L;s

II Transmission of Hog Cholera Virusby I-Iorseflies (Tabanidae: Diptera)Mac A. Tidwell,Ph.D.;W.D. Dean,D.V.M.;MargaretA. Tidwell,M.S.;Gary P. Combs, D.V.M.; D. W. Anderson, D.V.M., M.P.V.M.;W. O. Cowart, D.V.M., M.S.; R. C. Axtell, Ph.D.

SUMMARY

Epidemiologic evidence indicates that insects, specifically flies (Diptera) .may disseminate hog cholera virus (HCV). Two species of horseflies(Tabanidae), Tabanus lineola Fabricius and Tabanus quinquevittatusWiedemann, experimentally transroitted HCVto susceptible swine within 2 ,

hours after biting a virus-source pig. Three other Tabanus species were in-criminated. An apparent 24-hour 'delayed transmission of the virus by horse-flies occurred. Transmission attempts using 6 species of mosquitoes wereunsuccessful.

Laboratory diagnostic tests used for the detection of HCV were thefluorescent-antibody tissue section technique (FATST)for tonsillar tissue andthe fluorescent-antibody cell culture technique (FACCT)for splenic tissue.The fluorescent-antibody serum-neutralization test (FASNT)was used for thedetection of serum antibody against hog cholera (HC).

Received for publication March 3, 1971.From the Animal Health Diviaion, Agricultural Re-

search Service, U.S. Department of Agriculture, Raleigh,N. Car. 27601 (Dean and Andel'$On), Hyattsville, Md.20782 (Combs), and Elba, Ala. 36323 (Cowart); and theDepartment of Entomology, North Carolina State Uni-versity, Raleigh, N. Car. 'l:l6fY1 (Tidwell, Tidwell, andAxtell). Dr. and Ms. Tidwell's present address ia De-

'partment of Zoology and Physiology, Louisiana StateUniversity, Baton Rouge, La. 70803.

Paper No. 3402 of the Journal Series of the NorthCarolina Stat., University Agricultural Experiment Station.

Supported in part by the North Carolina Sea GrantProgram.

Presented before the Section on Regulatory VeterinaryMedicine, 108th Annual Meeting of the AVMA, Detroit,Mich., July 18-22, 1971.

The authors thank Dr. W. C, Stewart, Virology Section,DiagnOlltic Services. National Animal Disease Laboratory,Am@~. !QW!!, 19r n@lpM lnftlrmtUlollllftd tUlllfft08ltO 1M-sisli\n~, TII" IIY!l1gm at!!II 1I(!!m(fflII!~ lliIJ. T. D,Ryan, North Carolina l)lagnostic J,abo1'll~1'I@jj, Rl!14;ijJh,N. Car" and G. L. Hunnicutt, Ed1!uton Ditlgnostic Lab.Ot'dtory , Edcnton, N. Car" for diagnostic seiv:ices; Dr.J. M. Falter, Entomology Department, North CarolinaState University, Raleigh, and Mi. D. W. Anthony,Entomology Research Division, ARS, USDA, Gainesville,f'la.. for helpful Suggestions; and Mr. H. S. Hofler andSons, Sunbury, N. Car., for making available propertyfor use as the teat site.

March, 1972

Little research has been conducted onthe possible role that insects have in thetransmission of Hcv,1,a,6,7Dorset et al.4were able to contaminate houseflies andstable flies with HCVby placing them incontact with, or allowing them to feed on,infectious eye secretions or blood ofaffected pigs. Flies were homogenized insaline solution and injected in susceptiblepigs. Transmission took place most often

. during the first 24-hour period, althoughone transmission was achieved 7 daysafter a fly had become contaminated.Tl'anllmissian without injection was sue.cessful wh@1'\houseflies previously e]t~posed to eye secretions of affected pigswere touched within 24 hours to the eyesof susceptible pigs. The bite of the stablefly resulted in transmission for periodsup t{) 24 hours.

