Fulminant Cryptosporidiosis after Near-Drowning: a HumanCryptosporidium parvum Strain Implicated in Invasive GastrointestinalAdenocarcinoma and Cholangiocarcinoma in an Experimental Model

Gabriela Certad,a,b Sadia Benamrouz,a,c Karine Guyot,a Anthony Mouray,d Thierry Chassat,d Nicolas Flament,d Laurence Delhaes,a,e

Valerie Coiteux,f Baptiste Delaire,g Marleen Praet,h Claude Cuvelier,h Pierre Gosset,g Eduardo Dei-Cas,a,e and Colette Creusyg

Laboratory Biologie et Diversité des Pathogènes Eucaryotes Emergents (BDEEP), Centre d’Infection et d’Immunité de Lille (CIIL), Institut Pasteur de Lille, INSERM U1019,CNRS UMR 8402, Université Lille Nord de France, Lille, Francea; Cátedra de Parasitología, Escuela de Medicina José María Vargas, Universidad Central de Venezuela (UCV),Caracas, Venezuelab; Environnement et Santé, FLST, Université Catholique de Lille, Université Lille Nord de France, Lille, Francec; Plateau d’Expérimentation Animale,Institut Pasteur de Lille, Lille, Franced; Parasitologie-Mycologie, Centre Hospitalier Régional et Universitaire de Lille, Université Lille Nord de France, Lille, Francee; Servicedes Maladies du Sang, Hôpital Huriez, Centre Hospitalier Régional et Universitaire de Lille, Université Lille Nord de France, Lille, Francef; Service d’Anatomie et de CytologiePathologiques, Groupe Hospitalier de l’Université Catholique de Lille, Lille, Franceg; and Academic Department of Pathology, Ghent University, Ghent, Belgiumh

In the present work, we report the characterization of a Cryptosporidium parvum strain isolated from a patient who nearlydrowned in the Deule River (Lille, France) after being discharged from the hospital where he had undergone allogeneic stem celltransplantation. After being rescued and readmitted to the hospital, he developed fulminant cryptosporidiosis. The strain iso-lated from the patient’s stools was identified as C. parvum II2A15G2R1 (subtype linked to zoonotic exposure) and inoculatedinto SCID mice. In this host, this virulent C. parvum isolate induced not only severe infection but also invasive gastrointestinaland biliary adenocarcinoma. The observation of adenocarcinomas that progressed through all layers of the digestive tract to thesubserosa and spread via blood vessels confirmed the invasive nature of the neoplastic process. These results indicate for the firsttime that a human-derived C. parvum isolate is able to induce digestive cancer. This study is of special interest considering theexposure of a large number of humans and animals to this waterborne protozoan, which is highly tumorigenic when inoculatedin a rodent model.

Cryptosporidium parvum, an intracellular parasite ubiquitous innature, is a significant health risk to humans and animals. It

causes self-limited watery diarrhea in immunocompetent personsbut has devastating effects in immunocompromised patients withAIDS, hematological malignancies, organ transplantation, or can-cer or those undergoing chemotherapy (9). Contaminated wateris the major source of Cryptosporidium infections for humans.More than 160 waterborne outbreaks have been reported globally,implicating contaminated drinking and recreational water (14, 15,21). The ingestion of as few as 10 oocysts can cause infection inimmunocompetent persons (13). These parameters, together withthe well-known resistance of the parasite to chlorine disinfectionat concentrations typically applied in drinking water plants, are arisk for water transmission of Cryptosporidium spp. (15, 21).

Interestingly, C. parvum has been correlated with digestive car-cinogenesis. An epidemiologic study in Poland reported a fre-quency of 18% of cryptosporidiosis in patients with colorectalcancer (17). However, in this report it was unclear whether C.parvum behaved as a carcinogenesis factor or simply as an oppor-tunistic agent whose development was enhanced by host immu-nosuppression. More consistent with a potential tumorigenic roleof this parasite, we recently showed that IOWA and TUM1 strainsof C. parvum of animal origin induced digestive neoplasia in arodent model (4–6). We report herein the first evidence of theability of a human-derived C. parvum strain to induce gastroin-testinal cancer in mice.

