Specific Serum and Local Antibody Responses against Cryptosporidium parvum during Medication of

Transcription

1 INFECTION AND IMMUNrrY, Oct. 199, p /9/ $0/0 Copyright 199, American Society for Microbiology Vol. 61, No. 10 Specific Serum and Local Antibody Responses against Cryptosporidium parvum during Medication of Calves with Halofuginone Lactate JOHAN E. PEETERS,1* ISABEL VILLACORTA,' MURIEL NACIRI, AND EMMANUEL VANOPDENBOSCH' Section of Parasitology, National Institute of Veterinary Research, B-1180 Bnrssels, Belgium, and Station de Pathologie Aviaire et de Parasitologie, Institut National de la Recherche Agronomique, F-780 Nouzilly, France Received February 199/Returned for modification 6 May 199/Accepted 14 July 199 Fecal and serum anti-cryptosporidium parvum immunoglobulin A (IgA), IgM, and IgG were monitored by an enzyme immunoassay in C. parnum-infected calves after medication with halofuginone lactate. In a first experiment, four groups of five 1-day-old colostrum-fed calves were inoculated with 10' oocysts of C. parvum. They were medicated with 0, 0, 60, or 10 pg of halofuginone lactate per kg from days to 8 postinfection (p.i.). Unmedicated calves passed large numbers of oocysts between and 14 days p.i. Treatment with 0 pglkg did not completely inhibit oocyst output during medication, whereas 60 and 10 ug/kg did. The latter groups passed only a reduced number of oocysts when the drug was withdrawn. In a second experiment, - to 6-day-old colostrum-fed calves were divided into three groups of 16 or 17 animals each. All animals had acquired C. parvum infection before arrival at the fattening unit. They were medicated with 0, 60, or 10 pg/kg for 7 days beginning on the day of arrival. Unmedicated calves passed large numbers of oocysts from 0 to 1 days. Medication stopped oocyst output at day 7, but some of the calves again passed low numbers of oocysts 7 days after withdrawal of the drug. Experimental infection of unmedicated calves was followed by a rise in local anti-c. parvum IgA and IgM titers. Rising coproantibody levels coincided with falling oocyst output. In halofuginone-medicated and experimentally infected calves, only specific anti-c. parvum IgM levels rose during the first 5 days p.i. Specific IgA levels increased in association with oocyst output after withdrawal of the drug in the 60- and 10-pg/kg groups. In naturally infected calves, on the other hand, both specific IgA and IgM levels rose further during medication. Although titers were lower than in unmedicated controls, no significant differences were observed. Both medicated and unmedicated calves were equally protected from a challenge with 107 oocysts 16 weeks after the first contact with the parasite. Cryptosporidium parvum is a protozoan parasite found worldwide that is of considerable veterinary interest. It causes neonatal diarrhea in calves, goat kids, and lambs. C. parvum has been identified as the second most common infectious agent in outbreaks of diarrhea in calves (1, 7). As the parasite is not host specific, it can easily be transmitted from one mammalian species to another. The overall distribution of the parasite in ruminants causes contamination of surface and ground water. Contaminated drinking water was reported as a source of human cryptosporidiosis (5, 10). Prevention of cryptosporidiosis is difficult. Whereas most other species of enteric coccidia have a genetically programmed series of developmental stages in the life cycle and are incapable of recycling within the host, C. parvum has two stages that initiate autoinfectivity, type I merozoites and sporozoites derived from thin-walled oocysts. Both characteristics are believed to be the life cycle features of C. parvum responsible for the development of severe infections in hosts exposed to only a small number of oocysts (4). In the absence of effective drugs, only development of sufficient immunity is able to stop the cycle. At this time, no commercially available drugs are approved for treatment of infections caused by the parasite. In a review of the literature, Fayer and Ungar (7) reported that of 79 antibiotics and anticoccidial agents tested, none * Corresponding author showed sufficient activity. Sulfaquinoxaline was shown to reduce oocyst output in mice and rabbits (, 17). Although arprinocid was reported to reduce the rate of infection in experimentally infected rats and hamsters (1, 1), McDonald et al. (14) were not able to confirm such an effect in vitro. Lasalocid, however, was more promising in both in vitro cultures and naturally infected calves (8, 15, 0). Unfortunately, the drug is rather toxic. Better results were obtained with halofuginone lactate