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The effect of cotrimoxazole on experimentalCryptosporidium parvum infection in kids

B Koudela, A Boková

To cite this version:B Koudela, A Boková. The effect of cotrimoxazole on experimental Cryptosporidium parvum infectionin kids. Veterinary Research, BioMed Central, 1997, 28 (4), pp.405-412. �hal-00902490�

Original article

The effect of cotrimoxazole on experimentalCryptosporidium parvum infection in kids

B Koudela A Boková

Institute of Parasitology, Academy of Sciences of the Czech Republic, B!aMMOf.!! 31, 1,CZ-370 05 !eské Budijovice, Czech Republic

(Received 16 January 1997; accepted 13 March 1997)

Summary ― The prophylactic and therapeutic effects of the folic acid inhibitor cotrimoxazole(trimethoprim in combination with sulfamethoxazole) was tested in goat kids experimentally infectedwith Cryptosporidium parvum oocysts. Of the twenty-four 6-day-old kids inoculated with 6 x 106 oocystsof C parvum, ten kids were administered cotrimoxazole prophylactically at a dose 20 mg/kg per dayoftrimethoprim /100 mg/kg per day of sulfamethoxazole for 14 consecutive days beginning I day beforeinfection. Six kids were therapeutically treated at the same dose of cotrimoxazole for 9 consecutive daysbeginning 5 days post infection, and eight kids served as untreated controls. Experimental C parvuminfection caused a severe clinical disease with profuse watery diarrhea, oocysts shedding and intesti-nal lesions in all groups of kids. Total days and severity of diarrhea were similar for all groups ofkids. However, the mean duration of oocysts shedding, mean number of cryptosporidia per ileal vil-lus, and distribution of cryptosporidia in the intestine were increased in both groups of cotrimoxazole-treated kids. These findings indicate that cotrimoxazole failed to control cryptosporidiosis.

Cryptosporidium parvum / kid / experimental infection / therapy / cotrimoxazole

Résumé ― Effets du cotrimoxazole sur l’infection expérimentale des chevreaux par Crypto-sporidium parvum. Les effets prophylactiques et thérapeutiques d’un inhibiteur de l’acide folique,le cotrimoxazole (trimethoprim combiné au sulfamethoxazole) ont été testés sur un troupeau de che-vreaux infectés expérimentalement par les oocytes de Cryptosporidium parvum. Sur les 26 che-vreaux, âgés de 6 jours et inoculés avec 6 x 106 oocytes de C parvum, dix chevreaux ont reçu du cotri-moxazole dans un but prophylactique, à la dose de 20 mg/kg par jour de trimethoprim pour 100 mg/kgpar jour de sulfamethoxazole pendant 14 jours consécutifs à partir de la veille de l’infection ; sixchevreaux ont été traités dans un but thérapeutique à la même dose de cotrimoxazole pendant 9 joursconsécutifs à partir de 5 jours après infection et huit chevreaux non traités ont servi de témoins.L’infection expérimentale par C parvum a provoqué une maladie clinique grave avec diarrhée pro-fuse, une excrétion d’oocytes et des lésions intestinales dans tous les groupes de chevreaux. La gra-vité et la durée totale de la diarrhée ont été similaires pour tous les groupes de chevreaux. Cependant,

* Correspondence and reprintsTel: (42) 38 41 158; fax: (42) 38 47748; e-mail: koudela@paru.cas.cz

la durée moyenne de l’excrétion des oocytes, le nombre moyen de cryptosporidies par villosité dansl’iléon, et la distribution des cryptosporidies dans l’intestin ont été augmentés dans les deux groupesde chevreaux traités par le cotrimoxazole. Ce travail indique que le cotrimoxazole ne permet pas decontrôler la cryptosporidiose.