615

TABLE I-Tabanidae (Horseflies and Deerflies) and Culicidae (Mosquitoes) Collected onPremises Known To Have Herds Infected with Hog Cholera-

Species

Percentageof total"collected . Species

Percentageof total..collected

1.3

TabanidaeChlorotabanus

crepu.cularis (Bequaerl)

Chrysops{lavidus Wiedemannhinei Daeckereicherli FairchildvitlatU8 Wiedemann

1.30.40.60.6

Diachlo,u.lerrugalus (Fabricius)

TabanU8americanU8 ForsleEalrotu. Fabriciuscalens Linnaeusluscico.tatus Hinegladiator Stonelineola Fabriciusmolestu. Saymularis Stonepeliolalus Hinequinquevitlatus Wiedemann.ubsimili. Bellardi.uleilrons Macquartzythicolor Philip

0.2

1.50.50.45.26.2

43.60.71.42.9

16.914.51.70.1

CulicidaeAedes

atlanticU8 Dyar .& Knabcanaden.i. (Theobald).ollieiton. (Walker)vexano (Meigen)'

9.63.80.44.2

Anophelesc,ucions comple..quadrimaculdtU8 Say

Culexpipiens comple><sa.inarius Coquillett

2.50.8

J9.612.5

Culisetamelanura (Coqnillett)

Psorophoraciliata (Fabricius)con/innis (Lynch-Anibalzap)lero% (Humboldt)

0.8

0.840.4

4.6

'-'-.Gales and Perqnimans Counties, N. Car., Aug. 26-8ept. 3, 1970. .. Total number of tabanida, 839;total number of mosqnitoes, 210.

Recently, dissemination of HC by in-sects has been suspected due to an ap- .parent increase in the number of explosiveepizootics in localized areas. Character:'istically, this localized .spread cannot beassociated with any known transmissionfactor, and its peculiar nature promotesthe suspicion that other factors are re-sponsible. Hog cholera eradication teamsin North Carolina and. elsewhere fre-quently noted a pattern in the localized'spread of the disease. Cholera was oftenfirst detected in herds located at themargins of woods or swamps. Its spreadusually followed the courses of forestsand streams. Most area spread occurredduring June through September, theperiod of fly abundance. Suspicion ofinsect transmission was increased by ob-servations of horseflies, deerflies, andmosquitoes in the vicinity of swine herds.Composite suspensions .prepared fromsome of these flies were injected inspecific-pathogen-free swine. Of 3 swine,2 developed HC, and the viral isolatesfrom these swine were of low virulence.

Horseflies seemed to prefer larger swine,and most often, adult swine were the first

616

affected with HC. Horseflies seen feedingon swine in herds test positive for HCoccasionaUy included a specimen(}fTabanus american us Forster and Ta-banus atratus Fabricius. The flies wereseen feeding most frequently on the back,head, and ears. Closer examination re-vealed T. quinquevittatus (approx.4 perswine) on the ventral surfaces, especiallyon the udders of sows. Tabanus lineolaaveraged less than 1 per swine and wasnot observed to have a feeding site prefer-ence. Two specimens of Tabanus mo-lestus Say landed on the backs of theswine. These observations were made inSeptember during the final portion of thefly season. The abundance of flies atthis time would not be indicative of thenumbers or species during other portionsof the season. Horsefly and mosquitospecies collected from premises wherecholera-infected herds were located arelisted (Table 1).

In light of (1) the field observations,(2) the suspected role of insects, and (3)the substantial ,numbers of both indi-viduals and species of horseflies andmosquitoes in the vicinity of cholera-

Am. J. Vet. Res., Vol. 33, No.3

infected herds, t~ present study' wasmade to determine the capability ofhorseflies' and mosquitoes to trans!IlitHCV.

Materiafi and MethodsExperimental Housing.-The site of the

experiment was the former Naval AuxiliaryAir Station near Edenton, N. Car. On anisolated portion of the air base. a sedel;; of5 bomb bunkers were utilized as sheltersfor' some of the experimental pigs. Thepigs were individually kept in 14 pens 0.2by 1.2 by 1.2 m.) with fine 32-mesh screentops. One pen was in each of 3 bunkers.and 2 were in each of 2 large bunkers. Theremaining pens (with plastic rain covers)were placed outdoors 20 m. apart. An in-sectary and a fly-feeding house (2.4 m. longby 1.2 m. wide by 2.1 m. high) had fine-mesh screen on the top and on 3 sides. Aquarantine house (3 by 3 by 2.1 m. high)held the virus-source pigs and was located80 m. from the nearest experimental pigs.