in lambs (16). In naturally C. parvum-infected calves, the drug strongly reduces oocyst output and clinical signs (). Seven to 10 days after withdrawal of the drug, low numbers of oocysts may again appear in the feces, indicating reinfection or reactivation of inhibited stages. However, almost all animals passing oocysts after drug withdrawal remain free of clinical signs. As the number of animals which pass oocysts increases again at higher doses, it seems logical to accept that lower doses allow the development of immunity, as they do not completely inhibit development of the parasite. Currently, little is known about the immunobiology of Cryptosporidium spp. Enteric cryptosporidiosis in immunocompetent hosts is self-limiting, and the immune status of the host appears to determine the severity and duration of infection (4). Calves that recover from infection are resistant to a second challenge with the same isolate (9). Therefore, recovery depends on a specific, acquired immune response, although the exact mechanisms responsible for resistance have not yet been defined. Recent studies showed that

2 VOL. 61, 199 HALOFUGINONE AND LOCAL IMMUNE RESPONSE TO C. PARVUM 4441 increasing anti-c. parvum immunoglobulin A (IgA) antibodies in the gut are associated with decreasing oocyst output in lambs (1) and calves (19). This might indicate that IgA inhibits penetration of host cells by sporozoites, as has already been demonstrated for Eimeria tenella infection in chickens (16). As lower doses of halofuginone seem to allow the development of immunity during medication, we decided to study the specific serum and local antibody responses in C. parvum-infected calves during and after medication with halofuginone lactate. MATERIAILS AND METHODS Animals and husbandry. In a first experiment, 0 male 1-day-old calves of mixed Holstein-Friesian breed were purchased from traditional calf breeders. The animals received colostrum before transfer to the Institut National de la Recherche Agronomique. They were allocated to five individual pens with wooden grid floors, measuring 5 by 1.75 m, situated in the same room. Calves within adjacent pens were tethered to a side of the pen which was not shared to prevent contact between animals. The room was aerated with filtered air, and hygienic measures were taken to avoid cross-contamination of the animals. Animals were fed a semisynthetic milk substitute ad libitum. In the second experiment, a total of 50 male - to 6-day-old calves of red-and-white mixed Belgian breed, purchased from the local market and colostrum fed, were tested. Animals were not vaccinated and did not receive any vitamins, other nutritional supplements, or any type of medication during the experiments. Before the calves were admitted, the research unit was thoroughly cleaned and disinfected. The animals were allocated at random to individual wooden pens with wooden grid floors measuring 5 by 1.75 m. Solid walls between the boxes completely prevented contact between the animals. Animals were fed a semisynthetic milk substitute four times a day. For the first weeks after arrival, the amount of milk substitute was restricted, in contrast to the ad libitum-fed animals, and all animals were fed the same amount of milk substitute. The same animals were used for experiment. C. parvum. In the first experiment, a C. parvum isolate from a -year-old boy () that had been serially propagated for 8 years in lambs or calves without exposure to any anticoccidial drug was used. In the third experiment, a recent bovine isolate obtained from a fattening unit was used. Immunostaining of Western blots (immunoblots) of C. parvum antigens separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with bovine IgA obtained from naturally infected calves, recognizing C. parvum antigen in the enzyme-linked immunosorbent assay (ELISA), and with IgG obtained from rabbits hyperimmunized with a bovine C. parvum isolate did not reveal differences between the two isolates used for the experimental infections or among the isolates from the naturally infected calves (unpublished data). Oocysts were purified from fresh fecal material within 6 h of collection as described before (18). Briefly, feces were washed through 150- and 45-pum sieves (Endocotts Ltd.) and centrifuged at 500 x g for 5 min. Subsequently, the sediment was shaken in a 1:1 (vol/vol) mixture of water and diethyl ether. After a second centrifugation, the supernatant was decanted. After being washed, the sediment was resuspended in distilled water and passed on a discontinuous Percoll gradient (Pharmacia Fine Chemicals, Uppsala, Sweden) composed of four -ml layers with densities of 1.1, 