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Infection and Immunity, July 2006, p. 4387-4389, Vol. 74, No. 7
0019-9567/06/$08.00+0     doi:10.1128/IAI.02055-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Transient Neonatal Cryptosporidium parvum Infection Triggers Long-Term Jejunal Hypersensitivity to Distension in Immunocompetent Rats

Rachel Marion,1,{dagger} Asiya Baishanbo,1,2,{dagger} Gilles Gargala,1 Arnaud François,3 Philippe Ducrotté,1 Celia Duclos,3 Jean Fioramonti,4 Jean Jacques Ballet,5 and Loïc Favennec1*

Laboratoire de Parasitologie and ADEN UPRES EA-3234, CHU Charles-Nicolle, 76031 Rouen, France,1 College of Pharmacy, Xinjiang Medical University, Urumqi, China,2 Laboratoire d'Histopathologie, CHU Charles Nicolle, Rouen, France,3 INRA, 180 Chemin de Tournefeuille, BP 3, 31931 Toulouse Cedex 9, France,4 Laboratoire d'Immunologie et Immunopathologie, UPRES EA-2128, CHU Clemenceau, Caen, France5

Received 21 December 2005/ Returned for modification 30 January 2006/ Accepted 4 April 2006


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ABSTRACT
 
In 5-day-old immunocompetent Sprague-Dawley rats infected with either 102 or 105 Cryptosporidium parvum oocysts, transient infection resulted 120 days later in increased cardiovascular depressor response to jejunal distension and jejunal myeloperoxidase activity (P < 0.05). Nitazoxanide treatment normalized jejunal sensitivity (P < 0.001) but not myeloperoxidase levels (P > 0.05). Data warrant further evaluation of the role of early cryptosporidiosis in the development of chronic inflammatory gut conditions.


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TEXT
 
Hypersensitivity to visceral distension is a frequent feature of the irritable bowel syndrome (IBS), a common functional gastrointestinal disorder (25). Acute bacterial gastroenteritis was a recognized etiological factor in a subset of patients of which more than half remained symptomatic 6 years postinfection (p.i.) (15). Campylobacter and Salmonella spp. were found to be involved (13, 18). Experimental models of IBS triggered by infection are presently lacking (4). In otherwise healthy humans, enteric infection due to the waterborne protozoan parasite Cryptosporidium sp. is self-limiting; however, when acquired in early life, cryptosporidiosis may impair growth and development, and its long-term impact remains largely undetermined (7, 9, 10, 17, 24). The aim of this work was to explore in suckling rats the consequences of transient Cryptosporidium parvum gut infection on adult jejunal and rectal sensitivities to distension and investigate the effects of nitazoxanide (NTZ), a 5-nitrothiazolyl derivative found to be active against C. parvum development (6, 8, 11, 21).

Cryptosporidium sp.-free pregnant Sprague-Dawley rats were from IFFA CREDO (Lyon, France). C. parvum oocysts, a kind gift from R. Mancassola and M. Naciri, INRA, Nouzilly, France, were purified as described previously (12). Nineteen and twenty-nine 5-day-old suckling rats were gavaged with 100 µl of phosphate-buffered saline (PBS) containing either 102 or 105 oocysts, respectively. PBS alone was administered to 29 control rats. Another group of 29 rats infected with 105 oocysts was given oral NTZ (200 mg/kg body weight/day, twice daily; Romark Laboratories, Tampa, FL) for 14 days p.i. Rats were weighed weekly. Fecal oocyst shedding monitored on days 11 and 25 p.i. was expressed as oocyst numbers/10 microscopic fields (11). On day 14 p.i., four rats from each group were killed; their jejunal tissues were fixed in 10% formalin-PBS and embedded in paraffin. Four-micrometer sections were Giemsa stained and considered infected if at least one cryptosporidial developmental form was observed within one mucosal cell by three independent investigators. From day 20 p.i., all rats were individually housed in filtered cages and provided heat-sterilized food and water ad libitum. Animals were handled according to the regulations enforced by the French Ministry of Agriculture.

On day 120 p.i., 15 rats from each group were anesthetized with intraperitoneal sodium pentobarbitone (Abbott Diagnostic, Rungis, France), a midline abdominal incision was made to expose the small intestine, a cut was made on the antimesenteric side at one end of a jejunum segment, i.e., 7 cm from Treitz' ligament, a 5-cm-long part of a balloon was introduced, the intestinal segment was replaced in the peritoneal cavity, and the abdomen was closed. Rectal distension was performed by inserting a balloon by the anal route. Balloons were arterial embolectomy catheters (Fogarty-Edwards Life Sciences, Saint-Prex, Switzerland). Distending the jejunum or rectum by rapid inflation (0.1 to 0.4 and 0.4 to 1.2 ml, respectively; 25 s every 5 min) resulted in a stimulus-related decrease in systemic blood pressure, which was recorded from a side arm of the carotid cannula using a pressure transducer (P10EZ) connected to a window graph 240 (Gould, Courtaboeuf, France) (14).

With 10 animals from each group, day-120 p.i. myeloperoxidase (MPO) activity was measured in full-thickness, 2-cm jejunal fragments as described previously (5). MPO from human neutrophils (Sigma, L'Isle d'Abeau Chesnes, France) was used as a standard. One MPO unit was defined as the activity able to convert 1 µmol H2O2 to H2O min–1 at 25°C. Results were expressed in MPO units/g protein.