Pigs.-Ten susceptible pigs were obtainedfrom outside the areas containing knownHc-infected pigs. Blood samples and ton- .sillar biopsies from these 10 pigs were sub-mitted for FASNTand FATST. Additionalpigs were subsequently obtained from thesame source. Three infected pigs were ob-tained from a local herd determined to betest positive for HC. These 3 pigs hadclinical signs of HC. and tonsillar tissue wastest positive by the FATST. Additional in~fected pigs were acquired to provide a con-tinuing source of the virus.

Quarantine Security Measures.-All footgear was cleaned and disinfected, and dis-posable gloves were used or hands werewashed in disinfectant between the han-dling of each pig. Precautions were takento prevent the accidental entry of insects-into cages when doors were opened. Cover-alls worn while in contact with contam-inated pigs were removed and placed inplastic bags for laundering. All straw bed-ding and other wastes were incinerated orburied. A minimal distance of 20 m. wasmaintained between each pen, except whensusceptible pigs. designated as controls.were placed in the same bunker with aprincipal pig.

CoUection of Insects.-Only female horse-flies and mosquitoes were used in the trans-mission studies, as blood feeding is limited

March, 1972

to this sex. The flies were collected fromareas lacking swine. Horseflies were col-lected primarily by the USe of. a modifiedManitoba f;mp similar to one. described byCatta." The horseflies were transferred in-dividually to 155-Gm. vials with gauze lidsand were placed in an insulated cooler.Mosquitoes were collected with an aspiratorand transferred to a 450-ml. freezer cartonbefore being placed in the cooler. At theinsectary (24 C.). the mosquitoes were keptin individual glass vials (13.6 Gm.) withgauze covers and containing a cotton padsoaked in 10% sugar solution.

Insect Feeding Technique:-The pig usedas the source of virus was tied on a restrain-ing table. In' general, the infected pig usedfor insect feeding was one that had. thehighest fever and maximal leukopenia. Forfeeding, the horseflies were transferred fromthe 155-Gm. vials to shorter, wide-mouthplastic cups with index cards used as covers.The cup was inverted on the pig, and thecard was removed. To induce the fly tofeed on the pig, the surface of the skin wasmoistened with water. and occasionally, CO,was provided by holding a small piece ofdry ice near the plastic cup. After feeding,the fly was transferred to an ovipositionchamber. This chamber was a 155-Gm. via!a quarter filled with moist sand and coveredwith a piece of gauze. A cotton pad im-pregnated with a 10% sugar solution wasprovided. and a sprig of grass was placed.in the sand for use as a site for oviposition.After the eggs were deposited, the fly wasgenerally receptive for a second feeding.Approximately 20% of the flies fed whenfirst offered the blood source. Those fliesinitially refusing to feed were retained andrepeatedly offered a blood source until theyfed or were discarded after 5 or 6 unsuc-cessful attempts.

The individual mosquitoes were fed di-rectly from their vials which were invertedon the pig. allowing the mosquito to probethrough the gauze cover. After a bloodmeal, the mosquito was transferred to" a58-Gm. vial for oviposition and WaS pro-vided with moist sand, a layer of dampfilter paper, and a IIUllu' water pad.

Transmission Experiments.-Jnsects wereallotted to 3 experimental groups.

Group 1 (Immediate Transfer FeedingGroup) .-Insects in this group were inter-rupted during feeding on a diseased pigand were removed before a complete blood

617

meal was taken. Within 2 hours, msecls horseflies had fed contracted HC andwere allowed to complete their meal on a . were shown to be test positive by thesuscep?ble. pig. .Four pigs were ~edto FATST.One pig (No.6) on which mos-hors.efliesm this .manner, and .1 pIg was quitoes had fed' remained healthyexposed to mosqUItoes. throughout the study and later provedGroup 2 (Twenty-Four-Hour Delayed . . .. h IIFeeding Group).-Flies in this group were. ~usceptIble to the VU'USIn the ~ a engetreated like those in group 1, except that a moc~lum. . .24-hour delay was imposed before feeding PIg 4 was bItten by 34 horseflies (dayswas resumed on a susceptible pig. Three 0 through 5) representing 4 species, in-pigs were exposed to horseflies jp this eluding Tabanus calens Linnaeus (2

