48 J. Apr. Mosq. CoNrnol Assoc. Vor-.2, No. l

NATURAL TRANSMISSION OF DIROFILARIAIMMITIS BY AEDESAEGYPTII

C. M. HENDRIX', C. J. BRUNNER3 .qNoL. K. BELLAMY2

ABSTRACT. The Liverpool strain of the mosquito Ardasaegypti was infected with microfilariae of the canine heartworm, Dirofilaria immitis, and was used to transmit heartworm larvae to three dogs. Methods of confirm- ing heartworm infection in these dogs included the rnodified Knott's test, a commercial enzymelinked- immunosorbent assay(ELISA), an indirect fluorescent antibody (IFA) test, and post-mortem examination.

INTRODUCTION ously into the canine definitive host (Tulloch et al. 1970, Kotani and Powers 1982). The pur- Ludlam et al. (1970) listed 63 speciesof mos- "complete pose of this experiment was to assessthe ability quitoes in which larval development of Ae. aegypti (Liverpool strain) to transmit in- of Dirofilaria immitis has been reported." Since fections of D. immiti,sto the dog. then, an additional 9 mosquito species capable of supporting larval development of D. immitis to the infective third larval stage within the MATERIALS AND METHODS head or proboscis have been reported (Crans RrrrrNr; TEcHNreuEs.The Liverpool strain and Feldlaufer 1974, Seeleyand Bickley 1974, of Ae. aegyptiwas provided by Dr. McCall, Weinrnann and Garcia 1974, Bickley 1976, John Athens, Georgia. Eggs oviposited on moisr cot- Christensen1977, Mosha and Magayuka 1979, ton batting were hatched in 22 x I I x 5 cm Rogers and Newson 1979, Acevedo 1982, Wal- white enarnelware pans containing distilled ters and Lavoipierre 1982). However, the capa- water at a depth of 4 crn. Food for larval devel- bility of transmitting infective third stage larvae opment was a mixture of I part rat chow, I part of D. immitis to the canine definitive host by brewers' yeast and I part lactalbumin. Pupae rnosquito bite has been dernonstrated with only were collected with a llulb syringe and trans- 7 of these 72 species (Bancroft 1904, Kume and ferred to 185 rnl paper cups; the cups were then Itagaki 1955, Newton 1956, Bemrick and transferred to 30.5 x 30.5 x 30.5 cm mosquito Moorhouse 1968, Bickley er al. 1977, Christen- holding cages (Arnerican Biological Supply, sen 1977, Hendrix et al. 1980). Baltirnore MD, 21228) in an environmental Aedesaegypti (Linnaeus) has been one of the charnber at 26"C, 80% relative hurnidity. and most extensively studied rnosquito species with alternating l2-houl light and dark photo- regard to its role in the larval developrnent of D. periods. Ernerged rnosquitoes were rnaintained irnzitls (Ludlam et al. 1970). Both wlld and lab- in the holding cages on a 5% sucrose solution oratory strains of this rfosquito have been (in a vial with a cotton plug wick). studied with diverse results regarding their Ixpecrrox oF \.roseurrons. The donor dog suitabilities as interrnediate hosts (Buxton and was a 22.7 kg, rnixed-breed female exhibiting Mullen 1981, Tolbert and 1982, Johnson circulating rnicrofilariae of D. immitis on the Sauerrnan and Nayar 1983). rnodified Knott's test (Newton and Wright Although Ae. aegypti has been used exten- 1956). Her circulating rnicrofilaremia level av- sively to study the developrnent of D. immitis elaged 150 rnicrofilariae per 20 p.l of blood. To (Kershaw et al. 1953, Taylor 1960, Singh et al. encourage their feeding on the heartworrn- 1967, McGreevy et al. 1974, Lindernann 1977), infected donor dog, adult female rnosquitoes transrnission of D. immitis to dogs by bites frorn were denied blood rneals for 3 days following infectious Ae. aegypti in the laboratory has not their ernergence. Mosquitoes fed unhindered been demonstrated (Hinrnan 1935). on the donor dog's hind leg for periods ranging Researchers who have utilized Ae. aeglpti for frorn 30 min to I hr. Cages were then returned infecting dogs with the canine heartworrn have to the environrnental charnber to allow the lar- dissected infective third stage larvae fi-orn the val developrnent of D. inmitis to take place mosquito and injected the larvae subcutzrne- within the rnosquitoes -Ip.rxsrrrssroN over the next 14 days. sruDrEs. The test group of 4 litterrnate dogs, three recipients (dogs A, C and I Publication nurnber 1727. Supported in part by a D) and one control (dog B), had spent their research grant-in-aid from Auburn University Col- entire lives in an indool kennel environrnent (a lege of Veterinary Medicine. "bioclean" 2 Department of Pathology and Parasitology, Col- area). Prior to the initiation of the lege of Veterinary Medicine, Auburn University, AL study, blood sarnples fr-orn all dogs were exarn- 36849-3501. ined using the rnodified Knotr's resr and wer-e 3 Department of Microbiology, College of Veteri- negative for rnicrofilariae. Sera frorn each ani- nary Medicine, Auburn Universiry, AL 36849-3501. rnal exarnined for antibodies against adult anti- 49 Mencs, 1986 J. Apr. Mosg. CoNrnoL Assoc.