MATERIALS AND METHODSC. parvum isolate. The Cryptosporidium isolate was recovered from stoolsamples from a 51-year-old man with acute lymphoblastic leukemia typeB who nearly drowned after being discharged from the hospital where he

had undergone allogeneic stem cell transplantation (HSCT). Briefly, onday 19 posttransplantation, extensive skin lesions documented as graft-versus-host disease (GVHD) grade II occurred, requiring intense immu-nosuppression with methylprednisolone and tacrolimus. The skin lesionsevolved favorably. The patient left the hospital on day 49 posttransplan-tation. Five days later, he plunged into the Deule River (Lille, France) withhis car to commit suicide. He was rescued after nearly drowning andadmitted to the hospital. Two days after hospitalization, he developedabdominal pain and severe diarrhea (losing 1 to 2 liters/day). An endos-copy ruled out digestive GVHD. The presence of Cryptosporidium sp. wasdocumented in biopsy specimens of the stomach, duodenum, colon, andrectum, as well as in the stools. Stool microbiological tests for bacteria andviruses were negative. Nitazoxanide was administered, reducing the num-ber of evacuations per day. On day 85 posttransplantation, he developedabdominal pain and distension accompanied by fecal vomiting and inter-ruption of intestinal transit. An abdominal computed tomography scanwith contrast revealed pancolitis, without either fluid collection or free airin the peritoneal cavity. Intestinal GVHD was ruled out after colonoscopy.Intestinal transit began spontaneously 2 days later. Stool analysis revealedan uncontrollable cryptosporidial infection (about 500,000 oocysts/g offeces). Fecal cultures were negative for bacteria and viruses. At that time,white blood cell counts were as low as 1,610 cells/mm3. On day 91 post-transplantation, he developed acute respiratory failure with severe hypox-emia, which was treated with imipenem and amikacin. While microbio-

Received 8 August 2011 Accepted 5 January 2012

Published ahead of print 13 January 2012

Address correspondence to Gabriela Certad, gabriela.certad@pasteur-lille.fr.

G.C. and S.B. contributed equally to this article.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.06457-11

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logical tests (bacterial, fungal, and viral cultures, as well as PCRs of bloodand fluid samples) remained negative, diarrhea intensity increased againto more than 2 liters/day. He deteriorated rapidly and became confuseddespite intensive supportive therapies. The patient died on day 100 post-transplantation.

Molecular identification of the Cryptosporidium isolate. GenomicDNA was isolated from oocysts present in the patient’s stools, and a frag-ment of the 18S rRNA gene was amplified by nested PCR (20) and se-quenced for isolate identification. Subtyping of C. parvum was based onsequence analysis of the GP60 gene (1).

Inoculation of SCID mice with the C. parvum isolate. In order toevaluate the virulence and tumorigenic potential of this C. parvum isolateof human origin, oocysts purified from the patient’s feces were inoculatedin SCID mice treated or not with 4 mg/liter dexamethasone (Dex) (Merck,Lyon, France) via drinking water (4–6).

Seven-week-old CB17 SCID mice were obtained from a colony bredand regularly controlled for assessment of microbial (including Helicobac-ter spp.) or parasitological infections at the Pasteur Institute of Lille(France). Animals were housed in groups in covered cages and main-tained under aseptic conditions in an isolator with standard laboratoryfood and water ad libitum.

Before inoculation, the samples were screened for the presence of cul-turable bacteria by plating onto selective or nonselective culture media(Trypticase soy, Trypticase soy and blood, Hektoen, Tergitol 7 with tri-phenyl tetrazolium chloride [TTC], Difco Pseudomonas isolation agar,and Sabouraud agar). Oocyst viability was assessed by testing excystation(5, 6), and the infectivity of parasites was verified by a preliminary oralinoculation of four SCID mice. These animals developed C. parvum in-fection and were followed throughout the experiment.

