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2004

A Focus of Infection by taichui (: ) in the Southern Philippines

Vicente Y. Belizario Jr. University of the Philippines Manila

Winifreda U. de Leon University of the Philippines Manila

Mary Joan J. Bersabe Center for Health Development for Southern Mindanao

Purnomo U.S. Naval Medical Research Unit # 2

J. Kevin Baird U.S. Naval Medical Research Unit # 2, [email protected]

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Belizario, Vicente Y. Jr.; de Leon, Winifreda U.; Bersabe, Mary Joan J.; Purnomo; Baird, J. Kevin; and Bangs, Michael J., "A Focus of Human Infection by (Trematoda: Heterophyidae) in the Southern Philippines" (2004). U.S. Navy Research. 73. https://digitalcommons.unl.edu/usnavyresearch/73

This Article is brought to you for free and open access by the U.S. Department of Defense at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in U.S. Navy Research by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Authors Vicente Y. Belizario Jr., Winifreda U. de Leon, Mary Joan J. Bersabe, Purnomo, J. Kevin Baird, and Michael J. Bangs

This article is available at DigitalCommons@University of Nebraska - Lincoln: https://digitalcommons.unl.edu/ usnavyresearch/73 RESEARCH NOTES 1165

LITERATURE CITED HAMMOND, D. M., L. R. DAVIS, AND L. BOWMANN. 1944. Experimental AIKAWA, M., A. SCHWARTZ,S.UNI,R.NUSSENZWEIG, AND M. HOLLING- infections with Eimeria bovis in calves. American Journal of Vet- DALE. 1984. Ultrastructure of in vitro cultured exo-erythrocytic erinary Research 5: 303±311. stage of Plasmodium berghei in a hepatoma cell line. American HERMOSILLA, C., B. BARBISCH,A.HEISE,S.KOWALIK, AND H. ZAHNER. Journal of Tropical Medicine and Hygiene 33: 792±799. 2002. Development of Eimeria bovis in vitro: Suitability of several CHOBOTAR, B., H. D. DANFORTH, AND R. ENTZEROTH. 1993. Ultrastruc- bovine, human and porcine endothelial cell lines, bovine fetal gas- tural observations of host-cell invasion by sporozoites of Eimeria trointestinal, Madin-Darby bovine kidney (MDBK) and African papillata in vivo. Parasitology Research 79: 15±23. green monkey kidney (VERO) cells. Parasitology Research 88: DANFORTH, H. D., B. CHOBOTAR, AND R. ENTZEROTH. 1984. Cellular 301±307. pathology in mouse embryonic brain cells following in vitro pen- LONG,P.L.,AND C. A. SPEER. 1977. Invasion of host cells by Coccidia. etration by sporozoites of Eimeria papillata. Zeitschrift fuÈr Paras- In Parasite invasion, A. E. R. Tayler and R. Muller (eds.). Black- itenkunde 70: 165±171. well Scienti®c, London, U.K., p. 1±16. ÐÐÐ, R. ENTZEROTH, AND B. CHOBOTAR. 1992. Scanning and trans- MCNEIL, P. L., M. F. S. CLARKE, AND K. MIYAKE. 1999. Cell wound mission electron microscopy of host cell pathology associated with assays. In Current protocols in cell biology, I. J. S. Bonifacino, M. penetration by Eimeria papillata sporozoites. Parasitology Re- Dasso, J. B. Harford, J. Lippincott-Schartz, and K. M. Yamada search 78: 570±573. (eds.). Wiley, New York, p. 12.4.1±12.4.15. È DAUGSCHIES, A., H. J. BURGER, AND M. AKIMURA. 1998. Apparent di- MOTA, M. M., G. PRADEL,J.P.VANDERBERG,J.C.R.HAFALLA,U. gestibility of nutrients and nitrogen balance during experimental FREVERT,R.S.NUSSENZWEIG,V.NUSSENZWEIG, AND A. RODRIGUEZ. infection of claves with Eimeria bovis. Veterinary Parasitology 77: 2001. Migration of Plasmodium sporozoites through cells before 93±102. infection. Science 291: 141±144. DUBEY, J. P., A. L. HATTEL,D.S.LINDSAY, AND M. J. TOPPER. 1988. ÐÐÐ, AND A. RODRIGUEZ. 2001. Migration through host cells by ap- Neonatal Neospora caninum infection in dogs: Isolation of the icomplexan parasites. Microbes and Infection 3: 1123±1128. causative agent and experimental transmission. Journal of the NICHOLS, B. A., AND G. R. O'CONNER. 1981. Penetration of mouse peri- American Veterinary Medical Association 193: 1259±1263. toneal macrophages by the protozoon Toxoplasma gondii. New ev- DUBREMETZ, J. F., N. GARCIA-REQUET,V.CONSEIL, AND M. N. FOUR- idence for active invasion and phagocytosis. Laboratory Investi- MAUX. 1998. Apical organelles and host-cell invasion by Apicom- gation 44: 324±335. plexa. International Journal for Parasitology 28: 1007±1013. SABIN, A. 1941. Toxoplasmic encephalitis in children. Journal of the ENTZEROTH, R., F. R. MATTIG, AND R. WERNER-MEIER. 1998. Structure and function of the parasitophorous vacuole in Eimeria species. American Medical Association 116: 801±814. International Journal for Parasitology 28: 1015±1018. SPEER, C. A., L. R. DAVIS, AND D. M. HAMMOND. 1971. Cinemicro- FAYER, R., AND D. M. HAMMOND. 1967. Development of ®rst-generation graphic observations of the development of Eimeria larimerensis schizonts of Eimeria bovis in cultured bovine cells. Journal of Pro- in cultured bovine cells. Journal of Protozoology 18: 11. tozoology 14: 764±772. ÐÐÐ, J. P. DUBEY,J.A.BLIXT, AND K. PROKOP. 1997. Time lapse FIEGE, N., D. KLATTE,D.KOLLMANN,H.ZAHNER, AND H. J. BUÈ RGER. video microscopy and ultrastructure of penetrating sporozoites, 1992. Eimeria bovis in cattle: Colostral transfer of antibodies and types 1 and 2 parasitophorous vacuoles, and the transformation of immune response to experimental infections. Parasitology Research sporozoites to tachyzoites in the VEG strain of Toxoplasma gondii. 78: 32±38. Journal of Parasitology 83: 565±574.