Cryptosporidium parvum / chevreau / infection expérimentale / thérapie / cotrimoxazole

INTRODUCTION

Cryptosporidiosis is a disease caused by acoccidian parasite of the genus Cryp-tosporidium that inhabits the microvilli ofthe epithelial surface of the gastrointestinaland respiratory tracts of a wide variety ofvertebrates including humans. In mammals,Cryptosporidium parvum primarily infectsthe intestine and clinical features of the infec-tion include watery diarrhea, dehydrationand weight loss. The infection is self-limiting,except in immunodeficient hosts, in whichit may cause protracted and untreatable diar-rhea (Fayer et al, 1990, O’Donoghue, 1995).A variety of therapeutic and prophylactic

pharmaceuticals have been tested for effi-cacy against C parvum in humans or ani-mals but none of them produced satisfac-tory prophylactic or therapeutic benefits(Fayer et al, 1990; O’Donoghue, 1995).

Recently, Woods et al ( 1997) found thatC parvum infectious ability was reduced byincubation with folic acid inhibitors in

screening in vitro studies and supposed thatthis was due to the synergistic action oftrimethoprim in combination with one ofthe sulfonamides. This study was conductedto test the prophylactic and therapeutic effi-cacy of cotrimoxazole (trimethoprim/sul-famethoxazole) against a single experi-mental C parvum infection in kids.

MATERIALS AND METHODS

Inoculum

Feces of a naturally infected scouring calf( 12 days of age) were used as the source of

cryptosporidial oocysts. Morphologicallythe oocysts corresponded to the species Cparvum Tyzzer ( 1912). The fecal materialwas suspended in 2.5% potassium dichro-mate and, after removing large particles bypassage through a series of graded sieves,the suspension was kept at room temperaturefor I week. Oocysts were concentrated byflotation in Sheather’s sugar solution andthen washed with tap water to remove theflotation solution. To decontaminate the

oocysts, the suspension was treated in bleach(SAVO, Bochemie, Bohumin, CzechRepublic; 1.25°!o sodium hypochlorite) for15 min. The purified oocysts were stored in2.5% potassium dichromate at 4 °C for27 days until used for kid inoculation.Before inoculation, the C parvum oocystssuspension was washed three times by cen-trifugation in sterile phosphate-buffered-saline (PBS, pH 7.2) and counted by ahemocytometer. The viability of the oocystswas verified by inducing experimental infec-tion in 5-day-old suckling mice (Vitovecand Koudela, 1988).

Experimental design

Twenty-four 1-day-old kids (Saanen goat) ofboth sexes were obtained from a goat-cheeseproduction unit of 80 spring kidding goatswith no record of health problems in thenewborn kids. The kids were separated fromtheir dams immediately after birth. In thefirst 24 h, they were fed with goat colostrumand then transported to an isolator room andfed three times daily with commercial cow’s smilk replacer (Sano, Germany). All 6-day-old kids were orally inoculated with a

syringe containing 6 x 106 C parvum

oocysts in 10 mL of PBS. They were ran-domly separated into three groups. Eachgroup of kids was reared in a separate pen inthe same room and kids of all groups were

kept in the same management conditions.Of the twenty-four 6-day-old kids inocu-lated with 6 x 106 oocysts of C parvum, tenkids (group P) were prophylactically admin-istered with cotrimoxazole (BerlocidO,Berlin-Chemie, Germany) at a dose20 mg/kg per day of trimethoprim/100 mg/kg per day of sulfamethoxazole intwo divided doses for 14 consecutive daysbeginning I day before infection. Six kids(group T) were therapeutically treated at thesame dose of cotrimoxazole for 9 consecu-tive days beginning 5 days post-infection,and eight kids (group C) remained untreatedand served as an infected control.

Assessment of infection

All kids were monitored by observing twicedaily for diarrhea and other clinical signs,and collecting a daily sample of feces fromthe rectum of each kid. The severity of diar-rhea was scored as 0 for normal, I for looseand formless, 2 for semi-fluid, or 3 forwatery. Each day, for each animal, 0.5 g offeces was examined microscopically for Cparvum oocysts using Sheather’s sugar solu-tion. The oocyst shedding was semi-quan-titatively evaluated under a magnificationof x 250 after flotation, 0 for no oocysts, I

for less than one oocyst per field, 2 for oneto ten oocysts per field, and 3 for more thanten oocysts per field.

