Hinck, Lawrence C.

Meurer, Rogers D.

Stevens, James L.

English

UARK (University of Arkansas )

Journal of the Arkansas Academy of Science Volume 38 Article 26 1984 Survival of Trypanosoma cruzi in Dead Chronically Infected Mice Lawrence C. Hinck Arkansas State University James L. Stevens Arkansas State University Rogers D. Meurer Merck Institute for Therapeutic Research Follow this and additional works at: https://scholarworks.uark.edu/jaas  Part of the Parasitology Commons Recommended Citation Hinck, Lawrence C.; Stevens, James L.; and Meurer, Rogers D. (1984) "Survival of Trypanosoma cruzi in Dead Chronically Infected Mice," Journal of the Arkansas Academy of Science: Vol. 38 , Article 26. Available at: https://scholarworks.uark.edu/jaas/vol38/iss1/26 This article is available for use under the Creative Commons license: Attribution-NoDerivatives 4.0 International (CC BY-ND 4.0). Users are able to read, download, copy, print, distribute, search, link to the full texts of these articles, or use them for any other lawful purpose, without asking prior permission from the publisher or the author. This General Note is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Journal of the Arkansas Academy of Science by an authorized editor of ScholarWorks@UARK. For more information, please contact scholar@uark.edu. Arkansas Academy of Science Proceedings, Vol. XXXVIII,1984 91Arkansas Academy of ScienceThe following summarizes efforts made thus far to protect important Arkansas gray bat caves. Some of these efforts have been previouslyreported (Harvey, 1975, 1976a, 1976b, 1979, 1980; Harvey et al., 1979).The very important gray bat hibernaculum located in the Ozark National Forest of Baxter County was gated by the U.S. Forest Service in1975. The gate was subsequently redesigned tobetter fit the particular conditions at the cave and a replacement gate was installed during the sum-mer of 1980. Five Buffalo National River caves have been fenced by the National Park Service to protect gray bat (and Indiana bat) colonies.One cave located on a private housing development in Benton County has been fenced by the developer to protect a gray bat summer colony.The U.S. Army Corps of Engineers recently constructed an artificual entrance into a gray bat cave located on Beaver Lake in Benton County,since the natural entrance is sometimes inundated during high lake levels.Inaddition to the above measures, the Arkansas Game and Fish Commission, U.S. Fish and Wildlife Service, U.S. Forest Service, and Na-tional Park Service have placed warning/interpretive signs at several gray bat caves. Other agencies and organizations involved in the gray batconservation effort include the Nature Conservancy, Arkansas Department of Parks and Tourism, Arkansas Natural Heritage Commission, Na-tional Speleological Society, Cave Research Foundation, Association for Arkansas Cave Studies, and Ozark Underground Laboratory.Important gray bat hibernacula and summer caves will be monitored for (he next several years. Additional measures will also be taken toprotect other important gray bat caves. Hopefully, the conservation effort willresult in removal of the gray bat from the endangered species list.LITERATURE CITEDBARBOUR, R. W., and W. H. Davis. 1969. Bats of America. Univ.Kentucky Press, Lexington. 286 p.HARVEY,M. J. 1980. Status of the endangered bats Myotis sodalis,M. grisescens, and Plecolus lownsendii ingens in the southernOzarks. Pp. 221,-223 inProc. Fifth Int. Bat Res. Conf. (D. E.Wilson and A.L. Gardner, eds.), Texas Tech Press, Lubbock,Texas. 434 p.IIVEY,M. J. 1975. Endangered Chiroptera of the southeasternUnited States. Proc. 29th Annu. Conf. S. E. Assoc. Game andFish Comm., 29:429-433. HARVEY,M.J., J. J. CASS1DY, and G.G. O'HAGAN. 1979. Statusof the endangered bats Myotissodalis, M. grisescens and Plecoluslownsendii ingens in Arkansas. Proc. Ark. Acad. Sci. 33:81.riVEY,M. J. 1976a. Status of endangered bats in the BlanchardSprings Caverns area. P. 63 in Natl. Cave Manage. Symp. Proc,1975. Speleobooks, Albuquerque, New Mexico.TUTTLE,M. D. 1975. Population ecology of the gray bat (Myotisgrisescens): factors influencing early growth and development.Occas. Pap. Mus. Natur. Hist., Univ. Kansas, 36:1-24.¦1VEY, M. J. 1976b. Status of endangered bats in the easternUnited States. Pp. 21-24 in Proc. 1976 Natl. Speleol. Soc.Convention.TUTTLE,M. D.,and D.E. STEVENSON. 1978. Variation inthe caveenvironment and its biological implications. Pp. 108-121 in Natl.Cave Manage. Symp. Proc, 1977 (R. Zuber, J. Chester, S. Gilbert,and D. Rhodes, eds.). Speleobooks, Albuquerque, New Mexico.fiVEY,M.J. 1979. Distribution, status, and ecology ofendangeredbats ofBuffalo National River, Arkansas. Pp. 76-84 (Vol. 8) inProc. Second Conf. on Sci. Res. in the Natl. Parks. 