J. Parasitol., 90(5), 2004, pp. 1165±1169 ᭧ American Society of Parasitologists 2004

A Focus of Human Infection by Haplorchis taichui (Trematoda: Heterophyidae) in the Southern Philippines

Vicente Y. Belizario, Jr., Winifreda U. de Leon, Mary Joan J. Bersabe*, Purnomo†, J. Kevin Baird†, and Michael J. Bangs†‡, College of Public Health, University of the Philippines, Pedro Gil, Ermita, Manila, Philippines; *Center for Health Development for Southern Mindanao, Department of Health, Davao City, Philippines; †U.S. Naval Medical Research Unit No. 2 (NAMRU-2), Kompleks P2M/PLP, Jl. Percetakan Negara No. 29, Jakarta 10560, Indonesia; ‡To whom correspondence should be addressed. e-mail: [email protected]

ABSTRACT: We report an exceptionally high rate of infection by Hap- infection rates not exceeding 3%. Intestinal parasite surveys were con- lorchis taichui (Nishigori, 1924) in human populations on Mindanao ducted in 1998 and 1999 in 2 communities in Monkayo, Compostela Island, southern Philippines. This intestinal ¯uke is seldom encountered, Valley Province, southern Mindanao in connection with an outbreak of and this is the ®rst report of high prevalence of infection (36%) in intestinal (Belizario et al., 2000). Survey results indicated by H. taichui in the Philippines. The likely source of haplor- heterophyid infection rates of 17% and 16%, respectively, in the general chine infection has been linked to consumption of raw or undercooked population (Belizario et al., 2000, 2001). Infections were reported as freshwater ®sh containing infective metacercariae. The most common heterophyid (Heterophyidae) ¯ukes on the basis of egg size and mor- clinical symptoms appeared as upper abdominal discomfort or pain and phology, without de®nitive species identi®cation. This report examined borborygmi. Praziquantel (75 mg/kg divided in 3 doses in 1 day) was adult worms recovered after treatment of patients given praziquantel a well-tolerated and effective treatment for infection by H. taichui. and provides conclusive identi®cation of Haplorchis taichui (Tremato- da: Heterophyidae) as the parasitic agent. Additional epidemiological Food-borne are an important cause of disease in hu- background on the investigations conducted in 2000 from infection foci, mans that create an adverse economic impact in endemic tropical areas including symptomatology and treatment of H. taichui infections, is (Rim et al., 1994; WHO, 1995). Until recently, the prevalence of het- provided. All clinical aspects of the investigation were conducted in full erophyidiasis in humans had been reported as very low in the Philip- accordance with Good Clinical Practice Guidelines and provisions of pines. For example, less than 1% of more than 30,000 stool specimens the World Medical Association Declaration of Helsinki (amended, examined in nationwide surveys during 1970±1980 were found to con- 1996). tain heterophyid ova (Cross and Basaca-Sevilla, 1984), with site-speci®c The barangay (village) of San Isidro is in the municipality of Mon- 1166 THE JOURNAL OF PARASITOLOGY, VOL. 90, NO. 5, OCTOBER 2004