Beginning 3 days post-inoculation(3 DPI), two prophylactically treated kids(group P) and two control kids (group C),and beginning 7 DPI, two therapeuticallytreated kids were euthanized with an over-dose of barbiturate (Thiopental°, Spofa,Czech Republic) every second or third day.At necropsy, the complete examination of all

organs and tissue was conducted. Samplesfor histopathology were collected immedi-ately after the animals were killed. The firstspecimen was taken from the ileum within5 cm of the ileocecal valve (ostium ileoce-cale - OIC). More samples were removed50 cm from OIC and then every 50 cm cra-nial from OIC so that the last jejunal speci-men originated from duodenum. In the largeintestine, specimens were collected fromapex ceci, colon near ansa centralis, and rec-tum. Additional samples for histology weretaken from abomasum, liver, kidneys,spleen, lung, pancreas, and regional mesen-teric lymphonodes. The material was fixedin 10% neutral formalin and processed byroutine histological methods. Histologicalsections were stained with hematoxylin-eosin, and with Wolbach’s modification ofGiemsa staining. On the basis of the histo-logical examination the distribution of cryp-tosporidia in the intestine and degree ofinfection were classified as follow:

- degree 0: no cryptosporidia found on themucosal surface;- degree I : moderate infection, sporadiccryptosporidia distributed on the intestinalsurface;- degree 2: medium infection, regularlydisseminated cryptosporidia on the intestinalsurface;- degree 3: massive infection, most of theepithelial surface covered by cryptosporidia.

The severity of the ileal infections,expressed as the mean number of cryp-tosporidia per villus, was determined bymicroscopic examination of the hema-toxylin-eosin stained ileal sections and enu-meration of the cryptosporidia, in variousdevelopment stages, that were attached tothe enterocytes of ten villi from the ileumwithin 5 cm from the ileocecal valve.

Statistical analysis

Each point in the graphs represents thedegree and distribution of C parvum infec-

tion in the intestine. The severities of ilealinfections were examined by analysis ofvariance (ANOVA) and comparisons ofmeans (Newman-Keuls test). Statistical sig-nificance was considered to be P < 0.05.

RESULTS

Clinical signs and oocyst shedding

None of the kids had cryptosporidia in theirfeces prior to inoculation. All inoculatedkids had a decreased appetite, and becamedepressed 3-4 days after inoculation. Theirfeces subsequently became watery withclumps of mucus, and color changed frombrown to yellow. Kids in group C, infectedbut untreated, had diarrhea for 3-9 days(mean duration of diarrhea 4.8 days) and Cparvum oocysts were shed for 4-1 1 dayswith a group average of 5.9 days. Prophy-

lactically treated kids (group P) had diar-rhea for 3- I 0 days (mean duration of diar-rhea 5.6 days) and they shed oocysts for 5-13 days with a group average of 7.8 days.Therapeutically treated kids had diarrheafor 3-10 days (mean duration of diarrhea4.8 days) and C parvum oocysts weredetected for 4-13 days with a group aver-age of 8.1 days. The severity of diarrheaand oocyst shedding, based on daily numer-ical scoring, were similar for all groups.

Histopathology

Pathological changes of cryptosporidiosiswere similar or identical in all groups ofanimals. The most severe lesions were seenin the posterior jejunum and ileum from 3 to7 DPI, and consisted of villus atrophy, vil-lus blunting and crypt hyperplasia (fig 1). ).Many jejunal villi were fused together and

cross-bridged. The epithelium covering theatrophic villi was basophilic and usuallylow columnar to cuboidal. The crypt epithe-lium remained tall and columnar. In infected

jejunal and ileal regions, the villus epithe-lium and lamina propria were infiltratedwith numerous mononuclear cells and neu-

trophils. Cryptosporidia were located in thebrush border of enterocytes (fig 2), primar-ily associated with villus enterocytes andwere rarely observed in the crypts.