12 vols.TUTTLE,M. D. 1979. Status, causes ofdecline, and management ofendangered gray bats. J. Wildl. Manage., 43:1-17.MICHAELJ. HARVEY, Ecological Research Center, Department ojBiology, Memphis State University, Memphis, 77V iHI52. (Present address,Department ofBiology, Tennessee Technological University, Cookeville, 77V 38505 JSURVIVAL OF TRYPANOSOMA CRUZl INDEAD CHRONICALLYINFECTED MICEITrypanosomacruzi (Chagas, 1909), the cause of Chagas' disease, is a flagellate protozoan found throughout most of Central and Southimerica where it infects approximately 12 million people (World Health Organization, 1960). Inthe United States this agent has been reportedlwildlifemammals in California, Arizona (Kofoid and Whitaker, 1936), Texas (Packchanian, 1941), New Mexico (Dias, 1951), Minnesota (Hedrick,955), Virginia (Tromba, 1951), Maryland (Walton et al., 1956), Georgia, Florida (McKeever el al.. 1958), Alabama (Olsen et al., 1964), and Loui-iana (Yeager and Bacigalupo, 1960). Cited reservoir hosts include such common mammals as the raccoon (Procyon lotor), opossum (Didelphisirginiana), grey fox (Urocyon cinereoargenteus), and striped skunk (Mephitis mephitis) (McKeever el al., 1958).The normal transmission of T.cruzi occurs when a bug (Hemiptera:Reduviidae) takes ablood meal and deposits feces containing trypomastigotesnthe skin. The parasites enter the bite wound and invade cells of many organs throughout the body as they circulate in the blood for approximate-y 30 days or longer. Upon entering a cell, the trypomastigote typically loses its flagellum and transforms into an amastigote which reproduces)y binary fission. During the acute phase amastigotes produced intracellularly are released by cell lysis and either reinvade new cells or transformnto trypomastigotes and reenter the circulation. The cells most often attacked are reticuloendothelial cells of the liver and spleen, cells of cardiac,mooth and skeletal muscle and certain cells of the nervous system. Animals surviving the acute phase typicallydevelop chronic infections in whichmastigotes form pseudocysts in the host's tissues producing only limited cell lysis. Inthe chronic infection, trypomastigotes in the blood are scantynd difficult to demonstrate by microscopic examination.Epidemiologic studies of zoonoses, such as Chagas' disease, contribute to the basic understanding of the biology of the etiologic agents andprovide information relating to both geographic distribution and transmission. Such information can be useful in prevention and control of thesediseases which may pose a threat toman, domestic animals and wildlife. Often studies of this nature have required the capture and, in some cases,the sacrifice ofpotential wildlife reservoirs.Meurer (1980), in a preliminary study, demonstrated that T.cruzi could be detected indead chronically infected mice by inoculating a modified91Journal of the Arkansas Academy of Science, Vol. 38 [1984], Art. 26Published by Arkansas Academy of Science, 198492Arkansas Academy of Science Proceedings, Vol. XXXVIII,1984General NotesTobie's diphasic medium (Tobie el ul., 1950) with a homogenate ofheart tissue. T.cruzi was cultured from two of three mice that had been heldfor 24 hours at 25 C while three out of three mice were positive after 24 hours at 5 C. The present study was conducted tomore accurately assessthe relationship between holding temperature and parasite survival in dead mice that were chronically infected and to compare the efficiency ofin vitro cultivation with animal inoculation for detecting T. cruzi in tissue homogenates of dead animals.The Calluromys LNX strain of T. cruzi was used in our current study since it typically produces a less intense acute phase parasitemia followedby a chronic infection in which mice have an extended survival time. Chronic infections were achieved by injecting mice subcutaneously with ap-proximately 6 x 104 trypomastigotes (determined by the method of Herbert and Lumsden, 1976) obtained from mice with acute infections. ASULilac-Wild cross mice were used for this purpose since these animals are hardy and tend to develop chronic infections consistently. Mice were con-sidered to be in the chronic phase when microscopic examination of tail blood from previously patent animals revealed very low or negative parasitemias.Inmost mice this was around 50 days postinoculation.Chronic phase mice were sacrificed and held at temperatures ranging from 5 C to 35 C. Holding periods ranged from zero to 48 hours forlower temperatures while 36 hours was maximum time for higher temperatures. The heart and liver were aseptically removed from each animaland homogenized in a Ten-Broeck tissue grinder with 2.0 ml sterile Locke's solution containing 18.5 fig/mlgentamicin to prevent bacterial growth.From each homogenate, two mice (ASU Piebald) were injected