of this new developement are highlighted. 2. Techniques used for vector identification a) Morphological techniques At least nine sibling species in the SinuLium dannosum s.1. species complex have been identified, based on the morphology of adultflies, and on the polytene chromosome patterns of mature Iarvae.(Procunier, 1989; Boakye, L993;.. Wilson & Post, t994). Morphological characters txonomj.c value like colour of basal wing tufts, antenna, fore coxae, tergal hairs on the 9th abdominal segment are routinely used in OCP to separate ,S. damnosun complexin three groups: savanna f lies ( S. srr.banum and S. damnosum s . str . ) ;females of S.yahense and females of Sou/Sq group including ^S. sanctipau-Z:. and S. squamosum sub-group. Morphometrc methods modified by WiIson ( 1993 ) a1lows the differentiation of S. damnosum s. s from S. sirbanum in the savanna species and S. squamosum from Sanctipauli subcomplex. Although this technic is an improvement over the morphological methods t a certain amount of overlap between S.squanosum and forms of S.soubrense are found in some areas. b) Technic of Electrophoresis. Starch-ge1 electrophoresis Meredith and Townson (1981) showed that adults of S.yahense and S. squamosum can be distinguished from each other and from the rest of S.damnosum species in West Africaby differences in the mobility of the enzymes trehalase and phosphoglucomutase trehalase. This technique is used in areas where S. squamosum shows morphological variation which overlap with those of both savanna species and members of the S.sanctipauli subcomplex(Garms and Cheke, 1985), thus confusing the separation of savanna and forest species. A field electrophoresis facility to identify S.squamosun, fu1ly portable, battery-operated system is available(Townson and Davies 1989). This technique is used on very rare occasion. c) Identification by DNA probes A recent work has shown that polymerase chain reaction (PCR)- based directed heteroduplex analysis (DHDA) may prove to be useful tool in distinguishing the sibling species of S.damnosum s.I. ( (Tang et a1. 1995). The method relies on the detection of sequenceheterogenicities present in a subregion of the mitochondrially encoded NADH dehydrogenase subunit 4 ( N D4 ) gene.The tehnique is based on the electrophoretic retardation of the complex formed by one chain of DNA considered as probe with an unknown chain to be identified ( figure 1 ) . Operationally used bythe OCP since may 1995, it allows the identification of six species of S.dannosum complex namely S.dannosum s.s., S.sirbanum (savanna spcies ) , S. leonense, S. sanctipauli, S. squamosun and S. yahense ( forest species ) using material derived from a1I stages of Sinuliunflies from egg to the adu1t, even from small fragments (part of antennae etc...). This technique is presently used to identifyfemales in the intermediate forest/savanna zones where morphologlcg,N/norphometric methods of identification methods are / l"/ not reliable because of the overlapdifferentiate S.squamosum from species of and to a less extent from savanna species. of criteria used to Sanct ipaul i sub-complex d) Cytotaxonomy S.dannosum complex, the main vectors of human onchocerciasisin West Africa have been found to be a complex of sibling species,described on the basis of the interspecific inversron differencesin the banding sequences of the rarvar sirk gland polytene chromosomes(Vajime and Dunbar, L975). Nine species are described inthe area covered by the World Health Organization's Onchocerciasis contror Programme in west Africa. These are, s, squamosum, s. yahense, s. sanctipauli, s. soubrense, s. damnosuln s. s, s . sirbanum, s. leonense, s. konkourense and s. diguerense ( vaj ime,19g9 . ,Post ,L986. , Boakye et aI.1993 ) . e ) Concordance between the results obtained with the varioustecniques. rn most cases, there is a satisfactory agreement on the results obtained using the different techniques described above( fisure 2) 3. Operational use of the techniques of identification of vectors a )-Morphological methodsThe geographic boundary of the operational zone of theprogramme was based on the distribution of the savanna speciesknown to be the most important vecto of human onchocerciaiis inWest Africa. A seasonnal variation of simulium flies population atall the catching of the entomological network is now available(Figure 3). It aIl-ows a more accurate prediction of seasonnal changes of various species and the periods when the transmission is1ikely to occur. The morphological identification of vector widety used in OCpsince 1993, is essential for: a better evaluation of control operations directed against S.dannosum complex vectors. It aIlows a constant recording of thesensitivity of the vectors to the various larvicides used, theresulted changes in the species composition. - an assessment of the vectorial role in natural conditions of allthe species involved in the transmission of humanonchocercisis(tabIel). This information is used to develop optimaltactics for vector control and possible combined insecticiae/arugtreatment strategies. b) Heteroduplex analysis of DNA The improvment of the morphological