1.09, 1.05, and 1.01 g/ml and centrifuged at 650 x g for 15 min. The band containing purified oocysts was removed, washed twice at 500 x g for 5 min each, and suspended in distilled water. The number of oocysts present was counted in a modified Neubai-er hemocytometer after mixing 0. ml of suspension with 0.8 ml of malachite green (0.16 g of malachite green, 0.1 g of SDS, 100 ml of distilled water). The suspension was then diluted to the desired concentrations. Animals were challenged with 106 (experiment 1) or 107 (experiment ) oocysts suspended in 0 ml of phosphate-buffered saline, ph 7. (PBS). Drug. Halofuginone lactate was supplied as a.97% (wt/vol) concentrate by Roussel Uclaf. A working solution was prepared daily by diluting 1 volume of concentrate in 46 volumes of distilled water in order to obtain 10,ug of halofuginone lactate per ml. Animals were medicated with 0, 0, 60, or 10,ug/kg of live weight. Experimental design. (i) Experiment 1. Twenty-one-dayold calves were divided into four groups of five animals and inoculated orally with 106 C. parvum oocysts at the station. Two groups were allocated per room. One group was not medicated, whereas the three other groups were medicated with 0, 60, or 10,ug of halofuginone lactate per kg from days to 8 postinfection (p.i.) (first treatment was 48 h after experimental infection). Fresh fecal material was collected daily for 1 month from each animal by rectal sampling, and the fecal consistency was noted. The samples were then stored immediately at 4 C. The presence of C. parvum oocysts in the samples was evaluated after sucrose flotation (d = 1.7) and scored semiquantitatively at a magnification of 50x (0, no oocysts; 1, fewer than 1 oocyst per microscopic field;, between 1 and 5 oocysts per field;, between 6 and 10 oocysts per field; 4, more than 10 oocysts per field). Coproantibodies were extracted from the feces at 4 C within 4 h of collection. For this, g of fecal material was homogenized by shaking with glass beads in 8 ml of PBS (ph 7.) and spun down at 650 x g for 15 min at 4 C. The supernatant was stored at -0 C and checked later for IgA, IgG, and IgM by ELISA. Fecal samples were examined for rotaviruses, coronaviruses, enterotoxinogenic Escherichia coli (K99), and Salmonella spp. by the Laboratoire de la Direction des Services Veterinaires d'indre et Loire before experimental infection. (ii) Experiment. At arrival, the 50 calves were divided into three groups of 16 to 17 animals each (mean body weight, 47.8 kg; coefficient of variation, 9.%) and allocated to individual boxes in a research unit. Only healthy-appearing calves in good shape were used. They were allocated alternatively to the different boxes. One group was not medicated, whereas the other two groups were medicated with 60 or 10 jig of halofuginone lactate per kg for the first 7 days after arrival. Animals were observed daily, and possible diarrhea was evaluated. Individual blood samples were taken from a jugular vein at arrival and 5, 6, and 11 days later. Serum was stored at -0 C until tested for circulating anti-c. parvum IgA, IgG, and IgM by ELISA. Over a period of 4 weeks, fresh fecal material was collected from each animal twice a week by rectal sampling. For the next 4 weeks, rectal samples were collected weekly and then every weeks for 8 weeks. As previous experiments () had showed a close relationship between the actual number of oocysts present and semiquantitative scoring of oocysts, fecal oocyst numbers were evaluated semiquantitatively with carbol fuchsin stain (11) at a magnification of 500x with a Leitz Laborlux 1 microscope. For each smear (100 pul), a surface of approximately 40 by 0 mm was examined, and the average quan-

3 444 PEETERS ET AL. titative score for 10 representative microscope fields was reported (0, no oocysts; 1, fewer than 5 oocysts per microscopic field;, between 5 and 5 oocysts per field;, more than 5 oocysts per field). After this step, fecal material was homogenized in PBS and spun down as described above. The sediment was checked for the presence of Salmonella spp. by standard biological procedures, and the supernatant was checked for the presence of rotavirus by immunodiffusion. An aliquot of supernatant was stored at -0 C until tested for IgA, IgG, and IgM by ELISA. (iii) Experiment. At 11 days of age, five calves in each group for experiment were challenged with 107 oocysts of C. parvum suspended in 0 ml of PBS (ph 7.). Five other calves per group served as unchallenged controls. For 4 weeks, diarrhea, elimination of C. parvum oocysts, rotavirus, and Salmonella spp., and kinetics of local and serum antibodies were evaluated. ELISA. Specific local and