subcutaneously with 0.5 ml portions and two tubes of modified Tobie's diphasic-medium (with human blood) containing gentamicin were inoculated with 0.2 ml each. ASU Piebald mice were used for this phase of the studybecause of their susceptibility to infection. Mice were checked for parasitemias periodically for a period of 30 days after which time they weredeclared negative. Invitro culture tubes were incubated at 25 C for 10 days before discarding.Results of the study indicate that survival time of T. cruzi in chronically infected mice is highlydependent on the holding temperature (Figure).At 5 C T. cruzi was detected in 100% of the mice held for as long as 24 hours when tested by in vitro cultivation or byanimal inoculation. Withincreasing temperature, the recovery rate of T. cruzi from dead animals decreased. At 10 C the recovery rate for24 hours was still 100% by animalinoculation and 93% by in vitro cultivation. The 24-hour positive fell to 60% and 87% for in vitro cultivation and animal inoculation respectiveat 25 C while at 30 C the rate dropped to 27% for both methods. The Table reveals that no parasites were detected in animals held at 35 C for 24 hours.Table. Recovery of Trypanosoma cruzi from chronically infected miceby in vitro cultivation and mouse inoculation.hours) (°C) invitro cultivation8 mouse inoculation^0 5 15/15 15/156"15/15 15/1512"15/15 15/1524"15/15 15/1536"1/15 2/1548"0/15 0/150 10 15/15 15/156"15/15 15/1512"14/14 14/1424"14/15 15/1536"1/15 1/1548"0/14 0/140 25 15/15 15/156" 15/15 15/1512"15/15 15/1524"9/15 13/1536"0/16 1/1548"0/15 0/1515/15 14/156"14/14 14/1412"10/15 14/1524"4/15 4/1536"0/15 0/150 35 15/15 15/156-14/15 15/1512 ¦ 2/15 3/1524"0/15 0/1536"0/15 0/15Amodified Tobie's diphasic medium was inoculated with heart-liverlomogenate and incubated at 25 C.hMice were injected subcutaneously with heart-liver homogenate.Figure. Relationship of holding time and holding temperature torecovery rate of Trypanosoma cruzi from chronically infected mice.C = Per cent recovery by inoculation of a modified Tobie's diphasicmedium with heart-liver homogenate.A = Per cent recovery by subcutaneous injectionof mice with heari-liver homogenate.The results also suggest that animal inoculation is a somewhat more sensitive method than in vitro cultivation for detecting T. cruzi in thetissue of dead animals. It was clear, however, from trial runs with several strains ofmice that a highlysusceptible line of mice is important forthis purpose.In the epidemiology of T.cruzi it appears likelythat dead chronically infected animals might serve as a source of infection for scavenginganimals for a period ofat least 24 hours at moderate temperatures and possibly as long as 36 hours at cool temperatures. The results also pointto in vitro or in vivo cultivation from heart-liver homogenates as an acceptable method for assessing the distribution and incidence of T. cruziin wildlife. These animals could be road kills or those taken by fur trappers.LITERATURE CITEDCHAGAS, C. 1909. Nova tripanosomiaze humana. Estudos sobre amortolojia e ociclo evolutivo do Schizotrypanus cruzi, n. sp. Mem.Oswaldo Cruz. 1:159-218.DIAS,E. 1951. Chagas' disease in the United States. J. Parasitol41:629-634.92Journal of the Arkansas Academy of Science, Vol. 38 [1984], Art. 26https://scholarworks.uark.edu/jaas/vol38/iss1/26Arkansas Academy of Science Proceedings, Vol. XXXVIII,1984 93Arkansas Academy of Science¦RICK, R. M. 1955. A trypanosome from the big brown bat,iptesicus fuscus fuscus (Beavois), in Minnesota. J. Parasitol.H:629-624.¦BERT, W. J., and W. H. R. LUMSDEN. 1976. Trypanosomairucei: A rapid "matching" method for estimating the host's>arasitemia. Exp. Parasitol. 40:427-431.IOID, C. A., and B. G. WHITAKER. 1936. Natural infection ofAmerican human trypanosomiasis in two cone-nosed bugs,Triatoma protracta and Triatoma uhleri Neiva, in the WesternUnited States. J. Parasitol. 22:259-263.IEEVER, S., G. W. GORMAN, and L.NORMAN. 1958. Occur-rence ofa Trypanosoma crwz;-like organism in some mammals fromSouthwestern Georgia and Northwestern Florida. J. Parasitol.44:583-587.¦JRER, R. D. 1980. A study of Trypanosoma cruzi and its apparentabsence in Northeast Arkansas. Unpubl. M.S. Thesis, ArkansasState Univ., State Univ.,AR. 27 p.¦EN, P. F., J. P. SHOEMAKE, H.F. TURNER,and K.L.HAYS.1964. Incidence of Trypanosoma cruzi (Chagas) in wild vectorsand reservoirs inEast Central Alabama. .1. Parasitol. 50:599-603.PACKCHANIAN,A. 1941. Reservoir hosts of Chagas' disease in thestate of Texas. J. Parasit. Suppl. 27:30.TOBIE, E. J., T.von BRAND,and B.MEHLMAN.1950. Cultural andphysiological observations on Trypanosoma rhodesiense Trypane-soma gambiense. .1. Parasitol. 36:48-54.TROMBA,F. G. 1951. Trypanosoma vesperiilionis and Lilomosoidessp. in Eptesicus fuscus fuscus. J. Parasit. 