RESEARCH NOTES 185 human philophthalmosis was reported from the United States (Gutierrez 1981. Ein ausgereifter saugwurm der gattung unter et al., 1987). The patient was a 66-yr-old man, and the parasite obtained der Bindehaut des Menschen. Klinische Monatsblatter fur Augen- from his left eye was identi®ed as P. gralli, although the position of heilkunde 179: 373±375. the cirrus sac in the specimen was not the same as that described for KANEV, I., P. M. NOLLEN,I.VASSILEV,V.RADER, AND V. D IMITROV. this species. A case was also reported from Israel (Lang et al., 1993). 1993. Redescription of Philophthalmus lucipetus (Rudolphi, 1819) The patient was a 13-yr-old girl, and the worm obtained from her right (: Philophthalmidae) with a discussion of its identity and eye was apparently mature, but the species was not identi®ed. Our case characteristics. Annals of the Naturalist Museum, Wien. 94/95 B: is the ®rst in Mexico, the ®rst human case of P. lacrimosus infection 13±34. in the world, and the 24th case of human philophthalmosis overall. LANG, Y., Y. WEISS,H.GARZOZI,D.GOLD, AND J. LENGY. 1993. A ®rst The authors are grateful to Dr. A. Villegas-Alvarez, who extracted instance of human philophthalmosis in Israel. Journal of Helmin- the worm; QFB Javier Sedano Millan for his collaboration; and Dr. thology 67: 107±111. Scott Monks, Universidad Autonoma del Estando d Hidalgo, Dra. Vir- MARKOVIC, A. 1939. Der erste fall von Philophthalmose beim Mensch- ginia Leon Regagnon and M. en C. Luis Garcia Prieto, Instituto de en. Albrecht von Graefes Archives fur Ophthalmologie 140: 515± Biologia, UNAM, and Biol. Maria Antonieta Arizmendi, Facultad de 526. Ciencias, UNAM, for their valuable help and advice. MIMORI, T., H. HIRAI,T.KIFUNE, AND K. INADA. 1982. Philophthalmus sp. (trematoda) in a human eye. American Journal of Tropical Med- LITERATURE CITED icine and Hygiene 31: 859±861. NASIR,P.,AND M. T. DIAZ. 1972. Avian ¯ukes of Venezuela. Rivista di BRAUN, M. G. C. C. 1902. Fascioliden der Vogel. Zoologische Jahr- Parasitologica, Roma 33: 245±276. bucher 16: 1±162. NOLLEN,P.M.,AND I. KANEV. 1995. The and biology of DISSANIKE,A.S.,AND D. P. BILIMORIA. 1958. On an infection of a human Philophthalmid eye ¯ukes. Advances in Parasitology 36: 206±271. eye with Philophthalmus sp. in Ceylon. Journal of Parasitology 32: TEIXEIRA DE FREITAS, J. F. 1955. Sobre dois trematodeos parasitos de 115±118. aves Philophthalmus lacrimosus Braun, 1902 e Renicola miran- GUTIERREZ, Y., H. E. GROSSNIKLAUS, AND W. L. ANNABLE. 1987. Human daribeiroi n. sp. Arquivos do Museu Nacional 42: 585±610. conjunctivitis caused by the bird parasite. American Journal of YAMAGUTI, S. 1958. Systema Helminthum. The digenetic trematodes of Ophthalmology 104: 417±419. Vertebrates, Vol. I, Parts 1±2. Interscience Publications, New York, KALTHOFF, H., K. JANITSCHKE,S.MRAVAK,W.SCHOPP, AND H. WERNER. New York, 1575 p.