TABLE I. Age and sex distribution of study participants and those in- kayo, Compostela Valley Province, southern Mindanao (Region XI), fected with Haplorchis taichui infection in Barangay San Isidro, Mon- Philippines, and located inland ϳ100 km north of coastal Davao City. kayo, Compostela Valley, May 2000. Farming is the most common occupation, with corn as the major cul- tivated crop, followed by rice, coconut, and banana. Based on Rural Health Unit (RHU) records, nearly 35% of interviewed households (n Male Female Total ϭ 270) did not have dedicated toilet±latrine facilities. The people of Age No. No. (%) No. No. (%) No. No. (%) San Isidro obtain water from 2 primary sources, i.e., rainwater and the group examined infected examined infected examined infected Saug River. Rainwater is preferred for cooking and washing purposes. The river regularly provides freshwater ®sh, crabs, shrimps, and snails Ͻ5 13 3 (23.1) 23 3 (13.0) 36 6 (16.7) that supplement the diet with additional protein. A detailed description 5±14 36 11 (30.6) 26 6 (23.1) 62 17 (27.4) of the study site has been provided elsewhere (Belizario et al., 2000). 15±30 25 16 (64.0) 22 10 (45.4) 47 26 (55.3) A cross-sectional, active case detection survey was conducted in May 31±45 34 17 (50.0) 29 11 (37.9) 63 28 (44.4) 2000 in San Isidro. Health interviews and stool samples were obtained 46±60 8 2 (25.0) 9 2 (22.2) 17 4 (23.5) from residents with a history of recent bowel disturbance (abdominal discomfort or pain, or , or both). The principal complaints, Ͼ60 6 2 (33.3) 11 4 (36.4) 17 6 (35.3) signs, and symptoms were noted, and a fresh stool specimen was ob- Total 122 51 (41.8) 120 36 (30.0) 242 87 (36.0) tained from each patient. Stool was examined as wet mounts using either the Kato thick smear (Kato±Katz) technique or the formalin±ether sedimentation technique (FEST) (Ash and Orihel, 1987; WHO, 1998). Processed stool samples were initially examined under the microscope

FIGURES 1±2. Haplorchis taichui (Nishigori) 1. Pyriform adult, glycerin whole mount, unstained, showing transverse rows of scale-like spines on . Bar: 200 ␮m. 2. Carmine-stained anterior portion of adult worm showing subterminal oral and ventrogenital complex (small arrow) containing ventral sucker armed with prominent spines. Bar: 100 ␮m. RESEARCH NOTES 1167