37:324.WALTON, B. C, P. M. BAUMAN,L. S. DIAMOND,and C. M.HERMAN. 1956. Trypanosoma cruziin raccoons from Maryland.J. Parasit. 42:20.WORLD HEALTH ORGANIZATION.1960. Chagas' disease. Reportofstudy group. Technical Report Series No. 202, Geneva.Yl.AGER, R. G., and A. D. BACIGALUPO. I960. Further studieson Trypanosoma cruzi in Louisiana. J. Parasitol. 46:7.YL.AGER,R. 1971. Transmission of Trypanosoma cruzi infection toopossums via the oral route. J. Parasit. 57:1375-1376.¦RENCE C. HINCK and JAMES L. STEVENS, Department ofBiologicalSciences, Arkansas Slate University, Slate University, AR 72467;•OG R D. MEURER, Merck Institute for Therapeutic Research, Rahway, N.J.EFFECT OF THE PAYMENT-IN-KIND(PIK) PROGRAM ON THE PSOROPHORA COLUMBIAEMOSQUITO POPULATION OF A NORTHEAST ARKANSAS RICEFIELD COMMUNITYtSince 1904, when rice was initiallycultivated as a commercial crop within the state, Arkansas has developed into one of the five major rice-)ducing states in the U.S. with an annual production of ca. 6,000,000 ha (Meisch, et al., 1982). Although some rice is grown in central andahwestern counties, the majorityof the crop is planted in the eastern half of the state, particularly in the "Grand Prairie" area ofeast -centralkansas which includes Arkansas, Lonoke, Monroe, and Prairie counties.Ithas been well established bya number of investigators in Arkansas, and in other rice-growing states, that the rice agroecosystem providessuitable breeding sites for several mosquito species including the dark ricefield mosquito, Psorophora columbiae (Dyar and Knab). InArkansas,Schwardt (1939), Horsfall (1942), Whitehead (1951), Meisch and Coombes (1975), and Olson and Huggins (1983), among others, all have reportedP. columbiae tobe the dominant mosquito species wherever rice is cultivated. Whitehead (1951), in particular, noted that ricefield mosquito numbershave increased in direct proportion to the state's increased rice acreage.InCraighead County inNE Arkansas, land devoted torice production in 1981 and 1982 was 33,590 and 33,376 ha, respectively (Olson andHuggins, 1983). Light trap studies conducted in Jonesboro by these investigators indicated that, of 34,041 adult mosquitoes captured between 30May and 2 October of 1981, 21,085 (61.9%) were P. columbiae. During the same period in 1982, P. columbiae numbered 24,675 (72.3%) of the34,114 mosquitoes trapped.(In1983, a federally funded payment-in-kind (PIK) program was implemented in an attempt to reduce national agricultural surpluses andimprove market prices by paying growers to keep land out of commercial crop production. As a result, American farmers idled some 31,000,000includingsignificant rice acreage in NE Arkansas. Under the PIK program, the amount of Craighead-County rice land was lowered by 39.0%20,350 ha (Fagala, Craighead Co. Extension Service, pers. comm.).The primary aim of this study was to evaluate the effect which this acreage reduction had upon the relative abundance of P. columbiae inJonesboro. To accomplish this objective, a standard New Jersey light trap was placed at each of four locations within the city limits. Samplingdates and trap locations (two peripheral and two central) were identical to those described by Olson and Huggins (1983). Light trap catches werecollected dailyand adult mosquitoes sorted and identified. Dailytrap totals were summed for each week of the 18-week study period and comparedwith data obtained in 1981 and 1982.IItshould be notedthat, as in 1981 and 1982, all traps were in areas subjected to periodic applications of a mosquito adulticide by ground-:rated ULV cold-aerosol generators. This undoubtedly lowered the total number of mosquitoes collected, particularly in the central-city areaich was further from rice fields which served as the main source of P. columbiae reinfestation.[Atotal of 18,232 adult mosquitoes was captured between 30 May and 2 October of 1983, with the PIK program in effect. This representedoverall decrease in the general mosquito population of46.5% in comparison with 1981 and 1982 pre-PIK totals reported by Olson and Huggins'83). As anticipated, P. columbiae, with 13,394 trapped individuals, was the dominant species and it comprised 73.5% of the season's catch,tier genera contributing to the remainder of the total were Anopheles (16.0%), Aedes (6.5%), Culex (4.0%), and Culiseta (less than 0.1%). Its observed that the 39.0% decrease in Craighead-County rice acreage in1983 was accompanied by a 43.4% reduction in the P. columbiae popula-n trapped within the Jonesboro city limits. This was believed to have been a direct result of the lessening of available breeding sites normally93Journal of the Arkansas Academy of Science, Vol. 38 [1984], Art. 26Published by Arkansas Academy of Science, 1984

Survival of Trypanosoma cruzi in Dead Chronically Infected Mice