serum antibodies were detected by ELISA as described before (19). Briefly, microtitration plates were coated with crude sonicated oocyst antigens in carbonate buffer (ph 9.6). After incubation for 1 h at 7 C and then at 4 C overnight, the plates were washed with PBS containing 0.05% Tween 0 (PBS-T). Wells were blocked with 1% horse serum (Life Technologies, Gaithersburg, Md.) at 7 C for 1 h. Fecal extracts were diluted 1:5 (= original supernatant), 1:5, 1:15, and 1:65. After a wash with PBS-T, 100,ul of fecal extract or serum dilution was added to the wells and incubated with affinity-purified IgG raised in rabbits against bovine IgA (Nordic Pharma, Tilburg, The Netherlands) or against bovine IgM (ICN Biomedicals, Costa Mesa, Calif.). After further washing, the wells were subsequently incubated with peroxidase-conjugated affinity-purified goat anti-rabbit IgG (Sigma Chemical Co., St. Louis, Mo.). To test for the presence of anti-c. parvum IgG in fecal extracts and in serum dilutions, samples were treated immediately after incubation with peroxidase-conjugated affinity-purified rabbit anti-bovine IgG. Finally, tetramethylbenzidine (Kirkegaard & Perry, Gaithersburg, Md.) was added and allowed to react at room temperature (+ C) for 15 min. The reaction was stopped by adding 1 M o-phosphoric acid, and the optical densities at 450 nm (OD450) were read in an EL-40 BioKinetics Reader (Bio- Tek Instruments, Winooski, Vt.). The last dilution of each sample giving an OD value larger than twice the mean OD of 10 negative-control samples was considered the end titer. Fecal extracts which gave titers of more than 65 were tested again at dilutions of 1:500, 1:1,000, 1:,000, and 1:4,000. Statistics. The mean oocyst output was tested by the nonparametric Wilcoxon-Mann-Whitney U test. Differences among antibody titers were evaluated by analysis of variance (). RESULTS Clinical findings and microbiology. In experiment 1, the experimental infection caused severe clinical symptoms in nonmedicated and medicated (0,g/kg) calves: anorexia, depression of weight gain, emaciation and/or dehydration, and death (three animals out of five in each group; see Fig. and 4). All calves in both groups exhibited diarrhea for several days. The surviving calves showed profuse diarrhea for 6 days (days 5 to 10 p.i.). No animals died in the 60- and 10-,ug/kg dose groups. In the 60-,ug/kg dose group, the calves did not exhibit watery diarrhea; only slightly pasty feces were noted for two animals. In the 10-,ug/kg dose group, no diarrhea was noted i 0;./ D D DD DDD D D D D D D D D D D D D D D D D D D D D D D FIG. 1. Influence of medication with halofuginone lactate on days 1 to 7 on mean oocyst output after experimental infection with 106 oocysts of C. parvum. *, no drug treatment; El, 0,ug/kg; x, 60,ug/kg; O, 10 plg/kg. Scoring is defined in the text. The kinetics of oocyst shedding is shown in Fig. 1. In unmedicated animals, oocyst output reached a peak at 7 days p.i. and lasted until day 14 p.i. Afterwards, no oocyst output was observed. In the 0-,ug/kg dose group, treatment did not completely inhibit oocyst output during medication, and no oocyst output was noted afterwards either. No passage of oocysts was noted during treatment in calves treated with 60 or 10,ug/kg. Oocyst output started 0 to 10 days after withdrawal of the drug. In some animals, other etiological agents of neonatal diarrhea were detected before inoculation; no coronaviruses were detected, but seven animals eliminated rotaviruses in the feces (two in the uninfected group, three in the 0-,ug group, and two in the 10-jg group). E. coli K99 was present in four calves (two in the 0-,ug group and one each in the 60- and 10-pug groups), of which one was also positive for Salmonella infection. This animal (0-,ug/kg group) died at days p.i. In experiment, all unmedicated calves passed cryptosporidial oocysts through the feces. Maximal passage occurred 7 days after arrival (Fig. ). Therefore, a 100% infection ratio at the arrival of the calves may be accepted. On day 1, oocyst output dropped to the detection limit of 104 to 105 oocysts per g of feces. Most animals shed oocysts again 5, 7, and 14 weeks after arrival. Medication with 60 or 10 pug of halofuginone lactate per kg of live weight stopped oocyst output completely at day 7. However, some of the medicated calves began to again pass low numbers of INFECT. IMMUN =q DO D4 D7 Dll D14 D18 D1 D5 D D5 04 D49 D05 D70 D84 D98 D11 FIG.. Influence of medication with halofuginone lactate on days 1 to 7 after arrival on mean oocyst output after natural infection with C. parvum. *, no drug; E, 60,ug/kg; *, 10 pg/kg. Scoring is defined in the text.