J. Parasitol., 89(1), 2003, pp. 185±188 ᭧ American Society of Parasitologists 2003

Infectivity of Spores Stored in Water at Environmental Temperatures

X. Li, R. Palmer, J. M. Trout, and R. Fayer*, Waste Pathogen Laboratory, Agricultural Research Service, United States Department of Agriculture, 10300 Baltimore Avenue, Beltsville, Maryland 20705-2350. *To whom correspondence should be addressed. e-mail: [email protected]

ABSTRACT: To determine how long waterborne spores of Encephali- al., 1998). Although the actual routes of transmission are not well tozoon cuniculi, E. hellem, and E. intestinalis could survive at environ- known, it is possible that the infective spore stage in urine or feces can mental temperatures, culture-derived spores were stored in water at 10, contaminate the environment and enter surface water used for recreation 15, 20, 25, and 30 C and tested for infectivity in monolayer cultures of or drinking water (Sparfel et al., 1997; Dowd et al., 1998; Cotte et al., Madin Darby bovine kidney (MDBK) cells. At 10 C, spores of E. in- 1999; Fournier et al., 2000). Isolation of spores and molecular detection testinalis were still infective after 12 mo, whereas those of E. hellem of microsporidia in ditch water and river water along with circumstantial and E. cuniculi were infective for 9 and 3 mo, respectively. At 15 C, evidence of waterborne transmission is accumulating (reviewed by Bry- spores of the same species remained infective for 10, 6, and 2 mo, and an and Schwartz, 1999). The longevity of -infecting microsporidia at 20 C, for 7, 5, and 1 mo, respectively. At 25 C, spores of E. intes- stored under different conditions has been studied (Oshima, 1964; Hen- tinalis and E. hellem were infective for 3 mo, but those of E. cuniculi ry and Oma, 1974; Fuxa and Brooks, 1979; Teetor-Barsch and Kramer, were infective for only 3 wk. At 30 C, the former 2 species were in- 1979), but data on the effect of environmental temperatures on longevity fective for 3 wk and 1 mo, respectively, and the latter species for only of spores of species that infect and humans are sparse. Such 1 wk. These ®ndings indicate that spores of different species of En- information can be extremely helpful in understanding the epidemiology cephalitozoon differ in their longevity and temperature tolerance, but at and evaluating the risk of transmission. The present study was designed temperatures from 10 to 30 C, all 3 have the potential to remain infec- to investigate the effect of temperature and storage in water on infec- tive in the environment long enough to become widely dispersed. tivity of spores of E. cuniculi, E. hellem, and E. intestinalis by testing in cultured mammalian cells. Of the 13 species of microsporidia that infect humans, Encephalito- Spores of E. cuniculi and E. hellem were purchased from the Amer- zoon cuniculi, E. hellem, E. intestinalis, and E. bieneusi have been re- ican Type Culture Collection, Manassas, Virginia (ATCC Nos. 50502 ported in a variety of domesticated animals (Deplazes et al., 1996; and 50451, respectively). Spores of E. intestinalis originally isolated Mans®eld et al., 1997; Mathis et al., 1999; Rinder et al., 2000) and from an acquired immune de®ciency syndrome (AIDS) patient and wildlife (Hersteinsson et al., 1993; Mathis et al., 1996; Thomas et al., grown in culture (Didier et al., 1996) were provided by Elizabeth Didier, 1997). Encephalitozoon cuniculi has been identi®ed in wild, laboratory, Tulane Regional Primate Research Center, Covington, Louisiana. En- and pet rabbits, wild and laboratory rats and mice, dogs, cats, foxes, cephalitozoon cuniculi and E. intestinalis were propagated in monolayer mink, and a variety of monkeys (reviewed by Bryan and Schwartz, cultures of MDBK cells, and E. hellem was propagated in human lung 1999; Deplazes et al., 2000). Encephalitozoon hellem and E. hellem± ®broblasts (WI-38) in T-75 ¯asks. The MDBK cells were cultured in like microsporidia have been found in psittacine birds in the United Dulbecco modi®ed Eagle medium (DMEM) supplemented with 1% States and Australia as well as in budgerigar chicks and wild, yellow- nonessential amino acids (NEAA), 2% N-2-hydroxyethylpiperazine-NЈ- streaked lory (reviewed by Bryan and Schwartz, 1999; Deplazes et al., 2-ethane-sulfonic acid (HEPES), 5% fetal calf serum (FCS), and 1%

2000). Encephalitozoon intestinalis spores were identi®ed in feces of penicillin or streptomycin in a 5% CO2 atmosphere incubator at 35 C. farm animals in Mexico (dog, pig, cow, and goat) (Bornay-Llinares et WI-38 cells were similarly cultured in minimum essential medium 186 THE JOURNAL OF PARASITOLOGY, VOL. 89, NO. 1, FEBRUARY 2003

TABLE I. Number of cultures with intracellular development of microsporidia out of 2 inoculated with spores of Encephalitozoon intestinalis (I), E. hellem (H), and E. cuniculi (C) after storage for 1 wk to 12 mo at environmental temperatures from 10 to 30 C.