The distributions of cryptosporidiathroughout the intestine of the untreated andcotrimoxazole treated kids are shown in fig-ures 3-5. No cryptosporidia or associatedpathological lesions were found in the largeintestine of untreated kids (fig 3). In con-trast, in kids treated with cotrimoxazole,small numbers of cryptosporidial develop-mental stages were found in the large intes-tine (figs 4 and 5). The differences between

the distributions of cryptosporidia through-out the intestine were found in cotrimoxa-zole treated kids. On 9 DPI, more cryp-tosporidia were located on the intestinalsurface in therapeutically treated kids than in

prophylactically treated kids. However,more cryptosporidia were found in prophy-lactically treated kids on 13 DPI. Data forthe severity of ileal infections, expressed asthe mean number of cryptosporidia per ilealvillus are presented in table I. These datashowed that the mean number of cryp-

tosporidia per ileal villus was significantlyhigher in cotrimoxazole therapeuticallytreated kids on 9 DPI.

DISCUSSION

A major objective of many workers involvedin Cryptosporidiuw research was to developreproducible experimental animal modelsof the infection and clinical disease. Acuteclinical infections have proven difficult toestablish in small laboratory animals. Aswith other ruminants, neonatal kids are sus-ceptible to C parvurn infection and clinicalinfections have been established in experi-

mentally infected kids (Tzipori et al, 1982;Current et al, 1983). Neonatal kids experi-mentally inoculated with C parvum oocystswere also used to test anticryptosporidialactivity of paromomycin (Mancassola et al,1995). Recently, we have characterized kidsas model for experimental cryptosporidiosis(Koudela and Vftovec, 1997).

Immunodeficient individuals who have

cryptosporidosis may develop life-threat-ening diarrhea (O’ Donoghue, 1995). Treat-ment with cotrimoxazole has provided someclinical improvement in immunodeficientpatients with cryptosporidiosis (Del Bonoet al, 1987). In the present study, we useddaily doses of cotrimoxazole which are rec-ommended for therapy in toxoplasmosis andPneumocystis cariuii pneumonia (PCP) inimmunodeficient patients.

Combinations of sulfonamides with folicacid inhibitors have long been used for thetreatment of intestinal coccidiosis in ani-mals (Long, 1993). However, studies on theefficacy of sulfonamides with folic acidinhibitors against cryptosporidiosis are few.Mirtschin and Ormerod ( 1990) reported thattrimethoprim/sulfamethoxazole-based prod-ucts used for snake cryptosporidiosisinduced oocyst-negative stools. In anotherstudy, trimethoprim/sulfadiazine treatmentalso induced Cryptosporidium serpentis neg-ative feces as determined by flotation, how-ever, histological examination of the gas-tric mucosa revealed cryptosporidialdevelopmental stages (Norton and Jacob-son, 1989).

Studies of treatment of experimentalcryptosporidiosis with sulfonamides pre-sented mixed results. Sulfadimethoxidinereduced cryptosporidial infection in

immunosuppressed rats (Regh et al, 1988;Regh, 1991 ), and sulfonamide prophylaxiswas obtained in another immunosuppressedrat model (Brasseur et al, 199 1 In contrast,the results of another study have shown thatsulfadimethoxidine did not have anticryp-tosporidial activity in experimentally

infected calves. Calves treated with 5 mgof sulfamethoxidine for 21 consecutive dayshad diarrhea on average 2.3 days longer thanuntreated calves, and more C parvumoocysts were shed per milliliter of feces bytreated than by untreated calves (Fayer,I 992).

Our results show that treatment with cot-rimoxazole did not significantly influencethe clinical course of experimental cryp-tosporidiosis. Total days and severity ofdiarrhea were similar for all groups of kids.