Infective doses of 105 oocysts were prepared as described previously(4–6) and inoculated by oral-gastric gavage. Control mice were inoculatedwith only phosphate-buffered saline (PBS). In addition, the possibilitythat biotic (e.g., virus) or abiotic factors present in the inoculum couldinduce neoplastic lesions was ruled out by administering to six Dex-treated SCID mice an inoculum from which oocysts were previously re-moved by filtration using a Nanosep MF tube with a 0.45-�m-pore-sizemembrane.

Fecal specimens were collected and processed as previously described(5). Periodically or when signs of imminent death appeared, mice wereeuthanatized by carbon dioxide inhalation.

Experiments were conducted in the animal facility of the Institut Pas-teur de Lille (research accreditation no. A59107). Animal protocols wereapproved by the French regional ethical committee (approval no. CEEA112011).

Histology and immunohistochemistry. Stomach, liver, duodenum,samples of proximal, medium, and distal parts of jejunum, the ileocecalregion, and colon were removed, fixed in 10% buffered formalin, pro-cessed using standard histological techniques, and embedded in paraffin.Sections 5 �m thick were stained with hematoxylin and eosin (H&E).Parasite load in digestive sections was scored as follows: 0, no parasites;�1, small number of parasites, focally distributed; �2, moderate numberof parasites, widely distributed; and �3, abundant parasites present,widely distributed throughout the section (4, 5). Lesions at different siteswere scored as described previously (4, 5), with slight modifications asfollows: 0, no lesion; 1, inflammation and/or regenerative changes; 2,low-grade intraepithelial neoplasia (LGIEN); 3, high-grade intraepithelialneoplasia (HGIEN); 4, suspicion of invasive adenocarcinoma or invasiveadenocarcinoma (penetration of dysplastic glands through the muscularismucosae with desmoplastic stromal response); and 5, adenocarcinomawith the invasion through the submucosa and deeper. (Note that in cate-gory 3, adenoma with HGIEN, carcinoma in situ [limited to the epithe-lium], and intramucosal adenocarcinoma [invasion of the lamina pro-pria] were also included.)

The Volgens-Gomori stain (Reticulin) (3) was employed for assess-ment of basement membrane integrity. A mouse monoclonal antibody to

cytokeratin (without dilution) (AM071-5 M; Biogenex, Netherlands) wasused to detect epithelial cells. An anti-alpha smooth muscle actin mono-clonal antibody (dilution 1:100) (M0851; Dako, Denmark) was used tostain muscle fibers. Sections were examined using a Leica DMRB micro-scope equipped with a Leica digital camera connected to an ImagingResearch MCID analysis system (MCID software, Cambridge, UnitedKingdom). Histological diagnoses were made independently by threepathologists.

In order to analyze results, mice were divided into four groups con-sidering postinfection (p.i.) delay at euthanasia or death, as follows: group1, days 40 to 50 p.i.; group 2, days 51 to 60 p.i.; group 3, days 61 to 90 p.i.;group 4, �90 days p.i. (Table 1).

RESULTS

Numerous oocysts and life cycle stages of Cryptosporidium sp.were observed in the patient’s stools and digestive biopsy speci-mens, respectively. Sequencing was performed, and C. parvumsubtype IIaA15G2R1was identified. After experimental inocula-tion of Dex-treated SCID mice with oocysts from this patient,numerous parasites were detected in mouse feces from day 1 p.i.until the end of the experiment. The mice presented clinical man-ifestations, such as bloody diarrhea, ruffled coat, hunched pos-ture, and lethargy, after day 40 p.i. One mouse had a rectal pro-lapse. A total of 10 Dex-treated mice out of 20 (50%) exhibitingthese signs died before planned euthanasia (Table 1). After spon-taneous or programmed death, organs were removed for histolog-ical examination.