Temperature 10 C 15 C 20 C 25 C 30 C Time IHC IHC IHC IHC IHC

1wk ND* ND 2 ND ND 2 ND ND 2 ND ND 2 2 ND 2 2wk ND ND 2 ND ND 2 ND ND 2 ND ND 2 2 ND 0 3wk ND ND 2 ND ND 2 ND ND 2 ND ND 2 2 ND 0 1mo 2 2 2 2 2 2 2 2 2 2 2 0 0 2 0 2mo 2 2 2 2 2 2 2 2 0 2 0 0 0 0 0 3mo 2 2 1 2 2 0 2 2 0 2 1 0 0 0 0 4mo 2 2 0 2 2 0 2 2 0 0 0 0 0 0 0 5mo 2 2 0 2 1² 0 2 1 0 0 0 0 0 0 0 6mo 2 2 0 2 1² 0 2 0 0 0 0 0 0 0 0 7mo 2 2 ND 2 0² ND 1 0 ND 0 0 ND 0 0 ND 8mo 2 2 ND 1 0² ND 0 0 ND 0 0 ND 0 0 ND 9mo 2 1 ND 2 0² ND 0 0 ND 0 0 ND 0 0 ND 10 mo 2 0 ND 2 0² ND 0 0 ND 0 0 ND 0 0 ND 11 mo 2 1 ND 0 0² ND 0 0 ND 0 0 ND 0 0 ND 12 mo 2 ND ND 0 ND ND 0 ND ND 0 ND ND 0 ND ND

* ND, not done. ² Results based on observation of 1 culture inoculated with spores.

(MEM) supplemented with 1% NEAA, 2%HEPES, 10% FCS, 1% pen- chromotrope method and examined by bright®eld microscopy. Enceph- icillin or streptomycin, 1% L-glutamine, and 1% sodium pyruvate. Mi- alitozoon cuniculi, E. hellem, and E. intestinalis had been stored for 2, crosporidia were harvested from culture supernatant by centrifuging at 8, and 10 mo, respectively, at the time that they were examined for 1,500 g for 15 min and were resuspended in deionized water. An aliquot ®lament extrusion. of organisms was stained with calco¯uor white (Becton Dickinson Mi- Spore infectivity for all 3 species decreased with increasing temper- crobiology Systems, Sparks, Maryland), pipetted into a well of heavy, ature or increased time of storage (Table I). Spores of E. intestinalis te¯on-coated 3-well glass microscope slide (Cel-Line, Erie Scienti®c, stored at 10 C were still infective after storage for 12 mo, but at higher Portsmith, New Hampshire), and counted with the aid of an epi¯uoresc- temperatures infectivity was lost more quickly until at 30 C, spores were ence microscope. Within each species examined, the bodies appeared not infective after 1 mo of storage. Similar patterns were observed for to be of fairly uniform size and shape, and with the exception of 2 or E. hellem and E. cuniculi. However, E. cuniculi lost infectivity more 3 lighter-staining bodies per 100 organisms, staining intensity also ap- rapidly than did E. hellem, which lost infectivity more rapidly than did peared to be uniform, suggesting that virtually all the forms examined E. intestinalis (Table I). Intracellular clusters of developing microspor- were in the spore stage. Spore concentrations were adjusted with de- idia were found in all positive control wells, but none was found in ionized water, and spores were pipetted into microcentrifuge tubes that negative control wells. were capped and held in each of 5 circulating water baths at 10, 15, Spores examined for extrusion of the polar ®lament by DIC micros- 20, 25, and 30 C, respectively. Temperatures were monitored twice dai- copy and quick-hot Gram-chromotrope stain did not show the presence ly, except during weekends. At weekly intervals for 4 wk, and then at of extruded ®laments. monthly intervals (28±30 days), 1 tube of each species of microsporidia Microsporidia spores found in different surface waters (Avery and was removed from each water bath, and an equivalent quantity of 5 ϫ Undeen, 1987; Dowd et al., 1998) exhibit resistance to environmental 10 5 spores was aspirated from that tube, and 2.5 ϫ 10 5 spores were conditions in such waters (Kucerova-Pospisilova et al., 1999) and have inoculated into each of the 2 wells of an 8-well Lab-Tek chamber slide been linked to waterborne infections (Cotte et al., 1999). Knowledge of (Nalge Nunc International, Naperville, Illinois), with each well contain- the in¯uence of time and temperature on infectivity of microsporidia is ing a monolayer of MDBK cells. After 6 days incubation at 35 C in a necessary for evaluating the risk of waterborne contamination and for