FIGURES 3±4. Haplorchis taichui 3. Ventral sucker with crescentic group of 14 hollow spines (sclerites). Bar: 25 ␮m. 4. Operculated eggs (30±35 ␮m long, 15±18 ␮m wide) from stool showing prominent ``shoulders'' at apex and thickened posterior prominence. Bar: 30 ␮m. by trained microscopists assigned to the local government health facil- yr of age (mean 27.2). Infection prevalence was greatest (55.3%) in the ities, with on-site cross-checking conducted by staff from the Diagnostic 15±30-yr-old age group, followed by the 31±45-yr-old cohort, together Parasitology Laboratory, College of Public Health, University of the representing 62% of the sampled population found infected among the Philippines Manila (UP-CPH). A subsequent independent quality con- 6 age groups. Infection prevalence was generally higher in age groups trol of sample results was conducted by UP-CPH. Using the Kato±Katz 5 yr and older, although 16.7% of children less than 5 yr of age had H. technique, heterophyid eggs in stool were counted along with other taichui infection. Overall, male subjects had a higher, but not statisti- helminths (, , Capillaria philip- cally signi®cant (chi-square, P Ͻ 0.05), infection prevalence compared pinensis, and eggs) (Belizario et al., 2001). Heterophyid eggs with females (30%). The majority of cases (71%) had moderate to heavy were counted on a standardized template grid and multiplied by a factor infection burdens, particularly patients Ͼ15 yr of age. Most cases in of 24 to approximate the number of eggs present per gram (epg) of age groups below 15 yr had light infections. The infection burden (es- feces. A classi®cation of infection intensity (`burden') was used to rep- timated egg density) ranged from 24 to 26,256 epg, with a mean (geo- resent light (1±100 epg), moderate (101±1,000 epg), and heavy (Ͼ1,000 metric) egg count of 256 epg. epg) infections (Belizario et al., 2001). In San Isidro, 83 patients were interviewed for clinical signs and Sixty-seven patients infected with H. taichui were given commer- symptoms associated with H. taichui infection. A review of their his- cially available praziquantel (Distocide௡, Shin Poong Pharm. Co., Ltd., tories showed that 38 (45.8%) patients complained of peptic ulcer±like Seoul, Republic of Korea), 75 mg/kg, divided in 3 doses in 1 day. Stool symptoms, with upper abdominal discomfort or pain reported by 35 specimens were collected from 35 patients between 7 and 14 days after (42.2%), followed by borborygmi in 20 (24.1%) cases. Other com- treatment and examined using the Kato±Katz technique. The Monkayo plaints included nausea, chronic diarrhea, and weight loss. Fourteen RHU microscopist performed the initial examination in the ®eld, and (16.9%) patients reported no complaints and were considered asymp- the 10% formalin-®xed specimens were later forwarded to the UP-CPH tomatic. Although this was not a case±control study, most symptoms Diagnostic Parasitology Laboratory for crosschecking and con®rmation. associated with H. taichui infection quickly resolved after completing Patient response to drug treatment was categorized as one of the fol- the 1-day praziquantel treatment. lowing: (1) clinical cure, i.e., absence of eggs on repeat stool exami- Thirty-®ve (52.2%) patients submitted specimens for follow-up stool nation; (2) clinical improvement, i.e., lower intensity of associated examination after treatment. Using the Kato±Katz technique, 11 patients symptoms and infection (eggs) on repeat examination; (3) clinical fail- (31.4%) were found to have at least 1 helminth species infection. Only ure, i.e., similar or higher intensity of infection on repeat stool exami- 1 patient (2.9%) had stool containing heterophyid eggs. The arithmetic nation; and (4) indeterminate, i.e., no repeat stool examination possible and geometric means of Haplorchis egg reduction in stool were 99.3% (patient loss to follow-up or noncompliance). and 98.4%, respectively, with an overall cure of 97%. Similarly, com- Use of the Kato±Katz method or FEST revealed that 87 patients had plete clinical cure was seen in 34 of 35 (97%) patients, with signi®cant evidence of heterophyidiasis, i.e., an overall infection rate of 36.0% clinical improvement seen in the 1 patient who did not completely re- (Table I). From fresh stool, the Kato±Katz method revealed 31% of cover. None of the treated follow-up patients exhibited clinical failure. samples with heterophyid ova compared with 13.6% for FEST. Hap- Clinical outcome was indeterminate in 40 (53.3%) of the treated patients lorchis taichui infection was seen in all age groups from 19 mo to 73 because of loss to follow-up. In agreement with Radomyos et al. (1998), 1168 THE JOURNAL OF PARASITOLOGY, VOL. 90, NO. 5, OCTOBER 2004