Microscopic examination of tissue sections showed intensecryptosporidial presence (scored by microscopic analysis [Table1]) in the ileocecal region and between moderate and intense pres-ence in the stomach, duodenum, colon, and biliary tree.

The tumorigenic potential of this isolate was assessed: in 18 outof 18 (100%) Dex-treated or untreated mice submitted to histo-logical analysis, neoplastic lesions were found in one organ ormore (Table 1). Gastric neoplastic lesions located in the antropy-loric area were observed in 13 out of 13 (100%) Dex-treated oruntreated mice. Lesions evolved from low- and high-grade dys-plasia to invasive carcinoma (Fig. 1A to D). Variable C. parvumcolonization of the duodenum, intrahepatic bile ducts, and colonwas found, and the presence of parasites in these organs coincidedwith the presence of neoplasia. Particularly, a well-differentiatedcholangiocarcinoma associated with a high load of parasites wasobserved in two Dex-untreated SCID mice (Fig. 1E and F).

In agreement with our previous studies (4–6), the most severelesions were found in the ileocecal region. In this organ, lesionswere characterized by an irregularly thickened mucosa containingareas of low- and high-grade dysplasia (dysplasia-associated le-sion or mass [DALM]) observed mainly in group 1 (earliest eu-thanasia) (Fig. 2A), progressing to adenocarcinoma invading be-yond the submucosa (latest euthanasia) (Fig. 2E). After day 60 p.i.,all Dex-treated mice had invasive adenocarcinoma in the submu-cosa and deeper (Fig. 2D). In addition, one mouse from group 3exhibited vascular tumor emboli in the submucosa (Fig. 2F). Theinvasive nature of lesions was strengthened by the observation ofthe penetration of dysplastic glands through the muscularis mu-cosae associated with a desmoplastic stromal response (Fig. 2D).In the group of Dex-untreated mice, ileocecal neoplastic lesionsvaried in severity, confirming previous observations (4–6). In oneDex-untreated mouse, a well-differentiated adenocarcinoma in-vading the subserosa was detected (Fig. 2E and Table 1).

In addition to these neoplastic lesions, inflammation as well as

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degenerative and regenerative changes were observed. A diffuseinflammatory cell infiltrate with clusters of polymorphs involvingthe lamina propria adjacent to the crypts, the epithelium of thecrypts, the crypt lumen, and the submucosa was observed in theileocecal region. Congestion and dilatation of capillary blood ves-sels was noted. Ulcerations of the mucosa were covered by a fi-brinous exudate and inflammatory cells (similar aspects to thosefound in the patient’s biopsy specimens).

No parasites in the feces, tissues, or lesions were detected incontrol mice at any time.

DISCUSSION

We report herein the isolation and characterization of a virulentC. parvum strain. On the basis of clinical, parasitological, andepidemiological evidence, it can be assumed that this isolate in-duced a severe cryptosporidiosis in the HSCT recipient as a con-sequence of near-drowning. No diarrhea or other digestive symp-tom was noted before the submersion episode. Furthermore, thefinding of C. parvum subtype IIaA15G2R1 as a cause of the infec-tion is consistent with a possible ingestion of water contaminated

with stools from C. parvum-infected animals, even though amixed infection with other Cryptosporidium species cannot becompletely discarded. In fact, the subtype we identified is verycommon in many areas of the world, including Europe, and hu-man cryptosporidiosis cases due to this subtype are usually linkedto zoonotic exposure (7, 19). For example, some studies reportedthat IIaA15G2R1 is predominant in calves in Portugal, Slovenia,and The Netherlands and is also the major C. parvum subtype inhumans in these countries (19). Finally, Cryptosporidium wasidentified as a causal agent in 67% of recreational water outbreaksof gastroenteritis in the United States between 2005 and 2006 (21).