4 VOL. 61, 199 HALOFUGINONE AND LOCAL IMMUNE RESPONSE TO C. PARVUM ^ -.00 ~~~~~~~~~~~~~~~~~~ D0 D D5 D7 D9 D1 D14 D16 D19 D1 D D6 D8 D0 FIG.. Kinetics of mean oocyst output (score, left-hand scale) and mean titers of specific anti-c. parvum coproantibodies (log1o titer, right-hand scale) after experimental infection of 1-day-old calves with C. parvum. Bars, oocyst output; *, IgA; 5, IgG; *, IgM; *, day of death. oocysts 7 days after withdrawal of the drug. Afterwards, oocyst output followed the same pattern as in unmedicated calves. No Salmonella spp. were detected in any of the calves. In each group, 8 to 14 calves eliminated rotavirus in the feces, mainly between days 1 and 5. This was associated with liquid to mucous feces. One calf shed low numbers of an Eimeria sp. at 5 days p.i. In unmedicated calves, C parvum infection was associated with liquid to mucous feces in 11 of 17 animals between days 4 and 14, whereas no C. parvumassociated diarrhea was observed in medicated calves. In all groups, a peak of diarrhea was observed between days 1 and 5, associated with a mixed C. parvum-rotavirus infection. In experiment, both medicated and unmedicated animals remained completely refractory to massive infection: no oocyst output or clinical signs were detected. Kinetics of serum and coproantibodies. In experiment 1, 4 h after intake of colostrum at birth, fecal extracts were positive for specific anti-c. parvum IgA, IgG, and IgM isotypes at dilutions of 1:1,000, 1:500, and 1:650, respectively. After days, these titers dropped to.1:5. In unmedicated calves, rising specific immunoglobulin levels were closely associated with increasing oocyst output. Once immunoglobulins had reached their maximal level, oocysts 4 0 D0 D D5 D7 D9 D1 D14 D16 D19 D1 D D6 D8 D0 FIG. 4. Influence of halofuginone lactate (0 pg/kg) on fecal immunoglobulin levels (log1o titer, right-hand scale) and oocyst output (score, left-hand scale) after experimental infection with 106 oocysts of C parvum. Bars, oocyst output; U, IgA; 0, IgG; *, IgM; *, day of death DO D D5 D7 D9 D1 D14 D16 D19 D1 D D6 D8 D0 FIG. 5. Influence of halofuginone lactate (60,ug/kg) on fecal immunoglobulin levels and oocyst output after experimental infection with 106 oocysts of C. parvum. See Fig. 4 legend for details. were no longer detectable in the feces. Specific IgM titers rose very quickly from day to reach a peak on day 14 p.i. (Fig. ). Afterwards, IgM levels decreased. Specific IgA titers increased from day 5 p.i. and reached a peak on day 1 p.i. Fecal extracts were still positive for specific IgA 0 days after infection. Specific IgG levels rose slightly during oocyst output and had almost disappeared by weeks p.i. In the halofuginone lactate-medicated groups, only specific IgM levels rose during the first 5 days p.i. Depending on the dose, specific IgM titers rose further until day 16 (0,ug/kg), remained stable until day 1 (60,ug/kg), or decreased quickly (10,ug/kg) (Fig. 4, 5, and 6). In the 10-pg/lkg group, specific IgM levels rose again when the first oocysts appeared in the feces at 18 days p.i., or 10 days after withdrawal of the drug. Whereas specific IgM activity was present in all groups during treatment, specific IgA levels rose only in the 0-pg/kg group in association with oocyst output. In both the 60- and 10-p,g/kg groups, IgA levels increased only after drug withdrawal and in association with oocyst output; maximal levels were obtained at 14 and 6 days p.i., respectively. In experiment, on the day of arrival at the farm, low levels of specific anti-c. parvum coproantibodies were detected (titers, c1:5), yet specific serum anti-c. parvum serum IgA and IgG were detected by ELISA on the day of arrival of the calves (Table 1), indicating intake of immune colostrum. In unmedicated calves, local anti-c. parvum IgA and IgM levels rose simultaneously and reached maximal 4 0 D0 D D5 D7 D9 D1 D14 D16 D19 D1 D D6 D8 D0 FIG. 6. Influence of halofuginone lactate (10,ug/kg) on fecal immunoglobulin levels and oocyst output after experimental infection with 106 oocysts of C. parvum. See Fig. 4 legend for details