5% CO2 atmosphere incubator, culture medium was removed, and cul- maintaining laboratory stocks of organisms. Interest in spore survival tures in all 8 wells were ®xed in situ with 100% methanol for 30 min dates back nearly 9 decades (White, 1919). Most efforts to determine at room temperature. The plastic frame and silicon gasket that formed the effects of time against those of temperature on the viability of mi- the wells were removed, and each slide was air dried and stained by crosporidia spores have used spores of , a microsporidian the quick-hot Gram-chromotrope method (Moura et al., 1997), a cov- parasite of bees. One study concluded that storage of N. apis spores at erslip was af®xed, and the entire area of cells within each well was 4 C for 6±14 mo did not impair infectivity for honey bees (Malone et examined by bright®eld microscopy. For each species at each time and al., 2001). Other studies with N. apis spores have shown that storage temperature interval, spores were considered infective on ®nding intra- in clean water at approximately4Corinliquid nitrogen resulted in cellular clusters of developing microsporidia in either of the 2 wells. only a very slow decrease in viability over several years (Kramer, 1970; At each time period, 2.5 ϫ 10 5 freshly harvested spores of each species Vavra and Maddox, 1976). Nosema apis spores stored in distilled water also were inoculated into each of 2 wells as positive controls, and 2 at 5 C for 12 mo produced infections in 75% of inoculated bees, those other wells that received no spore served as a negative control. These stored for 24 mo produced infections in 72% of bees, and even after 7 were processed and examined in the same manner as were the wells yr, 60% of bees became infected (Revell, 1960). In contrast, naked and that received stored spores. dried spores of N. apis stored in a refrigerator, in bacteria-contaminated To determine if spores lost infectivity because elevated temperatures water, or in bee cadavers lost viability signi®cantly in 2 yr (Bailey, stimulated extrusion of the polar ®lament before they were inoculated 1972). Similar ®ndings have been reported for other microsporidia of into cell cultures, spores were examined after storage at 30 C. Droplets invertebrates (Vavra and Maddox, 1976). Nosema apis spores in water of spores were placed on glass microscope slides and examined by lost viability steadily during a period of 21 days at 33 C (Malone et al., differential interference contrast (DIC) microscopy, whereas others were 2001) and lost viability faster at temperatures above 35 C (Kramer, dried on glass microscope slides and stained by the quick-hot Gram- 1970). Nosema apis spores in water contaminated with bacteria and held RESEARCH NOTES 187 at 37.5 C lost viability after 2±4 days, whereas those held at room L. M. Weiss (eds.). American Society for Microbiology, Washing- temperature or under refrigeration survived for 6 days to 3 mo, respec- ton, D.C., p. 502±516. tively (White, 1919). COTTE, L., M. RABODONIRINA,F.CHAPUIS,F.BAILLY,F.BISSUEL,C. Compared with N. apis, only 1 report provides data on the effect of RAYNAL,P.GELAS,F.PERSAT,M.A.PIENS, AND C. TREPO. 1999. temperature and long-term storage on infectivity of E. hellem or E. Waterborne outbreak of intestinal microsporidiosis in persons with intestinalis (Kucerova-Pospisilova et al., 1999). Data on E. cuniculi is and without human immunode®ciency virus infection. Journal of sparse. After 2 yr of storage at 4 C, E. cuniculi spores were still infective Infectious Diseases 180: 2003±2008. for SCID mice (Koudela et al., 1999). Encephalitozoon cuniculi spores DEPLAZES, P., A. MATHIS,C.MULLER, AND R. WEBER. 