To our knowledge, this is the first evidence of near-drowningas a cause of cryptosporidiosis. In near-drowning events, in addi-tion to the complications directly caused by the submersion, thereare indirect causes of morbidity and mortality, including infec-tions. However, reports on the association between near-drowning and gastrointestinal infections are scarce, although ahigh risk for gut infection would be expected after swallowingwater from a contaminated aquatic environment.

The fatal outcome of this case, 6 weeks after the submersion

TABLE 1 Development of C. parvum II2A15G2R1 in SCID mice shown by parasite distribution and associated lesions

Mouse Dex treatmenta

Day of death p.i.b: Histopathological lesion score(s) (parasite score)c

By euthanasia Before planned euthanasia Stomach Duodenum Ileocecal region Colon Biliary tree

1 Yes 40 NDe ND ND ND ND2 Yes 40 ND ND ND ND ND3 Yes 40 ND 1 (�3) 1, 2 (�3) ND ND4 Yes 41 3 (�3) 1 (0) 1, 4 (�3) 0 (0) 0 (0)5 Yes 47 ND ND ND ND ND6 Yes 48 ND ND 1, 4 (�3) 2 (�2) ND7 Yes 48 2 (�2) 1 (�2) 1, 4 (�3) 1 (�2) 2 (�1)8 Yes 49 ND ND 1, 4 (�3) ND 2 (�1)9 Yes 49 ND ND ND ND ND10 Yes 52 ND ND ND ND ND11 Yes 54 3 (�3) 1 (�2) 1, 4 (�3) 3 (�3) 0 (�1)12 Yes 55 3 (�3) 1 (0) 1, 5 (�3) 1, 3 (�3) 0 (0)13 Yes 55 ND 1 (0) 1, 4 (�3) 0 (0) 0 (0)14 Yes 55 ND 1 (0) 1, 4 (�3) 0 (0) 0 (0)15 Yes 55 3 (�3) 1 (0) 1, 4 (�3) 0 (0) 2 (�1)16 Yes 59 ND ND ND ND ND17 Yes 59 2 (�2) 1 (0) 1, 4 (�3) 0 (0) 3 (�3)18 Yes 62 1, 5 (�3) 3 (�3) 1, 5 (�3) 3 (�3) 2 (�2)19 Yes 62 1, 3 (�3) 3 (�3) 1, 5 (�3) 1, 2 (�2) 2 (�1)20 Yes 62 1, 5 (�3) 2 (�2) 1, 5 (�3) 2 (�2) 2 (�1)21 Nod 91 1, 5 (�3) 1 (�3) 1, 3 (�3) 0 (0) 1, 2 (�1)22 No 91 1, 3 (�3) 0 (0) 1, 5 (�3) 3 (�3) 1, 4 (�3)23 No 91 1, 5 (�3) 0 (�2) 1, 3 (�3) 3 (�3) 1, 4 (�3)24 No 91 1, 3 (�2) 0 (0) 1, 3 (�3) 2 (�2) 0 (0)a Dexamethasone (Dex; 4 mg/liter of drinking water) administration commenced 2 weeks prior to inoculation and was maintained throughout the experiment.b For histopathological analysis, mice were divided into four groups considering p.i. delay at euthanasia or death, as follows: group 1, days 40 to 50; group 2, days 51 to 60; group 3,days 61 to 90; and group 4, �90 days.c Lesions at different sites were scored as follows: 0, no lesion; 1, inflammation and/or regenerative changes; 2, low-grade intraepithelial neoplasia (LGIEN); 3, high-gradeintraepithelial neoplasia (HGIEN); 4, suspicion of invasive adenocarcinoma or invasive adenocarcinoma (penetration of dysplastic glands through the muscularis mucosae withdesmoplastic stromal response); and 5, carcinoma with the invasion of the submucosa and deeper. (Note that in category 3, adenoma with HGIEN, carcinoma in situ [limited to theepithelium], and intramucosal adenocarcinoma [invasion of the basal membrane of glands] were also included.) Numbers in parentheses include the results of the histologicalsemiquantitative assessment of C. parvum organisms in the host tissues. The presence of parasites in the tissues was scored as follows: 0, no parasites; �1, small number of parasites,focally distributed; �2, moderate number of parasites, widely distributed; and �3, abundant parasites present, widely distributed throughout the tissue.d Oocyst viability before inoculation was assessed by an excystation test followed by a preliminary inoculation of four Dex-untreated SCID mice. These animals were infected andwere also followed until the end of the experiment. Control mice (uninfected Dex-treated SCID mice and Dex-treated SCID mice inoculated with an inoculum from whichCryptosporidium oocysts were removed by filtration) were euthanatized periodically as experimental mice. None of the mice in the control groups developed Cryptosporidiuminfection or histological gastrointestinal lesions.e ND, not done (organ not included in the histological examination).