1-day praziquantel administration appears well tolerated and effective itrix), dalag (O. striatus), and buriring (Poecilia sp). Other popular against H. taichui and could be considered for use in a mass treatment± methods of preparing and cooking ®sh, such as sabaw (boiling for sev- control campaign. eral minutes) and sugba (grilling over charcoal), may result in incom- Fresh stool specimens were obtained after treatment with praziquantel plete cooking of ¯esh and be an insuf®cient means to kill the encysted and ®xed in 10% formalin for later study. For the identi®cation of the metacecariae. The relatively lower prevalence of infection in sampled heterophyid specimens, formalin-preserved stool was prepared as a groups below 15 yr of age may re¯ect a lesser tendency to consume standard direct wet mount and stained with iodine for detection of ova. raw food preparations compared with adults. Egg morphology identi®ed a heterophyid-like origin but could not pro- The high prevalence of H. taichui found in the southern Philippines vide de®nitive species diagnosis. Formalin-preserved adult ¯ukes were would likely have gone undetected had it not been for investigations washed in physiological saline solution, mounted whole, either un- on intestinal capillariasis in the area. More recent investigations have stained or stained with carmine red, and mounted in Canada balsam shown the infection to have a wider distribution in Mindanao than pre- (Ash and Orihel, 1987). The condition of adult worms recovered from viously reported (V. Y. Belizario Jr., pers. comm.). The underreporting formalin-preserved stool varied from excellent to slight dis®gurement of unusual infections by clinicians is not uncommon because of a lack of the integument. In most cases, preserved worms expelled after treat- of familiarity with most trematode infections and the inexperience of ment with praziquantel could be adequately stained and identi®ed. Pho- laboratory diagnosticians detecting small helminth eggs. A number of tographic documentation was made using a Nikon UFX-DX microscope factors may have contributed to the high prevalence of H. taichui in system (Figs. 1±4). All adult worms examined (Figs. 1±2) conformed Monkayo. It has also been speculated that there may have been an to taxonomic descriptions for the genus Haplorchis Looss 1899 and increased risk of infection with this parasite, in addition to the C. phi- speci®cally to H. taichui (Pearson and Ow-Yang, 1982). Of particular lippinensis outbreak, a associated with the consumption of differential diagnostic importance is the ventrogenital complex with a raw or undercooked freshwater ®sh, as an indirect result of the severe distinctive ventral sucker containing a crescentric group of 12±16 hol- drought conditions during the 1997±1998 El NinÄo Southern Oscillation low sclerites, some ending in a laterodorsal notch (Fig. 3). Operculated, climatic event. During that time, food crops were in short supply, which thick-shelled eggs (30±35 ␮m long, 15±18 ␮m wide) examined from likely resulted in an increased reliance on nutrition derived from the stool were within the described size range for this species and clearly river. It would seem reasonable to conclude that periodic climatic anom- showed the prominent ``shoulders'' at apex and thickened posterior alies and conditions may in¯uence exposure risk to helminthic infec- prominence (Fig. 4). tions by changing human behavior and food habits. If such associations In Asia, H. taichui (Nishigori, 1924) has been reported in exist, the public's awareness of increased risk during times of environ- hosts from Iraq, India, Sri Lanka, Taiwan, Thailand, Malaysia, and the mental stress, together with community education on proper hygienic Philippines (Faust and Nishigori, 1926; Pearson and Ow-Yang, 1982; handling and preparation of freshwater ®sh and the sanitary disposal of Radomyos et al., 1983, 1998; Waikagul, 1991; Sukontason et al., 2000). human feces, should help diminish the risk of heterophyid infections In Southeast Asia, human infections of H. taichui have only been de- and other food-borne related parasitic zoonoses (WHO, 1995, 1998). scribed from Thailand and the Philippines (Cross, 1974; Waikagul, Department of Health efforts continue in Mindanao to identify, treat, 1991). This trematode species has been reported from natural and ex- and educate affected communities to combat this unpleasant and poten- perimental animal infections in the Philippines (Pearson and Ow-Yang, tially dangerous helminthic infection. 