ScholarWorks@UARK

Journal of the Arkansas Academy of ScienceVolume 38 Article 261984Survival of Trypanosoma cruzi in DeadChronically Infected MiceLawrence C. HinckArkansas State UniversityJames L. StevensArkansas State UniversityRogers D. MeurerMerck Institute for Therapeutic ResearchFollow this and additional works at: http://scholarworks.uark.edu/jaasPart of the Parasitology CommonsThis article is available for use under the Creative Commons license: Attribution-NoDerivatives 4.0 International (CC BY-ND 4.0). Users are able toread, download, copy, print, distribute, search, link to the full texts of these articles, or use them for any other lawful purpose, without asking priorpermission from the publisher or the author.This General Note is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Journal of the ArkansasAcademy of Science by an authorized editor of ScholarWorks@UARK. For more information, please contact scholar@uark.edu, ccmiddle@uark.edu.Recommended CitationHinck, Lawrence C.; Stevens, James L.; and Meurer, Rogers D. (1984) "Survival of Trypanosoma cruzi in Dead Chronically InfectedMice," Journal of the Arkansas Academy of Science: Vol. 38 , Article 26.Available at: http://scholarworks.uark.edu/jaas/vol38/iss1/26Arkansas Academy of Science Proceedings, Vol. XXXVIII,1984 91Arkansas Academy of ScienceThe following summarizes efforts made thus far to protect important Arkansas gray bat caves. Some of these efforts have been previouslyreported (Harvey, 1975, 1976a, 1976b, 1979, 1980; Harvey et al., 1979).The very important gray bat hibernaculum located in the Ozark National Forest of Baxter County was gated by the U.S. Forest Service in1975. The gate was subsequently redesigned tobetter fit the particular conditions at the cave and a replacement gate was installed during the sum-mer of 1980. Five Buffalo National River caves have been fenced by the National Park Service to protect gray bat (and Indiana bat) colonies.One cave located on a private housing development in Benton County has been fenced by the developer to protect a gray bat summer colony.The U.S. Army Corps of Engineers recently constructed an artificual entrance into a gray bat cave located on Beaver Lake in Benton County,since the natural entrance is sometimes inundated during high lake levels.Inaddition to the above measures, the Arkansas Game and Fish Commission, U.S. Fish and Wildlife Service, U.S. Forest Service, and Na-tional Park Service have placed warning/interpretive signs at several gray bat caves. Other agencies and organizations involved in the gray batconservation effort include the Nature Conservancy, Arkansas Department of Parks and Tourism, Arkansas Natural Heritage Commission, Na-tional Speleological Society, Cave Research Foundation, Association for Arkansas Cave Studies, and Ozark Underground Laboratory.Important gray bat hibernacula and summer caves will be monitored for (he next several years. Additional measures will also be taken toprotect other important gray bat caves. Hopefully, the conservation effort willresult in removal of the gray bat from the endangered species list.LITERATURE CITEDBARBOUR, R. W., and W. H. Davis. 1969. Bats of America. Univ.Kentucky Press, Lexington. 286 p.HARVEY,M. J. 1980. Status of the endangered bats Myotis sodalis,M. grisescens, and Plecolus lownsendii ingens in the southernOzarks. Pp. 221,-223 inProc. Fifth Int. Bat Res. Conf. (D. E.Wilson and A.L. Gardner, eds.), Texas Tech Press, Lubbock,Texas. 434 p.IIVEY,M. J. 1975. Endangered Chiroptera of the southeasternUnited States. Proc. 29th Annu. Conf. S. E. Assoc. Game andFish Comm., 29:429-433. HARVEY,M.J., J. J. CASS1DY, and G.G. O'HAGAN. 1979. Statusof the endangered bats Myotissodalis, M. grisescens and Plecoluslownsendii ingens in Arkansas. Proc. Ark. Acad. Sci. 33:81.riVEY,M. J. 1976a. Status of endangered bats in the BlanchardSprings Caverns area. P. 63 in Natl. Cave Manage. Symp. Proc,1975. Speleobooks, Albuquerque, New Mexico.TUTTLE,M. D. 1975. Population ecology of the gray bat (Myotisgrisescens): factors influencing early growth and development.Occas. Pap. Mus. Natur. Hist., Univ. Kansas, 36:1-24.¦1VEY, M. J. 1976b. Status of endangered bats in the easternUnited States. Pp. 21-24 in Proc. 1976 Natl. Speleol. Soc.Convention.TUTTLE,M. D.,and D.E. STEVENSON. 1978. Variation inthe caveenvironment and its biological implications. Pp. 108-121 in Natl.Cave Manage. Symp. Proc, 1977 (R. Zuber, J. Chester, S. Gilbert,and D. Rhodes, eds.). Speleobooks, Albuquerque, New Mexico.fiVEY,M.J. 1979. Distribution, status, and ecology ofendangeredbats ofBuffalo National River, Arkansas. Pp. 76-84 (Vol. 8) inProc. Second Conf. on Sci. Res. in the Natl. Parks. 