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event, may be due to the patient’s immunosuppression status,which could favor C. parvum infection. This diagnosis and itscourse are in agreement with (i) the fact that cryptosporidiosisremains an opportunistic infection causing significant life-threatening diarrhea (2), (ii) the observation of passage of morethan 2 liters of stool per day as a criterion of fulminant disease (9),and (iii) a rapid progression, comparable with the median of 5weeks reported in patients with fulminant cryptosporidiosis (2).

On the other hand, the extremely high parasite shedding in thispatient allowed us to purify a large amount of infective oocystsfrom stools and to infect SCID mice to test the tumorigenic po-

tential of a human-derived C. parvum isolate. We recently showedthat C. parvum strains of animal origin induce digestive cancer inSCID mice (4–6).

After experimental inoculation with the C. parvum clinical iso-late, high mortality was observed (Table 1). Additionally, as con-firmed by immunohistochemistry, several animals developed ad-enocarcinomas that progressed through all layers of the digestivetract to the subserosa and spread via blood vessels. This neoplasticprocess associated with a C. parvum isolate of high virulence seemsto be a good illustration of the well-known dysplasia-cancer path-way (11).

FIG 1 Gastric and hepatic neoplastic lesions in SCID mice infected with C. parvum IIaA15G2R1 isolated from an HSCT recipient patient. (A) High-gradeintraepithelial neoplasia in the antropyloric region of a Dex-treated SCID mouse 51 to 60 days postinfection (p.i.) showing important cellular atypias andpresence of numerous parasites (arrows) inside the glands. Stain, hematoxylin and eosin. Bar, 50 �m. (B) Intramucosal adenocarcinoma in the stomach of aDex-treated SCID mouse 51 to 60 days p.i. showing architectural distortion, gland fusion with buds of the glandular epithelium into the lamina propia, and lossof the basement membrane. Stain, Volgens-Gomori. Bar, 50 �m. (C) Well-differentiated adenocarcinoma in the gastric region of a Dex-treated SCID mouse 51to 60 days p.i. showing major cellular atypias (arrow) and numerous mitoses. Stain, hematoxylin and eosin. Bar, 100 �m. (D) Well-differentiated gastricadenocarcinoma invading (arrow) through the muscularis (m) in a Dex-treated SCID mouse 61 to 70 days p.i. Stain, hematoxylin and eosin. Bar, 100 �m. (E)Well-differentiated bile duct adenocarcinoma of the hepatohilar region of the liver (l) in a Dex-untreated SCID mouse 90 days p.i. Proliferation of glandsdisposed in desmoplastic stroma was observed. Stain, hematoxylin and eosin. Bar, 400 �m. (F) Well-differentiated bile duct adenocarcinoma of the hepatohilarregion in a Dex-untreated SCID mouse 90 days p.i. showing cellular atypias, architectural distortion, and parasites (arrow) in the lumen of the glands. Stain,hematoxylin and eosin. Bar, 100 �m.