pilini1er. .. .. . . ~pecimens),T. li~bla'n9'speC~mens),T.. Group 3 (~ostov~posltIon Feedmg quinquevittatus (11 specime'ns) , andG~up) .-Insects m this ~roup. were per- Tabanus sulci/rons Macquart (2 speci-~tted to fee~ to completion eIther ~n. a mens). These flies had been exposed tcivlrus-sou:ce pIg alone or ?n bo~h a VIrUS- d' sed igs 1 and 3 (Table 2). Firstsource pIg and a susceptIble pIg. In the !sea p .latter case, these were insects which had SIgnS of the dIsease were observed ~n daybeen used in other transmission experiments 9 when body temperature of the pIg was(group 1). After a complete blood meal 40.6 C. On day 11, th.e virus was de-was taken, all insects in this group were tected in tonsillar tissue by the FATST,placed in chambers to deposit eggs. After. and the white blood cell (WBC) count wasoviposi~ion, t.hey were alIo.wed to bite a 7,500jcmm. Necropsy on day '12 re-~usceptIb~e pIg. I~ a .few mstances, some vealed ecchymotic and petechial hem-msects did not OVIpoSItan~ were allow.ed orrhages of the diaphragmatic lobes of!o feed on the susceptible p~g after a mm- the lungs petechiation of the surface ofIIDal lapse of 4 days. One pig was exposed .' .., .to horseflies in the manner just described. the kidney, splemc mfarctIon~ and penp~-A 2nd pig was exposed only to the flies eral congestIOn of the cervICal, splemc,which had fed on the 1st pig and had ovi-superficial inguinal, and popliteal lymphposited a 2nd time. A 3rd pig was exposed nodes. The spleen contained HCV as "de-to 1 fly which had fed on the 2nd pIg an,cJ termined by FACCT; 4; plaque-forminghad oY!I>,~i~~ a 3rd time. A 4thi~!~'«~~himits (PFU) per IUi,' . .i~t~ detected.e~0~~';'U;~"ffl9squitoes, most off:~~!~~~~~iJ't'c-: Pig 9 was bitt~"'¥"i~: : If4eola (dayOVIpo§lfedOnce. . "0) which had fed'hnvli'us-source pig 16

ControlPigs.-Four pigs weremaintained (Table 2). At 4 days after transfer flya~ controls. .~o were.submitte~ on 6 oc~- feedings, the WBCcount was 8,lOOjcmm.,slOn~to a .sImilar regImen .as pigs ~ed m and on day 6, HCVwas detected in tonsil-the Im~edlate ~nsfer feedmg ~xperIm

.

e

.

~ts. lar tis.

sue.

bY the FATST. Ond8'.t~

..";,

.7, pig 9That IS, horseflIes and mos

. qUltOes whIch t f d t th qu rant1tte househad fed on or had otherwise had contact was rans, erre 0 e. a .with virus-source pigs, were placed in con.;." t~ repl~msh thesupp~y of vm~s-sourcetact with, but were not permitted to feed. pIgS. SignS of HC contmued until day 21on, these 2 control pigs. The 2 remaining. when body temperature decreased tocontrol pigs were not exposed to insects 38.9 C. A WBC count taken on day 26but were kept in pens adjacent to 2 of the was 20,lOOjcmm., and tonsillar tissueprincipal pigs. was test negative for HCV. This pig was

Challenge Inoculation.-All pigs remain- resistant to challenge inoculation withing at the termination of the study were the Ames strain of HCV, and splenic andmoved into a large bunker 33 days after tonsillar tissues were test negative forthe lOBitrAMf,1'1nHot feeelm,.. Eaohpf, Hav on the 7th day p()IIUnocu1ation~Tnt!was intramUijc~~rlY jf1t)culated ~ith 2 ml. postmortem examinatlo'l at this time re-of serum contammg the Ames stram of HCV. vealed slight pete~hiation of the heart

" and urinary blaflQ. JiL2~peripheral.Resu1ts . ., ~.ton(Yestion of thei£a aF@d lumbarPigs on Which Group 1 Insects Fed.- lym;h nodes, and a sPIeneticuinfarct.

Four pigs (No.4, 9, 10, and 13) on which. Pig. 10 was bitten by 5 T. lineola (day

biB Am. J. Vet. Res., Vol. 33. No.3

TABLE 2- Transmission Results Obtained by Transfer Feeding of Horseflies and Mosquitoes(Group 1) Within Two Hours of a Partial Blood Meal on a Hog Cholera-Infected Pig

No. ofdaysuntilfirst clini-cal signs

-Suscep-

White tible toblood cell c:hallenge

count inocula-(No./cmm.) tion.