However, the mean duration of oocystsshedding, mean number of cryptosporidiaper ileal villus, and distribution of cryp-tosporidia in the intestine were increased inboth groups of cotrimoxazole-treated kids.These findings can be explained by thechanges of intestinal microflora in cotri-moxazole-treated kids. It was noted that theintestinal microflora may stimulate non-

specific mechanisms contributing to resis-tance (Harp et al, 1992).

ACKNOWLEDGMENTS

We thank M Vachova and S Kucerova for theanimal care and V Schacherlova for her techni-cal assistance in laboratory. This study was sup-poiied by the Grant Agency of the Czech Repub-lic, grant no 508/93/2479.

REFERENCES

Brasseur P, Lemeteil D, Ballet JJ (1991) Anti-cryp-tosporidial drug activity screened with an immuno-suppressed rat model../ Proi<>;<><>1 38, 230S-23 IS S

Current WL, Rccse NC, Ernst JV, Bailey WS, Hey-man MB, Weinstein WM (1983) Human cryp-tosporidiosis in immunocompetent and immunod-eficient persons. Studies of an outbreak and

expcrimental transmission. N En,!l J Med 308,1252- I 257

Del Bono V, Anselmo M, Dodi F, Greco C, Rizzo F,Terranga A ( I 987) Pi-evaleiiza del la criptosporid-iosi in cordi di infezione da HTLV III. G Mol lnfPcireisit 39. I l46-1148

Fayer R (1992) Activity of sulfadimethoxine againstcryptosporidiosis in dairy calves. J Parasitol 78,534-537

Fayer R, Speer CA, Dubey JP ( 1990) General biologyof Cryptosporidium. In: Cryptosporidiosis in Manand Animals. (JP Dubey, CA Speer and R Fayer,eds), CRC Press, Boca Raton, USA, 2-29

Harp JA, Chen W, Harmsen AG (1992) Resistance ofsevere combined mice to infection with Cryp-tosporidium parvum: the importance of’ intestinalmicroflora. Infect IlI1l11un 60, 3509-3512 2

Koudela B, Vitovec J (1997) Experimental cryp-tosporidiosis in kids. Vet Parasitol (in press)

Long PL (1993) Avian coccidiosis. In: Para.ritic pro-tozoa. 2nd ed, (JP Kreier, ed) Academic Press Inc,San Diego, USA, 1-88

Mancassola R, Reperant JM, Naciri M, Chartier Ch(1995) Chemoprophylaxis of Cryptnsparidiumparvum infection with paramomycin in kids andimmunological study. Antimicrob Agent.sChemother 39,75-78

Mirtschin PJ, Ormerod S (1990) Indication for thetreatment of cryptosporidiosis in snakes. Aus HerpNews 5, 3

Norton TN, Jacobson ER (1989) Cyclic changes incryptosporidial infection in a San Diego mountainkingsnake, Lampropelti.s zonata pulchra. In: Proc3rd Int colloguium on the pathology of reptile.s andamphibians (ER Jacobson, ed) University of F7oridaPress, Gainesville, USA, 51-52

O’Donoghue P (1995) Cryptosporidium and cryp-tosporidiosis in man and animals. Int J Para.ritnl25, 139-195

Regh JE (1991) Anticryptosporidial activity associ-ated with specific sulfonamides in immunosup-pressed rats. J Parasitol77, 238-240

Regh JE, Hancock ML, Woodmansee DB ( 1988)Anticryptosporidial activity of’sulfadimethoxine.Antimicrob Agentv Chemother 32, 1907-1908

Tzipori S, Larsen J, Smith M, Leufel R (1982) Diarrheain goat kids attributed to Cryptosporidium infec-tion. Vet Rec lll, 35

Vftovec J, Koudela B (1988) Location and pathogenic-ity of Cryptosporidium parvum in experimentallyinfected mice. J Vet Med B 35, 515-524

Woods KM, Ncsterenko MV, Upton SJ (1996) Effi-cacy of 101 antimicrobials and other agents on

development of Cryptosporidium parvum in vitro.Ann Trop Med Para.sitol 90, 603-615