anti- gens of D. immitis using a commercial enzyme- 23 weeks postexposure and for (IFA linked immunosorbent assay(ELISA) (Dirotect' microfilarial antibodies test) at 26 weeks (4 Mallinckrodt, Inc., Bohemia, NY) and for an- postexposure. At necropsy 6 adult female tibodies against microfilariae using an indirect and 2 male) D. immitis were recovered frorn the fluorescent antibodv (IFA) test (Dawe et al. right ventricle and pulrnonary arteries of dog 1980) were antibody-negative. The dogs r,vere D. dogs transferred to individual cages in a confine- By 55 weeks following initial exposure rni- ment area with a screened entryway. Outside A, B and C had not exhibited circulating However, ELISA tests doors were locked and vents sealed to prevent crofilariae of D. immitis. A and C becatne accessof wild infectious mosquitoes to exper- revealed that sera from dogs against adult imental and control animals' weakly positive for antibodies and weeks, Transmission studies were conducted l4 days antigens of D. immitis at 30 29 sera frorn sentry following the mosquitoes' infectious blood respectively, postexposure: on the ELISA meal. One limb of each recipient dog was in- control dog B rernained negative (IFA test) serted through the accesssleeves of the holding test. Anti-microfilarial antibodies 3 dogs at any time cages and mosquitoes fed unhindered for pe- were not detected in these riods ranging from 30 min to I hr. On any during the study. I adult rnale D. immitis succeeding day following the initial 14 day de- At necropsy dog A had C had 2 adult velopmental period, if surviving mosquitoes in the right ventricle while dog No adult D. ira- would feed unhindered on the recipient dogs, fernales in the right ventricle. heart, puhnonary ar- they were perrnitted to do so. Dog A was ex- mitis were present in the of sentry control posed to mosquitoes on 7 occasions,dog C on 4 teries, lungs, or venae cava adult heartworrns occasions,and dog D on 3 occasions.Dog B was dog B nor were aberrant this control anirnal. never exposed to infected caged mosquitoes foJnd in ectopic siteswithin but rernained in the screened confinernent area with her littermates, serving as a sentry control. Pnn.pernxr pERIoD.After the initial exposure DISCUSSION period, all dogs were housed in individual cages of the in- in the rnosquito free environrnent. Beginning 3 Karnnan (1957) reviewed rnuch between weeks postexposure, sera from recipient and forrnation concerning the interactions that control anirnals were collected and frozen at D. immitis and the various rnosquito species hosts' weekly intervals for later analyses using the are capable of serving as interrnediate were ELISA and IFA tests.Three rnonths following \{uch of the data then and since then intricate the initial exposut'e, rvhole blood was collected gathet'ed in studies involving the 4e. ueg;pti at weekly intervals frorn recipient and contl'oL 6ost-p:rr':rsitelelationship between intake on anirnals and exatnined for- rnicrofilar-iae of D. and D. irnmitis: effect of rnicrofilarial 1953)' imrnitis using the rnodified Knotts test. the sulvival of the host (Kersharv et al. larvae NEcnopsr pRocEDURtls.All dogs were rnorphological changes occut-t'ing in reaction to euthanized by lethal injection of sodiurn pen- ('faylor- 1960), encapsulation, host 1977)' ef' tobarbital. Necropsy was perfortned on dog D developing larval stages(Lindernann incubation 29 weeks following initial exposure to infecti- fects o[ tetnperature on the extrinsic of infective ous rnosquitoes,and on dogs A, B and C at 55 (Singh et at. tSOZ;, and etnergence al. weeks postexposut-e. The hearts, pullnonal'y larvie florn the rnouthparts (Mc()r'eevy et of data has alteries, lungs and venae cavurof the recipient 1974). Although a large arnount surveys and control dogs were exarnined for adult D. been gatheled frorn epiderniological immitis. In addition, ectopic sites (brain, eye, (folbert and Johnson 1982, Sauerrnan and etc.) were exarnined for the presence of adult D. \ayar 1983), rnuch has been learned concern- immiti.; in the sently contt'ol anirnzll to ensule ing lalval developrnent within various labor:r- that aberlant adult healtwol'lns lvel'e not pl'e- tor:y stt'ains of Ae. aegypl1. These different' host sent (Otto 1974). str':rinscliffer- rnarkedly with regald to their efficierrcy, i.e. susceptibility velsus refi-actori- ness(Buxton and Mullen l98l). Due to these rvidesplead differ-ences, '4e. aegtpti has been RESUT-'fS utilized to study the genetic bases of suscepti- (Kaltrnan All dogs lernained healthy throughout the lrility to infections rvith D. irnmitis expelittrent. Dog D begatrto exhibit cilculating l95ii. Sulairnitnztnd'fownson 1980)' a rniilofilaliae of D. intmitis apploxirnately 28 Aedesaegypti has long been recognized.as namely in rveeks following the initial exposut'e. Set'utn model for developmental studies, larval developrnent of D. immilis to fi'orn this dog becarne positive fol antibodies suppolting 'fhese l:rlvae at'e againstadult D. irzrnlll.tar.rtigens (E[,ISA test) at the infective thircl lalval stage. 50 J. Au. Mosg. Coxrnol Assoc. Vor.2, No. I

usually dissected frorn rnosquitoes into various Nancy Hinkle of Auburn University,Alabama, (e.g., rnedia Ringer's solution, Hanks' balanced Dr. John W. McCall and Mrs. Jung Jun of salt solution or NCTC 109) and the released Athens, Georgia and the secretarialassistance infective larvae then collected and injected sub- of Ms. DianneTindell, Ms. Kris Kelly and Ms. cutaneously into recipient dogs. This was the Lisa Johnson. developmental role that,4e. aegyptiplayed in the past, a role of artificial transfer (Tulloch et al. Rderences Cited 1970, Kotani and Powers 1982). Until this re- port, this species had never been shown to Acevedo, R. A. 1982. Potential vectors of Dirofilaria transrnit the parasite successfully to the dog immitis in San Mateo Co. California. Moso. News (Hinrnan 1935). 42:272-274. Bancroft, T. L. 1904. The rnaturation of larvae to the infective On some further observations on the life history of Filaria immitis, Leidy. Br. Med. third larval stage within the proboscis does not J. l:822-823. guarantee the capacity of a mosquito species to Bemrick,W. J. and D. E. Moorhouse.1968. potential transmit the parasite (Crans to the dog and vectors of Dirofilaia immitis in the Brisbane area of Feldfaufer 1974, Seeley and Bickley 1974, Queensland, Australia. J. Med. Entomol. b:269- Bickley 1976). In Hinrnan's study (1935) with 272. an unknown strain of Ae. aegpti, the author Bickley, W. E. 1976. Failure of Culex salinarius ttt "rnaturation states that the of the larvae within transmit Dirofilaia immitis from dog to dog. Mosq. the insect host and its rnigration to the proboscis News 36:366-367. Bickley, W. E., has always been assumed to be prima evi- R. S. Lawrence, G. M. Ward and R. B. facie Shillinger. 