Parasites of the genus Cryptosporidium are intracellular parasites that occur throughout the animal kingdom and have been reported in many species of mammals, including human. Most infections in humans are caused by two C. parvum genotypes, genotype I and genotype II; these are the human and the bovine (zoonotic) genotypes, respectively. Successful experimental infection of Cryptosporidium parvum genotype I "human genotype" is described in four conventionally reared piglets and in a lamb. The inoculum was originally obtained from two diarrheic children, and the Cryptosporidium genotypes were determined by PCR and rDNA sequencing. The infective dose was between 106 and 2×106oocysts. No clinical signs were observed in the infected animals, except in a piglet that showed watery diarrhea. The oocyst shedding period in positive animals ranged between 4 and 10 days. Histopathologic examination of the gastrointestinal tract of two positive piglets revealed shortening of the villi and denudation of the villous tips of the jejunum. In one piglet, the colon mucosa revealed numerous Cryptosporidium oocysts. The storage time of the inocula (≤3 weeks in PBS at 4°C) and the age of the animal (newborn) were important for the successful induction of infectio

Deplazes, P.

Ebeid, M.

Mathis, A.

Pospischil, A.

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ORIGINAL PAPERInfectivity of Cryptosporidium parvum genotype I in conventionallyreared piglets and lambsReceived: 6 November 2002 / Accepted: 15 January 2003 / Published online: 12 March 2003 Springer-Verlag 2003Abstract Parasites of the genus Cryptosporidium are in-tracellular parasites that occur throughout the animalkingdom and have been reported in many species ofmammals, including human. Most infections in humansare caused by two C. parvum genotypes, genotype I andgenotype II; these are thehumanandthebovine (zoonotic)genotypes, respectively.Successful experimental infection of Cryptosporidiumparvum genotype I ‘‘human genotype’’ is described infour conventionally reared piglets and in a lamb. Theinoculum was originally obtained from two diarrheicchildren, and the Cryptosporidium genotypes were de-termined by PCR and rDNA sequencing. The infectivedose was between 106 and 2·106 oocysts. No clinicalsigns were observed in the infected animals, except in apiglet that showed watery diarrhea. The oocyst shed-ding period in positive animals ranged between 4 and10 days. Histopathologic examination of the gastroin-testinal tract of two positive piglets revealed shorteningof the villi and denudation of the villous tips of thejejunum. In one piglet, the colon mucosa revealed nu-merous Cryptosporidium oocysts. The storage time ofthe inocula ( £ 3 weeks in PBS at 4C) and the age ofthe animal (newborn) were important for the successfulinduction of infection.IntroductionHuman cryptosporidiosis is caused mainly by twogenotypes of Cryptosporidium parvum. Genotype I‘‘human genotype’’ has been detected almost exclu-sively in humans; the single non-human case that hasbeen identiﬁed was that of a dugong (Morgan et al.2000). Genotype II (‘‘bovine genotype’’) has a widerange of animal hosts, including humans. It has beenisolated particularly from calves, lambs and othernewborn ruminants (Peng et al. 1997; Spano et al.1998). Cryptosporidia are transmitted by the fecal-oralroute or indirectly through food or drinking water thatare contaminated with oocysts (Jokipii et al. 1983;MacKenzie 1994). C. parvum causes acute, self limitingdiarrhea in immunocompetent individuals and in do-mestic animals. However, cryptosporidiosis has achronic form and may have a fatal course in immu-nodeﬁcient individuals (O’Donoghue 1995).In the available literature, there is a paucity of in-formation about the propagation of C. parvum geno-type I oocysts. Despite the improvement of in vitrocultivation of C. parvum (Hijjawi et al. 2001), massproduction of oocysts depends on animal inoculation(Petry et al. 1995). Recently, experimental infection ofC. parvum genotype I in gnotobiotic piglets and in alamb was reported (Widmer et al. 2000; Giles et al.2001).The aimof this studywas to determine the infectivity ofC. parvum genotype I in conventionally reared piglets andlambs.Material and methodsAnimalsSix large white breed piglets and six Swiss white alp lambs wereused in this study. The piglets were obtained from Speciﬁc Patho-gen Free (SPF) conventional farms and were colostrum-deprivedby removing them from the sows immediately after birth. Theywere housed at 32C in pairs in plastic containers with wire meshtop and with wood shavings for bedding. They were fed six timesdaily with commercial piglet milk replacement.The lambs originated from the experimental sheep herd of theInstitute of Parasitology at the University of Zurich. The lambswere kept with their dams throughout the study.Parasitol Res (2003) 90: 232–235DOI 10.1007/s00436-003-0839-0M. Ebeid Æ A. Mathis Æ A. Pospischil Æ P. DeplazesM. Ebeid Æ A. Mathis Æ P. Deplazes (&)Institute of Parasitology, University of Zurich,Winterthurerstr. 