identification and thepossibility of using molecular biology techniques have contributedto: - provide the opportunity to identify all stages of the vectors. - increased the reliability of the parameters collected from theentomological network. It is a useful supptementary method used to,identify the infective flies in intermediate torest/savanna areas where there is an overrap in morphorogical characters. "jr{) /, )- a more accurate assessment species in natural condition. of vectorial role of differents c ) Cytotaxonomy The cytotaxonomic identification of larvae in the S. damnosum complex, is not only the most reliable method available, but alsothe reference for the development of alI other techniques. In factthe backbone of 1arval taxonomy is cytotaxonomy, to which morphology shourd arways be related. This technique irlows, - successful management of resistance depends on rapid cytotaxonomic identification of larvae which survive diagnosticdoses in susceptibility tests.Epidemiologically good results havebeen maintained in spite of resistance, but -at considerableincrease in the cost of treatment. Karyotyping may also reveal characteristic inversion patterns which could be used to trace the movements of resistant poputation. - Full caryotyping of larvae is time consuming but has been usefulfor the detection of hybrids and backcrosses, and for tracingpopulation movements using polymorphic inversion frequencies. It i;particularly useful in the study of the spread oi insecticideresistance, and reinvasion. - a good assessment of the distribution of species in the programmearea, the seasonal variation as well as the changes inthe vectorpopulat ion due to the impact of rarvicides . ror instance , ,s.soubrense (Milo form) has been eradicated from Guinea after fiveyears of larviciding. 4.Technique of identification of parasites. Polymerase chain reaction (PCR) based methods have beendeveloped to separate infective O.ochengr from O.vojvulus that aredifficult to distinguish morphologicaly. In the past several years, DNA probes have been isolated which demonstrate varying degrees oispecificity for onchocerca parasites. Some of these probes arespecific for the genus Onchocerca, while ot,hers are specific forO.voivuius, or for distinct strains of o.volvulus namely the forest and the savanna strains ( Meredith et a j. 1999 . , l,teredith et df . ,199L; Zimmerman et df. , 1993 ) .The identification of infective larvae from infectious flies using DNA probes based on the Southern blot and ELISA tests ( figure4\ are being carried out in ocp since Lggz. Four DNA probes areavairable for operqlionnar use within the programme. Two arespecific for the genliBt\Onchocerca, one of which is specific foro.volvulus (probe ovsz) and the second, specific for o.ochengi(OCH). The last two are specific for the forest strain (probe pF!-1) and for the savanna strain (probePSS-1BT) of o.vojvujis. Variousstages of the parasite in the vectors are currently identified inthe Programme as well as parasites (micrifitariae fiom skin snipsand adults worms extracted from nodules ) using these probes. rF- Vrtl't T-L,"",,, -,/t y.r^ )5. Operational use of the identificatioi'^ of the parasites. The recently oevelopped technique of identification of theparasites by DNA probes has contributed to: - a refine evaluation of the effect of ivermecti-n chemotherapy on transmission and epidemiology of the disease. - More accurate estimation of ATP. The Onchocerciasis Control Programme (OCP ) ,monitors the effectiveness of its efforts by measurement of the annual transmission (ATP). Calculation of ATP invclves estimation of the number of parous flies found to be carrying O.volvulus infectivelarvae in a given area. Since O.voLvulus and O.ochengj are co-endemic in the control area of OCP/ are transmetted by the same species of S.dannosum s.1.(Omar et aI. 1979) and are often morphologically indistinguishable(Omar et. al-. L979.,McCaI1 et. aI.1992), O.ochengr presents a chal-1enge to an accurate estimation of the ATP for O.voTvulus. Thisproblem is IikeIy to become more acute as active vector control isphased out within the OCP control aera. In such areas, it is expected that the blackly population wiII rapidly increase and thepresence of O.ochengr in cattle found in these areas will lead to a concomitant increase in the transmission of this parasite. The ability to characterLze individual infective Iarvae using DNAprobes that hybridze specificaily to O.volvu1us has help to overcome this probleme. It will also have value in efficiently allocating scarce resoures during the final years of OCP's operation, as well as in the years following the end of large scale vector control efforts in the OCP area. - an estimation of two types of annual transmission potential(ATP)by a combination of morphological identification of the vectors and the identification of the parasit.e by DNA probes are currently been used to monitor the success of control opreations in the OCP area. The first of ATP takes into account only the transmission of onchocerca volvulus by all species of S.dannosum complex, and the second type with the transmission of O.vol-vuJus by the savanna flies (figure 5 and 6). a better assessment of the vectorial dannosum species in natural conditions. capacity of Sinuliun l/ the proportion of animal parasites that coexist with humanparasite on various river basins within the programme ( figue 7 ) . The confirmation of the correlation between classification of O. volvuius by DNA probes and epidemiological patterns of Onchocercal blindness.