1977. Dog-to-dog dence of ability to transmit the given filarial transmission of heartworm by Aedes canadensis. infection. Mosq. News However, it is entirely possible that 37:137-138. many blood-sucking insects, discovered to be Buxton, B. A. and G. R. Mullen. 1981. Comparative susceptible to experimental infection and even susceptibility of four strains of Aedesaegypti (Dip- found naturally infected, are unable to suc- tera: Culicidae) to infection with Dirofitaia inrnitis. cessfully transrnit the parasite. Practically all of J. Med. Entornol. 18:434-440. our knowledge of transrnission of filarial or- Christensen, B. M. 1977. Laboratory studies on the ganisrns rests upon such circurnstantial evi- developrnent and transmission of Dirofilaria immitis dence, which rnay be quite fallacious." by Aedestriaittatus. Mosq. News 37:367-372. Crans, W. and M. F. Feldlaufer. 1974. The likely In the present study, very few adultD. immitis J. rnosquito vectors of dog heartworm in a coastal were transferred to the dogs by bites fi-orn Ae. area of southern New Jersey. Proc. Calif. Mosq. aegypti.Two of 3 recipient dogs in the present Control Assoc.42:168. study were arnicrofilarernic because they har- Dawe, D. L., R. E. Lewis,J. W. McCall, C. A. Rawlings bored single sex infections. This type of infec- and B. A. Lindernann. 1980. Evaluation of the indi- tion is irnpossible to diagnose using the rnod- rect fluorescent antibody test in the diagnosis of ified Knott's technique and the IFA test as rni- occult dirofilaliasis in dogs, pp. 33-36. In: G. F. crofilariae are not produced in single sex infec- Otto (ed.), Heartworm Symposium'80. Veterinary Medicine Publishing Co., Edwardsville, tions. Single sex infections, however, rnay be KS. Hendrix, C. M., W.J. Bernrick andJ. C. Schlotthauer. detected in dogs by using the EI-ISA test which 1980. Natural rransmission ol Dirofilaria immitis by detects antibodies against antigens of adult D. Aedesaexans. Arn. J. Vet. Res. 4l:1253-1255. tmnntxs. Hinman, E. H. 1935. Studies on the dog hearrworm, This study has dernonstr-ared thar the bite of Dirdilaia immitis, with special reference to filarial Ae. aegpti (Liverpool strain) rnay be used to periodicity. Arn. J. Tlop. Med. l5:371-383. transmit D. immilis from mosquito to dog. The Kaltrnan, L. 1953. Factors influencing infection of primary role of Ae. aegypti in transmission the rnosquito with Dirofilaria irzrzitis (Leidy, 1856). Exp. studies, however, should remain lirnited to the Palasitol. 2:27-78. Kaltrnan, L. 1957. The vector production of infective third stage larvae for. of canine filariasis; a leview with special r.efer.enceto factol.s influencing use in inoculative transfer studies. These in- susceptibility. Rev. Br-asil. Malariol. Pub. Avulsas. oculative methods tend to produce greatel- 5:l-4 l. numbers of adult worrns in the right ventricle Ket'shaw, W. E., M. \{. J. Lavoipierre and T. A. and pulmonary arteries and are thus a rnore Chalrnels. 1953. Studies on the intake of rni- efficient utilization of Ae. aeglpti (Liverpool crofilaliae by theil insect vectors, their. survival. and strain) as an interrnediate host of D. immitis. theil effect on the sur.viv:rlof their vectors. I.- Di- rofilaria immitis and Aedesaegypti. Ann. Trop. Med. Palasitol. 47 :207-224. ACKNOWLEDGMENTS Kotani, T. and K. G. Power.s. 1983. Developrnental stiges of Dirofilain inrnitis in the dog. Arn. J. Ver. The authors gratefully acknowledge the Res.43:2199-2206. technical assistance of Dr. Gary Mullen and Ms. Kurne, S. and S. Itagaki. 1955. On the life-cycle of