266a, 8057, Zurich, SwitzerlandE-mail: pdeplaze@vetparas.unizh.chTel.: +41-1-6358501Fax: +41-1-6358907A. PospischilInstitute of Veterinary Pathology, University of Zurich,Winterthurerstr. 266a, 8057 Zurich, SwitzerlandIn order to facilitate the collection of fecal material, piglets werekept on perforated aluminium ﬂooring with an underlying trayduring the period of oocyst shedding. In lambs, fecal collectionbags held in place by a harness were used.The experiment was approved by the animal experimentcommission in accordance with the Swiss legislation on animalwelfare.The inoculaTwo C. parvum genotype I isolates (Crypto-11 and Crypto-26) wereobtained from diarrheic children treated at the Zurich UniversityHospital. The identiﬁcation of theC. parvum genotype was based onPCR and direct sequencing of part of the 18S rDNA (Ward et al.2002).Fecal homogenates were kept at 4C either in phosphate buf-fered saline (PBS) supplemented with penicillin G sodium 40 IU/ml, streptomycin 40 lg/ml and amphotericin-B 0.05 lg/ml (PSF)or in 2% (w/v) K2Cr2O7 solution (Table 1). Oocyst number wasdetermined semi-quantitatively by examining 10 ll aliquots of thefecal homogenate under the light microscope after staining with themodiﬁed Ziehl-Neelsen procedure (Casemore 1991). No bacterio-logical or virological examination of the fecal samples wasattempted.Before inoculation, the conserving medium was removed bywashing the fecal material four times in sterile distilled water andcentrifugation at 1000 g for 5 min.Experimental proceduresThe infective dose, which ranged between 106 and 2·106 oocystssuspended in 4 ml of sterile distilled water, was administered bystomach tube.Thepigletswere inoculated at onedayof age except forone piglet (Table 1). The piglets were kept for 20 days post-infectionand then euthanised with an overdose of barbiturates. Intestinal la-vagewas performed and the intestinal contents were collected. In twopiglets (numbers 5 and 6), samples from the internal organs (liver,spleen, kidney and gall bladder) and samples from the intestinal tract(duodenum, jejunum, ileum, cecum, colon, and rectum) were ﬁxed in10% neutral formalin for histological examination. Histologicalsections of 4 lm thickness were prepared and stained routinely withH&E and with Giemsa stain.Four lambs were inoculated few hours after birth and two wereinoculated at the age of 6 weeks (Table 1). The lambs were dis-charged at the end of the study to join the experimental herd.Clinical monitoringThe animals were examined clinically on a daily basis for changesin vital signs (body temperature, heart rate and respiratory rate)and for evidence of diarrhea. Body weight was recorded daily.Fecal examinationAll animals were examined for the presence of Cryptosporidiumspp. oocysts in their feces prior to inoculation. Rectal swabs weretaken from every animal daily until the end of the experiment andtwo smears thereof were examined microscopically.To determine the Cryptosporidium genotype, a pool of fecalsamples from every positive animal was subjected to PCR anddirect sequencing of the 18S rDNA (Ward et al. 2002).Results and discussionIn the present study, successful experimental infectionwith Cryptosporidium parvum genotype I was establishedin four conventionally reared piglets and in a lamb.Experimental Cryptosporidium infection has been re-ported in piglets (Moon et al. 1982; Arnault et al. 1994)and in lambs (Tzipori et al. 1982) using Cryptosporidiaoocysts from diarrheic patients. However, interpretationof these studies was diﬃcult because all the experimentswere established without prior determination of theisolate’s genotype. Recently, C. parvum genotype I wassuccessfully propagated in gnotobiotic piglets (Widmeret al. 2000) and in a lamb (Giles et al. 2001).The infective dose in this study of 106 –2·106 oocystswas similar to that of a previous report in gnotobioticpiglets (Widmer et al. 2000). Four out of six pigletsstarted to shed oocysts 4–5 days post-infection(Table 1). No clinical signs were observed except in onepiglet (no. 1) that showed watery diarrhea, and a changeof fecal color from brown to yellow. The piglets weresteadily gaining weight despite shedding oocysts (datanot shown). The duration of shedding ranged between4 and 10 days. In all the