(figure 8 shows the distribution of savanna and forest strains of O.volvulus in the OCP area). t/ DNA villages located in probe analysis of samples collected from both the rain forest and savanna bioclimes and e) in the transition zone between the two has shown a strong correlation ( P< . oo1 ) between disease severity and probe recognition, supportinng the hypothesis that pathogenicity is strain related ( Zimmerman et aI. 7992) . Results obtained by the same authors suggest that it may be possble to use the strain- specific DNA probes to predict the pathogenic potential of theparasite throughout much of West Africa. - Determination of the prevalence of Onchocerca volvuius infectionin the vector populations by polymerase chain reaction of pools of V/black fIies. It is often difficult to measure the prevalence of infectionin a vector population, because this value is often 1ow, requiring the examination of a large number of individual insects, specially in onchocerciasis freed zones. O.voJvu-Zus-specific PCR assay that is adapted to detect a single infected blackfly in a pool of 100 flies is been developedin OCP in collaboration with Dr Unnash from university of Alabamain Birmingham.Results obtained from several batches are tested with the binomial equation developed by Dr KatoIi (university of Alabama, Birmingham) for determining the estimated probability offinding females infected by Onchocerca sp. This parameter is usefulin the assessment of the effectiveness of control mesures and the recrudecsence of the infection in the onchocerciasis freed zones. The operational use is being tested on Bougouriba river basin in Burkina Faso where recrudescence of infection has been detected. the objective is to verify the leveI of transmission of O. volvulus in the area using the classical method (catches/dissection) and to compare the results obtained with this method to those provided by DNA probes. - Identiflcation of O. volvulus infection using specific DNA-probesis being tested on samples of skin scratchings and on skin snips in OCP.Preliminary results are promising but the protocoles need to be refined and standardized. It is planned to adapt the heteroduplex analysis of DNA to theidentification of Onchcerca parasites in order to undertake aphylogenetic analysis savanna (blinding) strain and forest (lessblinding) strain of O.voLvulus. DHDA may become a useful tool in studing the vector-parasite relationship. 6. Conclusions The important effort put into the identification of the vectors of onchocerciasis since the beginning of the OCP has Ied to a betterknowledge of the bioecology of the Simulium flies. Theidentification of the parasites of Onchocercq species and strains of O.volvulus strainsahas helped in making imbrtant saving without loosing the efficacy and the quality of the control activities. The set up of an operational molecular biology Iaboratory in OCP has been a major achievement in the sence that shows how an advanced technology can be applied to solve operational problemes in a large *Y control programme such as OCP. i{", ^^}'r , d'} *".* l*,J nr L", * I fl k r,lr-^^ .-, HJ ,,prs.[v..\ tt-,.<) th*]'it + /] ';r"i'L6) '*r\r\&t+t , ' a ,L-t-,L{-* iL,r*nd.Q., rT r!,v i7y* : ,.) t"'*J't t& 'r, .:.4. ,., ru\rt\l o.'r }l-' ,llo"'L't"'*^'iul,,,tr^ }t* l',""'n'L t; n, r*r. i- . , "ir*'+ \i li' * * "t" "" /fjri" \n r ca ,nn^ d, ^. +F il J *..*'"'j '.^ri V\.*rL In^ri+"'r" i'tv tt t "1 '^"^ "l i{^+ .-';. ..* ^r.*1 *L^,*, L,r, t...t . lVt( +& nr. , l*L oaa / *.1.r rr.r-r. I q..t r tJ d la ]r"/fnh t,r* ^{rtl tfl +t'! uttvl*l ai,"',],j . l" '", . $a"*L' , Ji *, l'-l "r. .*l,n+-rr t / 'ft d ur^ I ( C) P l-Ern o f E>re s errta_t ion operationnar use of vector identification techniques I. Cytotaxonomy Principle - L slideSpecies identified 1 slideOperationnal application - L slide 2. Morphology Principle - 1 slide species identified 1 slide operationnal application - 1 slide 3. HDA (Heteroduplex Analysis) Principle l- slideSpecies identified 1 slide operationnal application 1 628-634. 4. Summary of all techniques slide 1 slide Operationnal use of parasites identification techniques 1. Identification of O. volvulus and O. ochengi by DNA probes - Principle ( 1 slide )Operational use ( 1 slide Showing the distribution of each species throughout the Programme area ) - More accurate assessment of ATP 2. Identification of savanna and forest strain of O. volvulus strain forest 3 worms strain - Principle - 1 slide showing the distribution of savanna and forest of O. volvulus in the programme area. - 1 slide showing the coincidence with blinding oncho and ( less blinding oncho ) . . Identification of microfilatiae from skin snips and adult from excised nodules. - 1 slide showing the distribution of forest and savanna of O. volvulus.