1996. Molecular in Medium 199 survived for 98 days at 4 C, 6 days at 22 C, and 2 days epidemiology of Encephalitozoon cuniculi and ®rst detection of at 37 C, as determined by infectivity for canine kidney cells in vitro Enterocytozoon bieneusi in faecal samples of pigs. Journal of Eu- (Waller, 1979). Another study found that E. cuniculi spores stored in karyotic Microbiology 43: 93S. culture medium remained infective for at least 24 days at 4 and 20 C ÐÐÐ, ÐÐÐ, AND R. WEBER. 2000. Epidemiology and zoonotic as- but lost infectivity after 12 days at 37 C (Shadduck and Polley, 1978). pects of microsporidia of mammals and birds. In Cryptosporidiosis In the present study E. cuniculi spores in deionized water appeared less and microsporidiosis, F. Petry (ed.). Karger, New York, New York, vigorous than those reported by Waller (1979) and Koudela et al. p. 236±260. (1999), with a few remaining infective for only 3 mo at 10 C. However, DIDIER, E. S., L. B. ROGERS,J.M.ORENSTEIN,M.D.BAKER,C.R. direct comparisons are not possible because the spores used in the pre- VOSSBRINCK,T.VAN GOOL,R.HARTSKEERL,R.SOAVE, AND L. M. sent study were not held at 4 or 5 C. BEAUDET. 1996. Characterization of Encepalitozoon (Septata) in- Infection with microsporidia begins with germination, when the polar testinalis isolates cultured from nasal mucosa and bronchoalveolar ®lament is triggered, everts from the spore, and forms a tube through lavage ¯uids of two AIDS patients. Journal of Eukaryotic Micro- which the spore content (sporoplasm) is expelled (Vavra and Larsson, biology 43: 34±43. 1999). It is not clear if all spores that undergo germination have the DOWD, S., C. GERBA, AND I. PEPPER. 1998. Con®rmation of the human potential to be infective. Arti®cially induced germination is technique pathogenic microsporidia Enterocytozoon bieneusi, Encephalito- dependent and may either fail to initiate extrusion of polar ®laments zoon intestinalis, and Vittaforma corneae in water. Applied and from spores that are potentially infective or stimulate extrusion of ®l- Environmental Microbiology 64: 3332±3335. aments from spores that lack infectivity. One study found that after FAYER, R. 1994. Effect of high temperature on infectivity of Crypto- arti®cial stimulation the germination rate of the ®sh parasite Loma sal- sporidium parvum oocysts in water. Applied and Environmental monae decreased from 51 to 0% after 100 days of storage in seawater Microbiology 60: 2732±2735. at 4 C (Shaw et al., 2000). Microscopic appearances differentiating in- ÐÐÐ, AND T. N ERAD. 1996. Effects of low temperature on viability tact spores from recently germinated spores, and from spores that had of Cryptosporidium parvum oocysts. Applied and Environmental lost extruded polar ®laments, have been reported (Kucerova-Pospisilova Microbiology 62: 1431±1433. et al., 1999). Germination was induced in spores of E. intestinalis, E. ÐÐÐ, J. M. TROUT, AND M. C. JENKINS. 1998. Infectivity of Crypto- hellem, and E. cuniculi stored in culture medium at 4 C for 48 mo sporidium parvum oocysts stored in water at environmental tem- (Kucerova-Pospisilova et al., 1999). Spores that retained infectivity for peratures. Journal of Parasitology 84: 1165±1169. cultured mammalian cells were found in only 1 of 6 pools of spores for FOURNIER, S., O. LIGUORY,M.SANTILLANA-HAYAT,E.GUILLOT,C.SAR- each species in 24-mo-old samples (Kucerova-Pospisilova et al., 1999). FATI,N.DUMOUTIER,J.MOLINA, AND F. D EROUIN. 