5 4444 PEETERS ET AL. TABLE 1. Influence of halofuginone lactate dose on serum antibodies after natural infection of - to 6-day-old calves with C. parvum Serum Ig Day Logl0 ELISA titer at dose: isotype P'i 0 jig/kg 60 jg/kg 10 jig/kg IgA ± ± ± ± ± ± ± ± ± 1.08 ± 1.0 IgG ± ± ± 0.61 ± ± ± ± ± ± ± ± 0.86 IgM ± ± ± ± ± ± ± ± ± ± ± ± 0.90 titers of 1:15 to 1:,000 and 1:5 to 1:65, respectively, 14 days after the mean first day of contact (Fig. 7). IgM levels declined very quickly. Once the oocyst output had dropped, IgA titers declined slowly to reach low titers (.1:5) 11 days after arrival. Levels of anti-c. parvum IgG remained low and rose only on the days preceding the mixed fecal C. parvum-rotavirus release. In medicated calves, the kinetics of specific anti-c. parvum IgA, IgG, and IgM were similar to the kinetics detected in unmedicated animals. Although titers of specific serum and local antibodies were somewhat lower than in unmedicated calves, no significant differences were detected (Table 1, Fig. 8). In experiment, at 11 days of age, medicated and unmedicated calves were challenged with 107 oocysts of C. parvum (isolate Slooten) 16 weeks after the primary contact with the parasite. No significant differences were established between serum and local antibodies among the medicated and unmedicated groups. Only fecal IgA levels rose slightly in comparison with the levels in the unchallenged control animals from 8 days after challenge. DISCUSSION In these experiments, C. parvum caused clinical signs; experimentally infected calves showed high mortality, and D4 D7 Dll 14 DI801D5 D8 M D4D49D568D70 D84 D98D11 FIG. 7. Kinetics of mean oocyst output (score, left-hand scale) and mean titers of specific anti-c. parvum coproantibodies (log1o titer, right-hand scale) after natural infection of - to 6-day-old calves with C. parvum. Bars, oocyst output; *, IgA; O, IgG;*, IgM. INFECT. IMMUN. DO D4 D7 DIt D14 D18 D1 D D4 D49 D56 D70 D D11 FIG. 8. Influence of halofuginone lactate on kinetics of specific fecal anti-c. parvum IgA after natural infection of - to 6-day-old calves with C. parvum. Scale, log1o titer. *, no drug; El, 60,ug/kg; A, 10 jig/kg. 65% of the naturally infected calves contracted diarrhea. All unmedicated calves passed large numbers of oocysts. After experimental infection, peak oocyst release was reached at 7 days p.i., whereas similar kinetics were observed 7 days after the arrival of calves purchased from the market. Therefore, we may assume that virtually all the animals in experiment became infected during or before transit to the rearing unit. Medication with 60 or 10 jig of halofuginone per kg completely prevented mortality in experimentally infected calves and C. parvum-associated diarrhea in naturally infected calves. The same doses also completely prevented oocyst output after experimental infection and stopped oocyst release 7 days after the start of treatment in naturally infected calves. This confirms the anticryptosporidial activity of the drug, as described before (). In unmedicated calves, oocysts were no longer detectable in the feces weeks after experimental infection, which indicates the development of resistance. Also, in naturally infected animals, oocyst output stopped almost completely by weeks p.i., although low numbers of oocysts were detected in the following months. Medicated animals passed oocysts after drug withdrawal, reaching peak values at 1 or 18 days p.i. in the 60-,ug groups and at 1 or days p.i. in the 10-jig/kg groups, yet significantly fewer than the unmedicated calves. This suggests at least some development of immunity during medication. The analysis of fecal extracts established that IgA and IgM are the major immunoglobulins available for activity against