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In particular, dysplastic changes in the biliary tree after C. par-vum infection have been reported in experimental rodents (4, 16).However, this is the first time that well-developed cholangiocar-cinoma lesions were found in C. parvum-infected mice. Interest-ingly, a possible association between human cryptosporidiosisand liver cancer (bile duct carcinoma) was suggested in patientswith X-linked hyper-IgM syndrome (8, 18). Development of chol-angiocarcinoma, like most tumors, is probably a multistep processdependent on the interaction between host genetics and environ-mental factors, including infection. Thus, it is widely accepted thatmetazoan parasites such as Opisthorcis viverrini and Clonorchissinensis could act as triggers of such carcinoma processes (10).

In conclusion, C. parvum IIaA15G2R1 was isolated and iden-tified as a cause of fulminant cryptosporidiosis in a near-drowningvictim who had undergone HSCT before the submersion episode.Additionally, the results of this study showed for the first time theability of an isolate of C. parvum of human origin to cause gastro-intestinal and biliary adenocarcinomas in an experimental model,providing supplementary evidence of a direct role of this parasitein the induction of digestive cancer (4–6). Furthermore, thesefindings demonstrate that C. parvum-induced neoplasia is an in-vasive process that can evolve rapidly in immunosuppressedhosts.

We finally highlight that this study is of special interest, con-

FIG 2 Ileocecal lesions of SCID mice infected with C. parvum IIaA15G2R1 isolated from an HSCT recipient patient. (A) Intramucosal adenocarcinoma in aDex-treated SCID mouse 40 to 50 days PI. The mucosa is irregularly thickened, with intraluminal budding in an adenomatous fashion around the entirecircumference of the cecum. Stain, hematoxylin and eosin. Bar, 1,500 �m. (B) Well-differentiated adenocarcinoma in a Dex-treated SCID mouse 51 to 60 daysPI. Architectural distortion of glands and loss of gland differentiation with epithelial atypias consisting of loss of normal polarity, nuclear stratification,prominent nucleoli, and irregularly scattered chromatin were observed. The presence of parasites (arrows) inside the glands is shown. Stain, hematoxylin andeosin. Bar, 50 �m. (C) Dex-treated SCID mouse 40 to 50 days PI. Volgens-Gomori stain shows architectural distortion, fusion of glands, loss of the basalmembrane, and presence of the neoplastic cells in the lamina propria. Bar, 50 �m. (D) Dex-treated SCID mouse 40 to 50 days PI. Interruption (arrows) of themuscularis mucosae (mm) and invasion into the submucosa (sm) by the neoplastic glands are shown (immunohistochemical stain for alpha smooth muscleactin). Bar, 400 �m. (E) Dex-treated SCID mouse 90 days PI. A well-differentiated adenocarcinoma invading the subserosa (ss) through the muscularis (m) isshown (immunohistochemical stain for cytokeratin). Bar, 200 �m. (F) Dex-treated SCID mouse 51 to 60 days PI. Vascular tumor emboli (arrow) were detectedin the submucosa (sm). Stain, hematoxylin and eosin. Bar, 50 �m.

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sidering the exposure of large number of animals and persons tothis protozoan, which is highly tumorigenic when inoculated in anexperimental model. Since mouse models of cryptosporidiosismimic the course of human cryptosporidiosis (12), a serious ex-ploration of this type of process in humans is needed.

ACKNOWLEDGMENTS

This work was supported by the Ministry of Research, France (EA3609/4547 Université de Lille 2), Federative Institute of Research 142 (InstitutPasteur de Lille), and the French National Research Agency (grants ANR-09-ALIA-009 and ANR-10-ALIA-004). G.C. was supported by a scholar-ship from the Consejo de Desarrollo Científico y Humanístico of theUniversidad Central de Venezuela. S.B. was supported by a scholarshipfrom the Catholic Institute of Lille.

Special thanks are extended to David Buob and Emmanuelle Leteurtre(Centre of Biology, Pathology and Cytogenetics, CHRU Lille, France), toJean-Pierre De Cavel (Platform of Animal Experimentation, Institut Pas-teur de Lille, France), and to Elizabeth Baumelou (ICL, Lille, France) foradvice and discussion.

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