2000. Detection In the present study spores were examined to determine if those stored of microsporidia in surface water: A one-yr follow-up study. FEMS at 30 C had extruded polar ®laments, which would render them non- Immunity and Medical Microbiology 29: 95±100. infective when inoculated into cell cultures. Although aqueous suspen- FUXA, J. R., AND W. M. BROOKS. 1979. Mass production and storage of sions of spores of E. cuniculi, E. hellem, and E. intestinalis were ex- Varirimorpha necatrix (Protozoa: Microsporidia). Journal of Inver- amined by DIC microscopy, and chromotrope-stained spores were ex- tebrate Pathology 33: 86±94. amined by bright®eld microscopy, no extruded ®lament was detected, HARP, J. A., R. FAYER,B.A.PESCH, AND G. J. JACKSON. 1996. Effect suggesting that factors other than extrusion of the ®lament were in- of pasteurization on infectivity of Cryptosporidium parvum oocysts volved in the loss of infectivity. Different methods applied in the present in water and milk. Applied and Environmental Microbiology 62: study might account for differences in observations, i.e., the lack of 2866±2868. visible extruded ®laments. HENRY,J.E.,AND E. A. OMA. 1974. Effects of prolonged storage of The present study demonstrated that time and temperature of storage spores on ®eld application of (Microsporidia: No- affect infectivity of microsporidian spores and that longevity varies sematidae) against grasshoppers. Journal of Invertebrate Pathology among species. As demonstrated with the protozoan parasite Crypto- 23: 371±377. sporidium parvum, the period of time in which organisms remained HERSTEINSSON, P., E. GUNNARSSON,S.HJARTARDOTTIR, AND K. SKIRNIS- infective decreased as environmental temperatures increased (Fayer et SON. 1993. Prevalence of Encephalitozoon cuniculi antibodies in al., 1998). As temperatures were increased or decreased beyond those terrestrial mammals in Iceland, 1986 to 1989. Journal of Wildlife normally found in nature, C. parvum lost infectivity rapidly, so that Disease 29: 341±344. organisms were rendered noninfective within 5 sec at 71.7 C and within KOUDELA, B., S. KUCEROVA, AND T. H UDCOVIC. 1999. Effect of low and 24 hr at Ϫ10 C (Fayer, 1994; Fayer and Nerad, 1996; Harp et al., 1996). high temperature on infectivity of Encephalitozoon cuniculi spores Those extremes have yet to be examined for infectivity of E. cuniculi, suspended in water. Folia Parasitologia (Praha) 46: 171±174. E. hellem, and E. intestinalis. KRAMER, J. P. 1970. Longevity of microsporidian spores with special reference to Octosporea muscaedomesticae ¯u. Acta Protozoolo- LITERATURE CITED gica 15: 217±224. KUCEROVA-POSPISILOVA, Z., D. CARR,G.LEITCH,M.SCANLON, AND G. AVERY,S.W.,AND A. H. UNDEEN. 1987. The isolation of microsporidia S. VISVESVARA. 1999. Environmental resistance of Encephalitozoon and other pathogens from concentrated ditch water. Journal of the spores. 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Journal of Infectious Diseases 178: 820±826. MATHIS, A., J. AKERSTEDT,J.THARALDSEN,O.ODEGAARD, AND P. D E- BRYAN,R.T.,AND D. A. SCHWARTZ. 1999. Epiemiology of microspo- PLAZES. 1996. Isolates of Encephalitozoon cuniculi from farmed ridiosis. In The microsporidia and microsporidiosis, M. Wittner and blue foxes (Alopex lagopus) from Norway differ from isolates from 188 THE JOURNAL OF PARASITOLOGY, VOL. 89, NO. 1, FEBRUARY 2003