positive piglets, PCR and se-quence analysis conﬁrmed the presence of C. parvumgenotype I as in the original inoculum. In two piglets(numbers 5 and 6), histological examination of theintestinal tract revealed shortening of the villi and de-nudation of the villous tips of the jejunum. The colonmucosa of piglet no. 5 showed numerous Cryptospori-dium oocysts (Fig. 1). The ileum of piglet no. 6 showedatrophy of the lymphoid follicles. In piglet no. 6, noCryptosporidia were detected in any histological sectionbut there was shortening and denudation of the villousTable 1 Experimental infection of piglets and lambs with Cryptosporidium parvum genotype I (‘‘human genotype’’)Animal Age at timeof inoculationInoculum(isolate)StoragemediumStoragetime ofinoculumNumberofoocysts ininoculumFirstexcretion ofoocyst PI / Durationof sheddingPiglet 1 Newborn Crypto-11 PBS+PSF 2 days 106 day 4/9 daysPiglet 2 Newborn Crypto-11 PBS+PSF 2 days 106 day 4/4 daysPiglet 3 Newborn First passage from piglet 1 PBS+PSF 7 weeks 2 · 106 No sheddingPiglet 4 12days Crypto-26 PBS+PSF 2 days 106 No sheddingPiglet 5 Newborn Crypto-26 PBS+PSF 18 days 106 day 4/4 daysPiglet 6 Newborn Crypto-26 2%K2Cr2O7 18 days 106 day 5/6 daysLamb 1 Newborn Crypto-26 PBS+PSF 3 weeks 106 day 5/10 daysLamb 2 6weeks First passage from lamb 1 PBS+PSF 4 days 2 · 106 No sheddingLamb 3 6weeks First passage from lamb 1 PBS+PSF 4 days 2 · 106 No sheddingLamb 4 Newborn Crypto-26 PBS+PSF 10 weeks 106 No sheddingLamb 5 Newborn First passage from lamb 1 PBS+PSF 8 weeks 106 No shedding233tips along the small intestine (jejunum and ileum). Thesehistological changes were consistent with the previousdescription of intestinal cryptosporidiosis in piglets(Moon and Bemrick 1981; Tzipori 1981; Moon et al.1982; Argenzio et al. 1990; Vitovec and Koudela 1992).Two piglets (numbers 3 and 4) did not excrete theoocysts throughout the experiment. The failure to infectone piglet (no. 3) was possibly due to the prolongedstorage of the oocysts used as inoculum. The half life ofC. parvum genotype I was reported to be 1 week at roomtemperature (Widmer et al. 2000), in contrast to C. par-vum genotype II, which showed a half life of 112 days atroom temperature (Yang et al. 1996).Failure to infect another piglet (no. 4) might beattributed to its age (12 days old) at the time of theinoculation. This assumption was supported by the factthat the same isolate was infectious in a newborn lamb(no. 1) and two newborn piglets (numbers 5 and 6).Infection with C. parvum genotype I was also suc-cessful in a lamb that remained symptomless. Lambsnumbers 2 and 3 were inoculated with oocysts collectedfrom lamb no. 1. They were 6 weeks old at the time ofthe inoculation and remained symptomless, and nooocyst shedding was observed throughout the experi-ment. However, it should be noted that the detection ofCryptosporidia oocysts in formed stool with theZiehl-Neelsen method requires 500,000 oocysts per g ofstool for a 100% accurate detection of oocysts (Weber etal. 1991).Due to logistic constraints, we could not obtainnewborn lambs and piglets in the time required toinvestigate the viability of the oocysts. Therefore, itremains to be seen whether the failure to induce theinfection in other lambs was attributable to the age ofthe inoculum.Despite the fact that a small number of animals wasused in this study, it seems that the storage time of theisolate might play a role in the infectivity of the oocysts.Isolates stored longer than 3 weeks in PBS at 4C failedto induce infection in newborn animals. This point needsfurther investigations to explain waterborne outbreaksdue to C. parvum genotype I oocysts. Inoculation withfreshly collected oocysts was unsuccessful in 12-day-oldpiglets. It is plausible to assume that older animals re-quire immunosuppression for the successful induction ofthe infection.Acknowledgements Thanks are due to Dr. Naomi Oliel and Dr.Esther Bu¨rgi for provision of the piglets, Mr. Hanspeter Mu¨ller forthe care of the experimental animals, and to the laboratory tech-nicians at the Institute of Parasitology for their unfailing helpduring the experiment.ReferencesArgenzio RA, Liacos JA, Levy ML, Meuten DJ, Lecce JG,Powell DW (1990) Villous atrophy, crypt hyperplasia,cellular inﬁltration, and impaired glucose-Na absorption inenteric cryptosporidiosis of pigs. 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Infectivity of Cryptosporidium parvum genotype I in conventionally reared piglets and lambs

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Infectivity of Cryptosporidium parvum genotype I in conventionally reared piglets and lambs

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