No

y""

'....

No, in parentheses =No. of days after transfer fly feedings; T, = Tabanu.; A, = Aedes; P. ="P.orophora; ,'ATST= tluorescent-antibody tissue section techDlque.

.. Remained healthy,

0) which had fed on virus-source pig 2 (days 0 through 13) representing 6 spe-(Table 2). At 9 days after transfer fly cies, including Aedes atlanticus Dyufeedings, tonsillar tissue was determined and Knab (8 specimens), Aedes intir-to be test positive by the FATST,and mat us Dyar and Knab (2 specimens),clinical signs seen at this time included Aedes sollicitans (Walker) (35 sped-body temperature of 40.1 C. and WBC mens), Aedes taeniorhynchus (Wiede-count of 8,500jcmm. Necropsy on day mann) (10 specimens), Aedes triseriatus, ,10 revealed petechiation of the urinary (Say) (3 specimens), and Psorophorabladder, infarction of the spleen, and 1erox (Humboldt) (13 specimen~'iTheseperipheral congestion of the renal and mosquitoes had fed on virus-source pigshepaticlymph n()des. Hog cholera virus. 2, 3, and 16 {Table 2). Pig 6,11~mained(28 PFU/ml,) was isolated from the' healthy throughout the st:udy}~d ton-spleen bythecFAccT. "<sillar tissues examined by'the~A'l'§T were

Pig 13. WaS'bitten by 20 T. quinque-\"..test negative for the ~§;/-'~ pigvittatus (day 0) which had fed on virus-iji'~later proved susceptible to .me.viros usedsource pig 16 (Table 2). At 7 days after for challenge inoculation.transfer fly fee dings, HCV was detected Pigs on Which Group 2 Insects Fed.-in tonsillar tissue by the FATST.The WBC Pigs 21, 25, and 27 apparently remainedcounts decreased from 15,800/cmm. on healthy after transfer fly feedings. Pigday 0 to .7,600/cmm. on day 8. Tem- 21 was bitten by 6 flies (days 0 throughperatures mcreased from 39.6 C? on ~ay 3) representing 3 species, including T,7 t~ 40.6 C" on day? and remamed h~gh lineola (2 specimens), T. quinquevittatusuntIl the pIg ~as killed and necrops~ed (3 specimens), and Tabanus nigrQ.yit-on day.11. L:slOns seen on necropsy ~- tatus Macquart (1 specimen). TI1eseeluded mfarctIon of the spleen, petechIa- flies had fed on virus-source pigs 9 anddCnt gf thQ urinary bladder, plilripheral 22 (Table) 8). PiS 21 remained hGaltbyoongellt!C')nof the mandibultn.. and in.. and IIItel' proved BU/itioptibleto the virusguinal lymph nodes, and congestion of used for challenge inoculation.the. visceral and parietal surfaces of the Pig 25 was bitten by 2 T. quinquevit-stomach. The spleen contained HCV (8 tat us (day Q) which had fed on virus-PFu/ml.) 'as determined by the FACCT. source pig 23 (Table 3). Pig 27 was

Pig 6 was bitten by 71 mosquitoes bitten by 2 T.lineola (day 0) which had

March. 1972 619

Suscep- No. of Species Resultof Viros-libJe pig insects of in- PATIn souroe

No. biting sects (tnnsil) pig No.

{

2

T. Calens }

4 19 T. lineolaPositive 1 and 311 T. quinquevittalus

2 T. sulcifrons

9 5 T. lineola Positive 16

111 5 T. lineola Positive 2

13 20 T. quinquevittatua Positive 16

f

8 A. atlanticus

}

2 A, infirmatus

t; 3S A. soUicitansNegative 2. tI. and 16I 10 A. taeniorhynchus

;I A. triseriotusI 1:1 p, fero%

4409 7,600 (11)

.. 8.100 (4)

9 8,500 (9)

7 7,600 (8)

..