Swiss domestic rabbits (Oryctolagus cuniculus). Parasitology Re- SPARFEL, J. M., C. SARFATI,O.LIGUORY,B.CAROFF,N.DUMOUTIER,B. search 82: 727±730. GUEGLIO,E.BILLAUD,F.RAFFI,L.M.MOLINA,M.MIEGEVILLE, AND ÐÐÐ, A. C. BREITENMOSER, AND P. D EPLAZES. 1999. Detection of new F. D EROUIN. 1997. Detection of microsporidia and identi®cation of Enterocytozoon genotypes in fecal samples of farm dogs and a cat. Enterocytozoon bieneusi in surface water by ®ltration followed by Parasite 6: 189±193. speci®c PCR. Journal of Eukaryotic Microbiology 44: 78S. MOURA, H., D. A. SCHWARTZ,F.BORNAY-LLINARES,F.C.SODRE,S. TEETOR-BARSCH, G. E., AND J. P. KRAMER. 1979. The preservation of WALLACE, AND G. S. VISVESVARA. 1997. A new and improved infective spores of Octosporea muscadomesticae in Phormia re- ``quick-hot Gram-chromotrope'' technique that differentially stains gina,ofNosema algerae in Anopheles stephensi and of Nosema microsporidian spores in clinical samples including paraf®n-em- bedded tissue sections. Archives for Pathology in Laboratory Med- whitei in Tribolium castaneum by lyophilization. Journal of Inver- icine 121: 888±893. tebrate Pathology 33: 300±306. OSHIMA, K. 1964. Method of gathering and purifying active spores of THOMAS, C., M. FINN,L.TWIGG,P.DEPLAZES, AND R. C. A. THOMPSON. Nosema bombycis and preserving them in good condition. Anno- 1997. Microsporidia (Encephalitozoon cuniculi) in wild rabbits in tationes Zoologicae Japonenses 37: 94±101. Australia. Australian Veterinary Journal 75: 808±810. REVELL, I. L. 1960. Longevity of refrigerated nosema sporesÐNosema VAVRA, J., AND R. LARSSON. 1999. Structure of the microsporidia. In apis, a parasite of honey bees. Journal of Economic Entomology The microsporidia and microsporidiosis, M. Wittner and L. M. 53: 1132±1133. Weiss (eds.). ASM Press, Washington, D.C., p. 7±84. RINDER, H., A. THOMSCHKE,B.SENGJEL,R.GOTHE,T.LOSCHER, AND M. ÐÐÐ, AND J. V. MADDOX. 1976. Methods in microbiology. In Com- ZAHLER. 2000. Close genetic relationship between Enterocytozoon parative pathobiology, vol. 1, biology of the microsporidia, L. A. bieneusi from humans and pigs and ®rst detection in cattle. Journal Bulla and T. C. Cheng (eds.). Plenum, New York, p. 281±319. of Parasitology 86: 185±188. WALLER, T. 1979. Sensitivity of Encephalitozoon cuniculi to various SHADDUCK,J.A.,AND M. B. POLLEY. 1978. Some factors in¯uence the in vitro infectivity and replication of Encephalitozoon cuniculi. temperature, disinfectants and drugs. Laboratory Animal 13: 227± Journal of Protozoology 25: 491±496. 230. SHAW, R. W., M. L. KENT, AND M. L. ADAMSON. 2000. Viability of WHITE, G. F. 1919. Nosema disease. U.S. Department of Agriculture Loma salmonae (Microsporidia) under laboratory conditions. Par- Bulletin Number 780. U.S. Government Printing Of®ce, Washing- asitology Research 86: 978±981. ton, D.C., 54 p.