12 vols.TUTTLE,M. D. 1979. Status, causes ofdecline, and management ofendangered gray bats. J. Wildl. Manage., 43:1-17.MICHAELJ. HARVEY, Ecological Research Center, Department ojBiology, Memphis State University, Memphis, 77V iHI52. (Present address,Department ofBiology, Tennessee Technological University, Cookeville, 77V 38505 JSURVIVAL OF TRYPANOSOMA CRUZl INDEAD CHRONICALLYINFECTED MICEITrypanosomacruzi (Chagas, 1909), the cause of Chagas' disease, is a flagellate protozoan found throughout most of Central and Southimerica where it infects approximately 12 million people (World Health Organization, 1960). Inthe United States this agent has been reportedlwildlifemammals in California, Arizona (Kofoid and Whitaker, 1936), Texas (Packchanian, 1941), New Mexico (Dias, 1951), Minnesota (Hedrick,955), Virginia (Tromba, 1951), Maryland (Walton et al., 1956), Georgia, Florida (McKeever el al.. 1958), Alabama (Olsen et al., 1964), and Loui-iana (Yeager and Bacigalupo, 1960). Cited reservoir hosts include such common mammals as the raccoon (Procyon lotor), opossum (Didelphisirginiana), grey fox (Urocyon cinereoargenteus), and striped skunk (Mephitis mephitis) (McKeever el al., 1958).The normal transmission of T.cruzi occurs when a bug (Hemiptera:Reduviidae) takes ablood meal and deposits feces containing trypomastigotesnthe skin. The parasites enter the bite wound and invade cells of many organs throughout the body as they circulate in the blood for approximate-y 30 days or longer. Upon entering a cell, the trypomastigote typically loses its flagellum and transforms into an amastigote which reproduces)y binary fission. During the acute phase amastigotes produced intracellularly are released by cell lysis and either reinvade new cells or transformnto trypomastigotes and reenter the circulation. The cells most often attacked are reticuloendothelial cells of the liver and spleen, cells of cardiac,mooth and skeletal muscle and certain cells of the nervous system. Animals surviving the acute phase typicallydevelop chronic infections in whichmastigotes form pseudocysts in the host's tissues producing only limited cell lysis. Inthe chronic infection, trypomastigotes in the blood are scantynd difficult to demonstrate by microscopic examination.Epidemiologic studies of zoonoses, such as Chagas' disease, contribute to the basic understanding of the biology of the etiologic agents andprovide information relating to both geographic distribution and transmission. Such information can be useful in prevention and control of thesediseases which may pose a threat toman, domestic animals and wildlife. Often studies of this nature have required the capture and, in some cases,the sacrifice ofpotential wildlife reservoirs.Meurer (1980), in a preliminary study, demonstrated that T.cruzi could be detected indead chronically infected mice by inoculating a modified91Journal of the Arkansas Academy of Science, Vol. 38 [1984], Art. 26Published by Arkansas Academy of Science, 198492Arkansas Academy of Science Proceedings, Vol. XXXVIII,1984General NotesTobie's diphasic medium (Tobie el ul., 1950) with a homogenate ofheart tissue. T.cruzi was cultured from two of three mice that had been heldfor 24 hours at 25 C while three out of three mice were positive after 24 hours at 5 C. The present study was conducted tomore accurately assessthe relationship between holding temperature and parasite survival in dead mice that were chronically infected and to compare the efficiency ofin vitro cultivation with animal inoculation for detecting T. cruzi in tissue homogenates of dead animals.The Calluromys LNX strain of T. cruzi was used in our current study since it typically produces a less intense acute phase parasitemia followedby a chronic infection in which mice have an extended survival time. Chronic infections were achieved by injecting mice subcutaneously with ap-proximately 6 x 104 trypomastigotes (determined by the method of Herbert and Lumsden, 1976) obtained from mice with acute infections. ASULilac-Wild cross mice were used for this purpose since these animals are hardy and tend to develop chronic infections consistently. Mice were con-sidered to be in the chronic phase when microscopic examination of tail blood from previously patent animals revealed very low or negative parasitemias.Inmost mice this was around 50 days postinoculation.Chronic phase mice were sacrificed and held at temperatures ranging from 5 C to 35 C. Holding periods ranged from zero to 48 hours forlower temperatures while 36 hours was maximum time for higher temperatures. The heart and liver were aseptically removed from each animaland homogenized in a Ten-Broeck tissue grinder with 2.0 ml sterile Locke's solution containing 18.5 fig/mlgentamicin to prevent bacterial growth.From each homogenate, two mice (ASU Piebald) were injected subcutaneously with 0.5 ml portions and two tubes of modified Tobie's diphasic-medium (with human