endogenous stages of C. parvum in the gut. Specific local IgG levels rose only during and shortly after oocyst release (experiments 1 and ) and during the episode of rotavirus shedding (experiment ), probably because of C. parvumand/or rotavirus-related alteration of the mucosa and subsequent leakage of serum antibodies. IgA and IgM were detected early during infection, probably reflecting preferential stimulation of the mucosal IgA response via the Peyer's patches. The secretion of maximal IgA levels specific to C. parvum occurred during the same period of time that oocyst levels in the feces had begun to drop noticeably. Thus, a good temporal association was found between C. parvum expulsion and the amount of specific IgA in secretions. Similar evidence was obtained in lambs after experimental infection with C. parvum (1). This might indicate antibodymediated inhibition of sporozoite and/or merozoite penetration of host cells, as demonstrated for E. tenella infection in chickens (6). Specific fecal IgA levels were maintained for at

6 VOL. 61, 199 HALOFUGINONE AND LOCAL IMMUNE RESPONSE TO C. PARVUM 4445 least 4 weeks after experimental infection and for at least 1 weeks in naturally infected calves. As the half-life of bovine IgA in intestinal secretions is short, the long-term presence of specific IgA in intestinal secretions reflects a continuous antigenic stimulation of calves in fattening units by reingestion of oocysts or recolonization of the intestinal tract. Analysis of specific fecal IgA, IgM, and IgG kinetics showed that halofuginone lactate reduces the specific local immune response to a certain extent. This may be explained by the lower numbers of C. parvum (reduced antigenic stimulation) in the gut. The immune response was closely related to the dose of drug used. Whereas specific IgM titers increased in all experimentally infected calves during treatment, IgA levels rose only in the 0-p.g/kg group in association with oocyst output. In both the 60- and 10-pg/kg groups, specific IgA levels only started to rise when the first oocysts were eliminated in the feces after withdrawal of the drug; maximal levels were obtained at 14 and 6 days p.i., respectively, in association with maximal oocyst output. This suggests that IgA requires a stronger antigenic stimulation than IgM and that a certain level of IgA is required in order to stop primary infection. In naturally infected animals, however, specific anti-c. parvum IgA levels rose further during drug treatment. This indicates that most animals had acquired the primary infection several days before treatment was started (treatment of experimentally infected animals began at days p.i.). After withdrawal of the drug, no differences in specific anti-c. parvum IgA, IgG, and IgM responses were observed between medicated and unmedicated animals. Moreover, both groups were completely refractory to a massive reinfection. We may conclude that medication with 60 to 10,ug of halofuginone lactate per kg not only strongly reduces oocyst output during the clinical phase of the disease, but also allows the development of a specific serum and local antibody response similar to that observed in the absence of treatment. ACKNOWLEDGMENTS This work was partly supported by a grant from Roussel Uclaf, Paris, France. We thank Danielle Vandergheynst, Riet Geeroms, Clement Derijck, and Luc Van Muylem for skillful technical assistance. REFERENCES 1. Angus, K. W Mammalian cryptosporidiosis: a veterinary perspective, p In K. W. Angus and D. A. Blewett (ed.), Cryptosporidiosis: Proceedings of the First International Workshop. Moredun Research Institute, Edinburgh.. Angus, K. W., G. Hutchison, I. Campbell, and D. R. Snodgrass Prophylactic effects of anticoccidial drugs in experimental murine cryptosporidiosis. Vet. Rec. 114: Arnaud-Battandier, F., M. Naciri, A. Fisher, C. Ricou, C. Griscelli, and P. 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