TABLE 3-Transmission Results Obtained by Transfer Feeding of Horseflies (Group 2) After~ T wenty-Four-Hour Delay Following a Partial Blood Meal o.n a Hog Cholera-Infected Pig

Suscep-tible to

chaUengeinocula tiOQ

Suscep. No. o!tible pig insecta

No. bitingSpeciee

of insect

Feedingperiod

in days

VirwJ..8OUlC8

pig No.Healthstatus

TABLE 4-Transmission Results Obtained by Transfer Feeding of Horseflies and Mosquitoes(Group 3) After Oviposition Delay Following a Blood Meal on a Hog Cholera-Infected Pig

fed on virus-source pig 23 (Table 3).Pigs 25 and 27 both apparently remainedhealthy; however, they later resiste(tchallenge inoculation with the Amesstrain of HCVat 7.days after inoculation.Tonsillar tissue from both pigs was testpositive for HCV by the FATST5 daysafter. inoculation. However, body tem-peratures remained .normal, and spleenwas test negative for HCVby the FACCT.Before challenge inoculation, WBCcountsfor pigs 25 and 27 were 26,700 and19,500jcmm., respectively. At the timeof necropsy, WBCcounts for both pigswere 16,200jcmm. Blood serums of thesepit~#!At A 1:4 dilytitm (FMNT) t1AY!!l@dapproximf,ltely A 76% J'~d\lgtit.min theplaque count of the test dose of HCV.Although this reduction was less thanthe 90% required to satisfy the criterionof the test, it indicated the presence ofvery low antibody levels.

620

Pigs on Which Group 3 Insects Fed.-Three pigs (No. 12, 19, anq 2Q) on whichhorseflies fed and 1 pig (Na?"7i;)on whichmosquitoes fed all remained healthythroughout the study and .later provedsusceptible to challenge inoculation. .

Pig 12 was bitten by 39 horseflieswhich had fed on virus-source pigs 1, 2,3, and 16 (Table 4). Five species ovi-posited and subsequently fed on pig 12.Oviposition occurred from 5 to 12 days(mode, 7 days) after the initial bloodmeal. In general, most flies accepted ablood meal within 36 hours after ovipo-sition. Species feeding on this pig in-(!ly(;h~dT. tJa~nI (l jjp~(;Jtmtn),'f'. lint?oltl(10 specimens), T. nigrovittatus (2specimens), T. quinquevittatus (24 spec-imens), and T. sulci/rons (2 specimtms).Pig 12 remained healthy throughout thestudy and later proved susceptible tochallenge inoculation.

Am. J. Vet. Res., Vol. 33. No.3

{

2 T. lineola

}21 3 T. quinquevittatus 4> 9 and 22 Remained healthy Yes

], T. nigrovittatus

25 2 T. quinquevittatus 1 23 Remained healthy No

27 2 T.lineolo. ! 23 Remained healthy No

7'. = Tabanus.

Av. pe- Suscep-Suscep- No. of riod of Virus- tible totible pig insecta Species delay source Health challenge

No'. biting of insect (days) pig No. status inoculation

{

1

hkM 1

10 T. lineola12 2 T. nigrovittatus 8 1. 2. 3. and 16 Remained healthy . Yes

24T. quinquevittatus.

2 T. sulci/TOns

{

3

T. lineola }

20 1 T. nigroviltatus 15 1. 3. and 16 Remained healthy Yes6 T. quinquevittatus .1 T. sulcifrons

19 1 T. nigTOvittatus 20 16 Remained healthy Y'j9

{

7 A. sollicitans

}7 1 A. triseriatus 10 3 and 16 Remained healthy Yes

5 P. ferox.-.'-.-----' --.

T. = Taba1lUB;A. = Aedes; P. == Psorophoro.

Pig 20 was bitten by 11 ho!'Seffiesrep-resenting 4 species, including T. lineola(3 specimens), T. nigrovittatus (I speci-men), T. quinquevittatus (6 specimens),andT. sulci/rons (1 specimen). Theseflies had fed on virus-source pigs 1, 3,.and 16 (Table, 4). Pig 20 remainedhealthy and later proved susceptible tochallenge inoculation.

Pig 19 was bitten by a single fly, T.nigrovittatus, which had fed on a virus-source pig (No. 16) 20 days previously.This pig remained healthy and laterproved susceptible to challenge inocula-tion.

Pig 7 was bitten by 13 mosquitoes rep-resenting 3 species, Including A. sollici-tans (7. specimens), A. triseriatus (1specimen), and P. ferox (5 specimens).These mosquitoes had fed on virus-source pigs 3 and 16 (Table 4). Pig 7remained healthy and later proved sus-ceptible to challenge inoculation.