J. Parasitol., 89(1), 2003, pp. 188±189 ᭧ American Society of Parasitologists 2003

STAT-6 Is an Absolute Requirement for Murine Rejection of Hymenolepis diminuta

Derek M. McKay and Waliul I. Khan, Intestinal Disease Research Programme, McMaster University, Hamilton, Ontario, Canada L8N 3Z5. e-mail: [email protected]

ABSTRACT: Infection with helminth parasites typically evokes a T help- because Th2 cells orchestrate immune events aimed at combatting ex- er 2-cell response in the mammalian host. Interleukin (IL)-4 and IL-13 tracellular parasites, i.e., drive humoral immunity and activate or pro- are important in the rapid expulsion of parasitic nematodes, with the mote mast cell and eosinophil activities. absence of either cytokine being compensated for by the other. How- Interleukin (IL)-4 and IL-13 are Th2-type cytokines that share a com- ever, the IL-4 and IL-13 common signaling molecule, signal transducer mon peptide chain in their receptors, i.e., that designated the IL-4 re- and activator of transcription 6 (STAT-6) appears to be mandatory for ceptor ␣ chain, and mobilize common, e.g., signal transducer and ac- the spontaneous expulsion of enteric helminths. Mice genetically de®- tivator of transcription (STAT-6), and unique intracellular signaling mol- cient in IL-4, IL-13, or STAT-6 were infected with the cestode Hyme- ecules in their target cells. These cytokines display closely related and nolepis diminuta and worm infectivity and jejunal goblet cell responses overlapping biological activity, as well as nonredundant functions assessed 12±18 days postinfection (PI). Only the STAT-6 knockout (McKenzie, 2000). Moreover, IL-4 and IL-13 have been implicated as (KO) animals harbored adult worms; neither gravid adult nor stunted key players in the expulsion of parasitic nematodes (Urban et al., 1998, H. diminuta was obtained from the infected IL-4 KO or IL-13 KO mice 2000), but considerably less data have been presented with respect to Ն12 days PI. Also, the establishment of worms in the intestine of STAT- their importance in the immunologically mediated loss of ¯atworm par- 6 KO animals was associated with a reduced goblet cell response. These asites. ®ndings support the hypothesis that increased mucin production is an Using gene knockout (KO) animals, this study examined the ability important part of the host response to tapeworm infection and that func- of mice lacking IL-4, IL-13, or STAT-6 to expel H. diminuta. Male tional STAT-6 signaling may be an absolute requirement for the rejec- mice de®cient in IL-4 (BALB/c background), IL-13 (BALB/c back- tion of intestinal cestodes and thus, helminth parasites in general. ground), or STAT-6 (C57/Bl6 background) and the appropriate parent stains were infected by oral gavage with 5 H. diminuta cysticercoids in Many parasitic helminth±rodent infections have been used to de®ne 100 ␮l of sterile saline (n ϭ 4 to 8) (McKay et al., 1991). BALB/c and the immunological (and physiological) events that lead to the rejection C57/Bl6 expel H. diminuta with similar kinetics. Noninfected, age- of the worm from a nonpermissive host. A variety of effector mecha- matched BALB/c and C57/Bl6 mice were also included in this study. nisms are now known to contribute to helminth expulsion, including Mice were killed 12±18 days postinfection (PI), a time at which im- increased mucus production, mast activation, eosinophil attack, anti- munocompetent mice have expelled their worms (McKay et al., 1990; body-dependent cytotoxicity, and altered peristalsis and water secretion Palmas et al., 1997). The entire small intestine was removed, ¯ushed (Andreassen et al., 1999); the relative importance of these responses with cold saline, and a portion of midjejunum ®xed in formalin, pro- re¯ects the complexity of the host±parasite relationship. However, it is cessed to wax, and sections stained with periodic acid±Schiff (PAS) clear that the rapid spontaneous rejection of helminth parasites, for ex- reagent for the enumeration of goblet cells. Goblet cell hyperplasia is ample Hymenolepis diminuta from mice, is a T cell±dependent phenom- characteristic of the murine gut response to this parasite (McKay et al., enon (McKay et al., 1990). Moreover, the majority of parasitic helminth 1990). The intestinal contents were examined for the presence of H. infections in mammalian hosts is accompanied by a T helper 2-cell diminuta. (Th2)±dominated response (Bancroft et al., 2000) and appropriately so On opening the abdomen of STAT-6 KO H. diminuta±infected mice