blood) containing gentamicin were inoculated with 0.2 ml each. ASU Piebald mice were used for this phase of the studybecause of their susceptibility to infection. Mice were checked for parasitemias periodically for a period of 30 days after which time they weredeclared negative. Invitro culture tubes were incubated at 25 C for 10 days before discarding.Results of the study indicate that survival time of T. cruzi in chronically infected mice is highlydependent on the holding temperature (Figure).At 5 C T. cruzi was detected in 100% of the mice held for as long as 24 hours when tested by in vitro cultivation or byanimal inoculation. Withincreasing temperature, the recovery rate of T. cruzi from dead animals decreased. At 10 C the recovery rate for24 hours was still 100% by animalinoculation and 93% by in vitro cultivation. The 24-hour positive fell to 60% and 87% for in vitro cultivation and animal inoculation respectiveat 25 C while at 30 C the rate dropped to 27% for both methods. The Table reveals that no parasites were detected in animals held at 35 C for 24 hours.Table. Recovery of Trypanosoma cruzi from chronically infected miceby in vitro cultivation and mouse inoculation.hours) (°C) invitro cultivation8 mouse inoculation^0 5 15/15 15/156"15/15 15/1512"15/15 15/1524"15/15 15/1536"1/15 2/1548"0/15 0/150 10 15/15 15/156"15/15 15/1512"14/14 14/1424"14/15 15/1536"1/15 1/1548"0/14 0/140 25 15/15 15/156" 15/15 15/1512"15/15 15/1524"9/15 13/1536"0/16 1/1548"0/15 0/1515/15 14/156"14/14 14/1412"10/15 14/1524"4/15 4/1536"0/15 0/150 35 15/15 15/156-14/15 15/1512 ¦ 2/15 3/1524"0/15 0/1536"0/15 0/15Amodified Tobie's diphasic medium was inoculated with heart-liverlomogenate and incubated at 25 C.hMice were injected subcutaneously with heart-liver homogenate.Figure. Relationship of holding time and holding temperature torecovery rate of Trypanosoma cruzi from chronically infected mice.C = Per cent recovery by inoculation of a modified Tobie's diphasicmedium with heart-liver homogenate.A = Per cent recovery by subcutaneous injectionof mice with heari-liver homogenate.The results also suggest that animal inoculation is a somewhat more sensitive method than in vitro cultivation for detecting T. cruzi in thetissue of dead animals. It was clear, however, from trial runs with several strains ofmice that a highlysusceptible line of mice is important forthis purpose.In the epidemiology of T.cruzi it appears likelythat dead chronically infected animals might serve as a source of infection for scavenginganimals for a period ofat least 24 hours at moderate temperatures and possibly as long as 36 hours at cool temperatures. The results also pointto in vitro or in vivo cultivation from heart-liver homogenates as an acceptable method for assessing the distribution and incidence of T. cruziin wildlife. These animals could be road kills or those taken by fur trappers.LITERATURE CITEDCHAGAS, C. 1909. Nova tripanosomiaze humana. Estudos sobre amortolojia e ociclo evolutivo do Schizotrypanus cruzi, n. sp. Mem.Oswaldo Cruz. 1:159-218.DIAS,E. 1951. Chagas' disease in the United States. J. Parasitol41:629-634.92Journal of the Arkansas Academy of Science, Vol. 38 [1984], Art. 26http://scholarworks.uark.edu/jaas/vol38/iss1/26Arkansas Academy of Science Proceedings, Vol. XXXVIII,1984 93Arkansas Academy of Science¦RICK, R. M. 1955. A trypanosome from the big brown bat,iptesicus fuscus fuscus (Beavois), in Minnesota. J. Parasitol.H:629-624.¦BERT, W. J., and W. H. R. LUMSDEN. 1976. Trypanosomairucei: A rapid "matching" method for estimating the host's>arasitemia. Exp. Parasitol. 40:427-431.IOID, C. A., and B. G. WHITAKER. 1936. Natural infection ofAmerican human trypanosomiasis in two cone-nosed bugs,Triatoma protracta and Triatoma uhleri Neiva, in the WesternUnited States. J. Parasitol. 22:259-263.IEEVER, S., G. W. GORMAN, and L.NORMAN. 1958. Occur-rence ofa Trypanosoma crwz;-like organism in some mammals fromSouthwestern Georgia and Northwestern Florida. J. Parasitol.44:583-587.¦JRER, R. D. 1980. A study of Trypanosoma cruzi and its apparentabsence in Northeast Arkansas. Unpubl. M.S. Thesis, ArkansasState Univ., State Univ.,AR. 27 p.¦EN, P. F., J. P. SHOEMAKE, H.F. TURNER,and K.L.HAYS.1964. Incidence of Trypanosoma cruzi (Chagas) in wild vectorsand reservoirs inEast Central Alabama. .1. Parasitol. 50:599-603.PACKCHANIAN,A. 1941. Reservoir hosts of Chagas' disease in thestate of Texas. J. Parasit. Suppl. 27:30.TOBIE, E. J., T.von BRAND,and B.MEHLMAN.1950. Cultural andphysiological observations on Trypanosoma rhodesiense Trypane-soma gambiense. .1. Parasitol. 36:48-54.TROMBA,F. G. 1951. Trypanosoma vesperiilionis and Lilomosoidessp. in Eptesicus fuscus fuscus. J. Parasit. 37:324.WALTON, B. C, P. M. BAUMAN,L. S. DIAMOND,and C. M.HERMAN. 1956. Trypanosoma cruziin raccoons from Maryland.J. Parasit. 