1982). Natural molluscan (Melania juncea Lea) and picine (Puntius spp. We extend special thanks for assistance provided by the Center for and Ophiocephalus striatus Bloch) species serving as intermediate hosts Health Development for Southern Mindanao, Department of Health, the of this parasite have been described from Luzon and Mindanao (Tub- local government units of Monkayo, Compostela Valley, and Salcedo angui, 1947; Velasquez, 1973a, 1973b). The common source of infec- Eduardo of the University of the Philippines, Los BanÄos. This study tion for humans likely involves a variety of freshwater ®sh that serve was supported by the Essential National Health Research, Department as a second intermediate host and harbor infective metacercaria en- of Health, RP and the Committee on Research Implementation and De- cysted in muscle tissue. Humans, domestic dogs, cats, swine, and the velopment, College of Medicine, University of the Philippines Manila, Bubulcus ibis coromandus (Boddaert) have been reported as de®n- with assistance from the U.S. Naval Medical Research and Develop- itive hosts of H. taichui in the Philippines (Velasquez, 1973a). The full ment Command, Navy Department. The opinions of the authors do not range of intermediate hosts and parasite distribution in the Philippines purport to re¯ect the positions of the U.S. Navy or the Department of requires more investigation. Health of the Republic of the Philippines. Haplorchine infections have generally been rare in humans but re- main a concern because of occasional reports of severe pathologic LITERATURE CITED changes in tissue (e.g., myocarditis) resulting from the minute eggs of heterophyids penetrating through the intestinal wall and lodging in ex- AFRICA, C. M., W. E. D DE LEON, AND E. Y. GARCIA. 1935. Intestinal traintestinal sites (Africa et al., 1935, 1940). High prevalence (63%) of heterophyidiasis with cardiac involvement: A contribution to eti- H. taichui in humans has been reported as the most common intestinal ology of heart failure. Journal of the Philippine Islands Medical ¯uke in northern Thailand (Radomyos et al., 1998), indicating that this Association 15: 358±361. parasite can pose a signi®cant health risk in certain endemic foci. In ÐÐÐ, ÐÐÐ, ÐÐÐ. 1940. Visceral complications in intestinal het- 1998, intestinal was identi®ed as the second leading cause erophyidiasis of man. Acta Medicine Philippina Monograph Series of morbidity in Monkayo. Hookworm was the most common helminth 1: 1±132. identi®ed followed by C. philippinensis, heterophyid species, A. lum- ASH,L.R.,AND T. C. ORIHEL. 1987. Parasites: A guide to laboratory bricoides, and T. trichiura (Monkayo RHU Records, 1998). Intestinal procedures and identi®cation. American Society Clinical Patholo- parasite surveys conducted after the discovery of capillariasis in the gists Press, Chicago, Illinois, 328 p. area in 1998 identi®ed 17 barangays in Mindanao with heterophyid BELIZARIO, V. Y., M. J. BERSABE,W.U.DE LEON,V.Y.HILOMEN,G.V. infections (Belizario et al., 2001), representing the largest cluster of PALLER,A.D.DE GUZMAN, AND M. G. BUGAYONG. 2001. Intestinal endemic heterophyidiasis so far detected in the Philippines and outside heterophyidiasis: An emerging food-borne parasitic zoonosis in Thailand. Based on these ®ndings, we suspect many of the infections southern Philippines. Southeast Asian Journal of Tropical Medicine were likely the result of H. taichui, although 5 other members in the and Public Health 32: 36±42. Haplorchinae (Haplorchis Looss, Procerovuom Onji and Nishio, and ÐÐÐ, W. U. DE LEON,D.G.ESPARAR,J.M.GALANG,J.FANTONE, Onji and Nishio) have also been reported to infect hu- AND C. VERDADERO. 2000. Compostela Valley: A new endemic fo- mans in the Philippines (Waikagul, 1991). cus for capillariasis philippinensis. Southeast Asian Journal of Local eating habits almost certainly contributed to the high preva- Tropical Medicine and Public Health 31: 479±481. lence of H. taichui. The Saug River is a major source of fresh food for CROSS, J. H. 1974. Diagnostic methods in intestinal ¯uke infections: A the local residents. Most people are fond of consuming a variety of review. In Diagnostic methods for important and am- freshwater ®sh, shrimps, crabs, and frogs. In particular, many freshwater oebiasis in Southeast Asia and the Far East, C. Harinsuta and D. ®sh are eaten raw and seasoned only with salt and vinegar, a local C. Reynolds (eds.). SEAMEO-TROPMED Project, Bangkok, Thai- preparation called kinilaw. Among the local varieties of ®sh eaten are land, p. 87±108. species of tilapia (Oreochromis sp.), paitan (Hypophthalmichthys mol- ÐÐÐ, AND V. B ASACA-SEVILLA. 1984. Biomedical surveys in the Phil- RESEARCH NOTES 1169