Values were expressed as means ± 1 standard deviation. Significance of differences between groups was evaluated using Student's t tests, thus assuming normal distributions of data.

Initial mean rat weights were not different in the control and infected groups (10.1 g ± 0.9 g and 9.7 g ± 0.6 g, respectively; P > 0.05). From day 6 to day 76 p.i., lower weights were observed in untreated p.i. rats than in controls (P < 0.01), while NTZ-treated p.i. animals did not differ from controls (P > 0.05). From day 83 to day 120 p.i., weight differences between the three groups were not significant (P > 0.05).

Intestinal infection was ascertained at day 11 p.i. by the presence of fecal oocysts (mean of 48 infected rats: 16.3 ± 5.6 oocysts/10 microscopic fields) and at day 14 p.i. by the presence of jejunal parasitic developmental forms in 4/4 and 2/4 rats administered 105 or 102 oocysts, respectively. NTZ treatment suppressed oocyst shedding (mean of 29 rats, 4.1 ± 5.7; P < 0.001 versus untreated) with no developmental form in the jejunum of 4/4 rats (Fig. 1). On day 25, oocyst shedding was abolished in all animals.


Figure 1
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  		FIG. 1. Histology of jejunal mucosa in untreated and nitazoxanide-treated postinfection rats and control rats. Typical patterns of jejuna sampled at day 14 postinfection (magnification, x200). (A) Jejunal mucosa, infected rat. Two parasitic forms are seen on the outer surface of epithelial cells. (B) Jejunal mucosa, infected rat treated with nitazoxanide. (C) Jejunal mucosa, control noninfected rat.

On day 120, depressor responses to jejunal distension volumes of 0.2, 0.3, and 0.4 ml were significantly higher in untreated p.i. rats than in controls (Fig. 2A; P < 0.01). No difference was observed between responses in control and NTZ-treated p.i. rats (P > 0.05). Rectal distension-induced responses did not differ between all groups of rats (Fig. 2B) (P > 0.05). In untreated and NTZ-treated p.i. rats, an increase in jejunal MPO activity was observed (289.7 ± 36.2 and 290.3 ± 39.9 units/g protein, respectively) compared with controls (113.3 ± 48.6 units/g protein; P < 0.05).


Figure 2
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  		FIG. 2. Depressor response to graded jejunal and rectal distension in infected and control anesthetized rats. (A) Jejunal distension; (B) rectal distention. Results are expressed as mean values. One bar represents one standard deviation from the mean. {blacklozenge}, control noninfected rats (n = 15); {blacksquare}, rats infected with 102 oocysts (n = 15); {blacktriangleup}, rats infected with 105 oocysts (n = 15); X, rats infected with 105 oocysts and treated with NTZ (n = 15); *, P < 0.05; **, P < 0.01.

Infection was transient with the present suckling-rat model (2, 6, 21, 22). Initial p.i. body weight reduction was previously mentioned (20, 23). The nociceptive response was investigated using balloon distension and measuring the decrease in blood pressure, a depressor, pseudoaffective response corresponding to brain stem/spinal reflexes which cease when the noxious stimulus is terminated (26). Compared to controls, increased sensitivity was observed at any jejunum-distending volume on day 120 in p.i. rats. Similar findings were reported for rats previously infected with Nippostrongylus brasiliensis (14). Increase in jejunal, not rectal, sensitivity suggests that it was restricted to the preferred site, i.e., small bowel, of C. parvum infection (16). Increased jejunal MPO activity in p.i. rats is consistent with local neutrophil attraction (3). NTZ has been found to be active with several cryptosporidiosis models (11, 19). The present regimen suppressed oocyst shedding and mucosal infection. At day 120 p.i., sensitivity to distension did not differ between NTZ-treated and control rats, consistent with NTZ control of the Cryptosporidium-induced increase in gut sensitivity.

Present data suggest that neonatal cryptosporidiosis results in increased jejunal sensitivity to distension in adults, regardless of MPO activity. Immune mechanisms may be involved, since with a post-Trichinella spiralis infection model, resident Th2 CD4+ T lymphocytes mediated long-term effects on neuromuscular function (1). The present infection model mimicked some key features of IBS patients, in which visceral pain represents a major symptom and increased sensitivity to gut distension is common; however, limited information is presently available on the long-term impact of human cryptosporidiosis on the development of intestinal disorders (9, 17, 20, 25). Present results permit speculation that cryptosporidiosis may be causative of IBS and warrant further studies of postcryptosporidiosis bowel disturbances.


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ACKNOWLEDGMENTS
 
This study was supported in part by grants from the Romark Research Foundation.  We are indebted to Véronique Tonerie for her help in the preparation of the manuscript.


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratoire de Parasitologie and ADEN UPRES EA-3234, CHU Charles-Nicolle, 1, rue de Germont, 76031 Rouen, France. Phone: 33 (0)2 32 88 66 39. Fax: 33 (0)2 32 88 68 75. E-mail: loic.favennec{at}univ-rouen.fr. Back

Editor: W. A. Petri, Jr.

{dagger} Both authors contributed equally to the work. Back


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Infection and Immunity, July 2006, p. 4387-4389, Vol. 74, No. 7
0019-9567/06/$08.00+0     doi:10.1128/IAI.02055-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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