42:20.WORLD HEALTH ORGANIZATION.1960. Chagas' disease. Reportofstudy group. Technical Report Series No. 202, Geneva.Yl.AGER, R. G., and A. D. BACIGALUPO. I960. Further studieson Trypanosoma cruzi in Louisiana. J. Parasitol. 46:7.YL.AGER,R. 1971. Transmission of Trypanosoma cruzi infection toopossums via the oral route. J. Parasit. 57:1375-1376.¦RENCE C. HINCK and JAMES L. STEVENS, Department ofBiologicalSciences, Arkansas Slate University, Slate University, AR 72467;•OG R D. MEURER, Merck Institute for Therapeutic Research, Rahway, N.J.EFFECT OF THE PAYMENT-IN-KIND(PIK) PROGRAM ON THE PSOROPHORA COLUMBIAEMOSQUITO POPULATION OF A NORTHEAST ARKANSAS RICEFIELD COMMUNITYtSince 1904, when rice was initiallycultivated as a commercial crop within the state, Arkansas has developed into one of the five major rice-)ducing states in the U.S. with an annual production of ca. 6,000,000 ha (Meisch, et al., 1982). Although some rice is grown in central andahwestern counties, the majorityof the crop is planted in the eastern half of the state, particularly in the "Grand Prairie" area ofeast -centralkansas which includes Arkansas, Lonoke, Monroe, and Prairie counties.Ithas been well established bya number of investigators in Arkansas, and in other rice-growing states, that the rice agroecosystem providessuitable breeding sites for several mosquito species including the dark ricefield mosquito, Psorophora columbiae (Dyar and Knab). InArkansas,Schwardt (1939), Horsfall (1942), Whitehead (1951), Meisch and Coombes (1975), and Olson and Huggins (1983), among others, all have reportedP. columbiae tobe the dominant mosquito species wherever rice is cultivated. Whitehead (1951), in particular, noted that ricefield mosquito numbershave increased in direct proportion to the state's increased rice acreage.InCraighead County inNE Arkansas, land devoted torice production in 1981 and 1982 was 33,590 and 33,376 ha, respectively (Olson andHuggins, 1983). Light trap studies conducted in Jonesboro by these investigators indicated that, of 34,041 adult mosquitoes captured between 30May and 2 October of 1981, 21,085 (61.9%) were P. columbiae. During the same period in 1982, P. columbiae numbered 24,675 (72.3%) of the34,114 mosquitoes trapped.(In1983, a federally funded payment-in-kind (PIK) program was implemented in an attempt to reduce national agricultural surpluses andimprove market prices by paying growers to keep land out of commercial crop production. As a result, American farmers idled some 31,000,000includingsignificant rice acreage in NE Arkansas. Under the PIK program, the amount of Craighead-County rice land was lowered by 39.0%20,350 ha (Fagala, Craighead Co. Extension Service, pers. comm.).The primary aim of this study was to evaluate the effect which this acreage reduction had upon the relative abundance of P. columbiae inJonesboro. To accomplish this objective, a standard New Jersey light trap was placed at each of four locations within the city limits. Samplingdates and trap locations (two peripheral and two central) were identical to those described by Olson and Huggins (1983). Light trap catches werecollected dailyand adult mosquitoes sorted and identified. Dailytrap totals were summed for each week of the 18-week study period and comparedwith data obtained in 1981 and 1982.IItshould be notedthat, as in 1981 and 1982, all traps were in areas subjected to periodic applications of a mosquito adulticide by ground-:rated ULV cold-aerosol generators. This undoubtedly lowered the total number of mosquitoes collected, particularly in the central-city areaich was further from rice fields which served as the main source of P. columbiae reinfestation.[Atotal of 18,232 adult mosquitoes was captured between 30 May and 2 October of 1983, with the PIK program in effect. This representedoverall decrease in the general mosquito population of46.5% in comparison with 1981 and 1982 pre-PIK totals reported by Olson and Huggins'83). As anticipated, P. columbiae, with 13,394 trapped individuals, was the dominant species and it comprised 73.5% of the season's catch,tier genera contributing to the remainder of the total were Anopheles (16.0%), Aedes (6.5%), Culex (4.0%), and Culiseta (less than 0.1%). Its observed that the 39.0% decrease in Craighead-County rice acreage in1983 was accompanied by a 43.4% reduction in the P. columbiae popula-n trapped within the Jonesboro city limits. This was believed to have been a direct result of the lessening of available breeding sites normally93Journal of the Arkansas Academy of Science, Vol. 38 [1984], Art. 26Published by Arkansas Academy of Science, 1984

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Survival of Trypanosoma cruzi in Dead Chronically Infected Mice

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