Control Pigs-All 4 pigs remained freeof cholera throughout the study and laterproved susceptible to challenge inocpla-tion.

Challenge lnoculation.-Pigs 2, 9, and23 (virus-source pigs) from the quaran-tine house had apparently recovered fromHC. Tonsillar tissue collected at thistime was test negative for all pigs (con-trol, principal, and virus-source pigs) bythe FATST,and WECcounts were morethan 19,OOO/cmm. Pigs, with the excep-tion of No.2, 9, 23, and No. 25 and 27('on which group 2 insects fed) devel-oped HC. All pigs which contracted thedisease had HCVin tonsils, body temper-atures greater than 40.6 C., and leuko-penia within 7 days after inoculation.

Typical lesions of HC (peripheral con-gestion of lymph nodes; petechiation ofthe epiglottis, heart, lungs, and urinarybladchu'i inf~r(.1ti()nof the spleen; andpneuIDQnia) Wliff@Ob~~rv0don th~jJ~pig~at necropsy. Spleen of all pigs containedHCVas determined by the FACCT,andserums were test negative at the 1:4dilution for HC antibodies by the FASNT.

Pigs 2, 9, and 23 which had recovered

March, !972

from HCwere resisting challenge infectionat 7 days after inoculation, at which timethey we!'e killed. . These pigs had highWHCcounts, and tonsillar and splenic tis-sues were test. negative for HCVby theFATSTand FAccT,respectively. Serums ofthese pigs had antibodies against HCV,asdetermined by the FASNTboth before andafter challenge inoculation.

Discussion

The results of this investigation indi-cate that tabanids are capable of trans-mitting hog cholera. Tabanus lineolaand T. quinquevittatus transmitted thedisease, and 3 other Tabanus spp.. wereincriminated; The results of the 24-hour delayed transmission experimentwere not conclusive. The resistance of 2of these pigs to the challenge virus givesevidence that these pigs had probablybeen exposed to the HCVby the bite ofthe tabanids.

The results of the postovipositionfeeding group represent S011).eevidencethat biological transmission of HCVdoes

, not occur in the species used in the pres-ent experiment. However, the data aretoo meager for final analysis. Most of theinsects in this group had obtained only apartial meal on the virus-source ...pig,since these flies were alsoHused in theimmediate transfer experiments. Thepossibility therefore exists that theseflies may not have obtained a sufficientquantity of virus to become infected.The amount of virus in the s~igsand available to the flies at the time offeeding was not determined. It is possi-ble that some of the source pigs were notviremic when exposed to the flies.

Transmission by mosquitoes was notdemonstrated; however, only 3 speciesparticipated in the postoviposition feed-ing test, In view of the frequency withwhich al'bovirumell are found 11U.~ociatfdwith mosquitoes, further studies areneeded to clarify their potential as bio-logical vectors of HC.

Other. kinds of flies may be capable ofdisseminating HCV.Culicoides spp., vec-tors of bluetongue virus, are suspects for

621

biological transmission. Hippelate$ spp.,or eye gnats, xpay be carriers of HCYbe-cause of their predilection for mucoussecretions; and as previously mentioned,houseflies and stable flies should also beconsidered.

When mosquitoes or other bloodsuck-ing flies are extremely numerous in na-ture, mechanical transmission may con-tribute to explosive dissemination of avirus.5 This type of transmission couldoccur in the ecologic situations in whichlocalized epizootics of HCare found. Ta-ban ids are usually numerous throughoutmuch of the fly season; they readily feedon swine, are strong fliers, and are capa-ble of experimentally transmitting thevirus. Tabanids are prime suspects fornatural dissemination of HCY.

622

References

1. Bernasky, N.: The Louse of the Pig asan Agent in Transmitting Infectious Diseasesof the Pig. Vet. Rec.. 23, (1910): 31-88.

2. Catts" E. P.: A CaI\Q~ Trap for Col-lecting TaDanidae. Mosquito News, 30, (1970):472-474.

. 3. Cole, C. G.. Henley, R. R. Dale, C. N~,Mott, L. 0., Torrey, J. P., and Zinober, M. R:History of Hog Cholera Research in. the U.S.Department of Agriculture 1884-1960. Agric.Info. Bull., 241, (1962): 44-45.

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