ippines. Monograph SP-47. United States Naval Medical Research SUKONTASON, K. L., K. SUKONTASON,B.KUNTALUE,N.BOONSRIWONG, Unit 2, Manila, Philippines, 117 p. S. PIANGJAI,U.CHAITHONG, AND P. V ANITTANAKOM. 2000. Surface FAUST,E.C.,AND M. NISHIGORI. 1926. The life cycles of two new ultrastructure of excysted metacercariae of Haplorchis taichui species of heterophyidae, parasitic in and . Journal (Trematoda: Heterophyidae). Southeast Asian Journal of Tropical of Parasitology 13: 91±132. Medicine and Public Health 31: 747±754. PEARSON,J.C.,AND C. K. OW-YANG. 1982. New species of Haplorchis TUBANGUI, M. A. 1947. A summary of the parasitic worms reported from Southeast Asia together with keys to the Haplorchis-group of from the Philippines. Philippine Journal of Science. 76: 225±322. heterophyid trematodes of the region. Southeast Asian Journal of VELASQUEZ, C. C. 1973a. Observations on some heterophyidae (Trem- Tropical Medicine and Public Health 13: 35±60. atoda: ) encysted in Philippine ®shes. Journal of Parasitol- ogy 59: 77±84. RADOMYOS, B., D. BUNNAG, AND T. H ARINASUTA. 1983. Haplorchis pum- ilio (Looss) infection in man in northeastern Thailand. Southeast ÐÐÐ. 1973b. Intramolluscan stages of Haplorchis taichui (Nishigori) in Melania juncea Lea in the Philippines. Journal of Parasitology Asian Journal of Tropical Medicine and Public Health 14: 223± 59: 281. 227. WAIKAGUL, J. 1991. Intestinal ¯uke infections in Southeast Asia. South- ÐÐÐ, T. WONGSAROJ,P.WILAIRATANA,P.RADOMYOS,R.PRAEVANICH, east Asian Journal of Tropical Medicine and Public Health 22 V. M EESOMBOON, AND P. J ONGSUKSUNTIKUL. 1998. Suppl: 158±162. and intestinal ¯uke infections in northern Thailand. Southeast WHO. 1995. Control of food-borne trematode infections. Technical Re- Asian Journal of Tropical Medicine and Public Health 22: 123± port Series 849. World Health Organization, Geneva, Switzerland, 127. 168 p. RIM, H.-J., H. F. FARAG,S.SORNMANI, AND J. H. CROSS. 1994. Food- WHO. 1998. Guidelines for the evaluation of soil-transmitted helmin- borne trematodes: Ignored or emerging? Parasitology Today 10: thiasis and at community level. WHO/CTD/SIP/ 207±209. 98.1. World Health Organization, Geneva, Switzerland, 48 p.

J. Parasitol., 90(5), 2004, pp. 1169±1171 ᭧ American Society of Parasitologists 2004

Observations on Myiasis by the Calliphorids, Bufolucilia silvarum and Bufolucilia elongata, in Wood Frogs, Rana sylvatica, From Southeastern Wisconsin

Matthew G. Bolek and John Janovy Jr., School of Biological Sciences, University of Nebraska–Lincoln, Lincoln, Nebraska 68588. e-mail: [email protected]

ABSTRACT: Larvae of certain species of blow¯ies (Calliphoridae) can In North America, 2 species of Bufolucilia have been reported to cause myiasis in frogs and toads, but there are few reports from North cause myiasis in 3 species of amphibians. Bufolucilia elongata caused American amphibians. Of these, most are from toads (bufonids). In this myiasis in 1 boreal toad, Bufo boreas boreas, and 6 American toads study, we observe primary myiasis in a population of juvenile wood from Colorado and Wisconsin, respectively, whereas B. silvarum was frogs, Rana sylvatica, collected on 22±23 August 2003, from south- reported from 48 bullfrogs, Rana catesbeiana, in California, 1 American eastern Wisconsin and compare our observations with previous studies toad from Nova Scotia (Canada), and 1 American toad from Ontario, on myiasis from toads. Two (5%) of 39 frogs were infected by the blow Canada (James and Maslin, 1947; Hall, 1948; Anderson and Bennett, ¯y Bufolucilia silvarum, with an intensity of 28 and 31, whereas 1 1963; Bleakney, 1963; Briggs, 1975). More recently, studies on the life (2.5%) of 39 frogs was infected by the blow ¯y Bufolucilia elongata history of B. silvarum from 9 American toads examined by Bolek and with an intensity of 14. We found that (1) B. silvarum lay eggs on Coggins (2002) indicate that these ¯ies deposit eggs on the back and healthy wood frogs, (2) eggs hatch, with ®rst-instar maggots penetrating ¯anks of the amphibian host. The larvae hatch and migrate under the under the skin, (3) maggots develop to mature third instars within 13± skin where they form a single lesion in the paratoid glands, back, neck, 16 hr of egg hatching, (4) maggots kill the host within 7±47 hr of egg and front or hind legs, where development takes place, and all infected hatching, and (5) maggots consume the entire frog carcass reducing it toads die within 1 day to 2 wk of infection (Bolek and Coggins, 2002). to bones within 42±59 hr of egg hatching. Our observations on the time No other information is available on these ¯ies infecting North Amer- of death and how quickly carcasses of wood frogs were consumed by ican amphibians. In their review on the life history of B. silvarum, these maggots compared with previous studies on toads suggest that Bolek and Coggins (2002) hypothesized that juvenile terrestrial anurans ®nding infected juvenile wood frogs may be uncommon. Therefore, that are diurnal and overlap in their ecology with this ¯y species may myiasis by these ¯ies on wood frogs and other small terrestrial anurans be more prone to parasitism by B. silvarum than is currently known. In may be a phenomenon that is much more common than is currently this study, we report observations on the life history of B. silvarum and observed. This is the ®rst report of B. silvarum and B. elongata causing B. elongata in juvenile wood frogs, R. sylvatica, from southeastern Wis- myiasis in wood frogs. consin and compare these data with infections by these 2 species of ¯ies in American toads examined by Briggs (1975) and Bolek and Cog- Myiasis in amphibians is caused by larvae of dipterans from Sarco- gins (2002) from Wisconsin. Our observations indicate that these ¯ies phagidae, Calliphoridae, and Chloropidae, some of which can cause may kill small frogs in the genus Rana and consume the carcass more substantial mortality in their amphibian hosts (Dasgupta, 1962; Crump rapidly than toads and therefore may be less commonly observed par- and Pounds, 1985; Schell and Burgin, 2001; Bolek and Coggins, 2002). asitizing these hosts in nature. These observations may account for the During 2003, the blow¯ies, Bufolucilia silvarum and Bufolucilia elon- numerous reports of myiasis of Bufolucilia spp. and other calliphorids gate, were found infesting juvenile wood frogs, Rana sylvatica,in such as Phaenicia sericata and Lucilia illustris in toads and rarely in southeastern Wisconsin. Members of this genus have been reported as other species of North American frogs (Anderson and Bennett, 1963; obligate or facultative parasites of amphibians, particularly species of Stewart and Foote, 1974; Bolek and Coggins, 2002). Bufo, but there are few reports of these ¯ies causing myiasis in North Thirty-nine juvenile wood frogs were collected by hand in the woods American ranid frogs (see Bolek and Coggins, 2002). Our observations during the day on 22±23 August 2003 at the University of Wisconsin± on the rapid consumption of wood frog carcasses by these maggots Milwaukee ®eld station, Ozaukee County, Wisconsin (43Њ23ЈN, suggest that myiasis on small terrestrial anurans may be more common 88Њ2ЈW). All frogs were measured (2.97 Ϯ 0.27 cm) and examined for in North America than is currently observed. external lesions, eggs, or maggots. Frogs suspected of being infected