Bovine antibody against Cryptosporidium parvum elicits a circumsporozoite precipitate-like reaction and has immunotherapeutic effect against persistent cryptosporidiosis in SCID mice.

Control of cryptosporidiosis is currently hampered by the absence of drugs or vaccines proven consistently effective against Cryptosporidium parvum. On the basis of observations that anti-C. parvum antibody has therapeutic effect against cryptosporidiosis, cows were immunized with C. parvum to produce hyperimmune colostral antibody. An antibody-rich fraction was prepared and differentiated from control (nonhyperimmune) antibody by enzyme-linked immunosorbent assay, immunofluorescence assay, immunoelectron microscopy, and in vitro neutralizing titer against DEAE-cellulose-isolated C. parvum sporozoites. Oocyst, purified sporozoite, and merozoite antigens recognized by hyperimmune antibody were defined by Western blot (immunoblot). Hyperimmune antibody recognized antigens common to oocysts, sporozoites, and merozoites, as well as stage-specific antigens. Upon incubation with hyperimmune antibody, sporozoites underwent distinct morphologic changes characterized by progressive formation and eventual release of membranous sporozoite surface antigen-antibody complexes, similar to the malaria circumsporozoite precipitate reaction. The infectivity of sporozoites having undergone this reaction was neutralized. The reaction was minimal or absent on sporozoites incubated with control antibody. To determine therapeutic effect in vivo, persistent C. parvum infection was established in adult severe combined immune-deficient (SCID) mice by oral inoculation with 10(7) oocysts. At 5 weeks postinfection, infected mice were treated for 10 days with hyperimmune or control antibody by inclusion in drinking water and daily gavage. Fecal oocyst shedding and infection scores in the gastrointestinal tract and gall bladder/common bile duct in hyperimmune antibody-treated mice were significantly lower than those in the control antibody-treated mice. Hyperimmune bovine antibody prepared against C. parvum may provide a first-generation therapy for control of cryptosporidiosis. Additionally, the defined antigens can be evaluated as subunit immunogens to produce better-characterized polyclonal antibody for control of cryptosporidiosis or as targets for monoclonal antibody-based immunotherapy.     

Lacteal immunity to enteric cryptosporidiosis in mice: immune dams do not protect their suckling pups.

The susceptibilities of passively immunized principal and nonimmunized control suckling mice to orogastric challenge with Cryptosporidium parvum oocysts were compared. Principals were suckled by dams that had recovered from C. parvum infection. Controls were suckled by dams reared free of C. parvum infection. Principals and controls were equally susceptible to challenge. Principals were susceptible even when their dams were hyperimmunized by oral and parenteral booster inoculations with C. parvum oocysts. Immune dams produced serum antibody against C. parvum, while nonimmune dams did not. Anti-cryptosporidia immunoglobulin G (IgG) and IgA were demonstrated in whey extracted from the stomachs of principals that had suckled immune dams but not in whey extracted from the stomachs of controls. It was concluded that passive lacteal immunity is not an efficient means of protection against cryptosporidiosis in mice. As in other coccidian infections, protective immunity against cryptosporidiosis may depend more on immune cells than on antibody.     

Beta-lactoglobulin as a potential modulator of intestinal activity and morphology in neonatal piglets

Studies were conducted to determine the effects of the whey protein beta-lactoglobulin (blg) on the intestinal development and intestinal morphology in neonatal piglets. Two trials (5 and 28 days) were conducted on piglets in three separate groups. One group served as a control group while the remaining two were experimental groups based on diets. The two experimental diets were a bovine colostrum powder, one with supplementation with blg and the other without. The control group remained suckling on a sow. All groups were weaned on day 20 and placed onto a solid commercial piglet diet. Enzymatic activity, total DNA synthesis, crypt depth, and villus height were all parameters used to evaluate the effect of blg. Piglets fed a diet supplement with blg had the greatest total DNA after 5 days. Sow-reared piglets had the greatest intestinal morphology development with regards to villus height. No significant differences were found in enzymatic activity between treatments.     

Effect of orally administered monoclonal antibody on persistent Cryptosporidium parvum infection in scid mice.

Scid mice, persistently infected after exposure to 10(7) Cryptosporidium parvum oocysts, were treated daily for 14 to 17 days with 0.4 mg of monoclonal antibody (mAb) 17.41 administered by the oral route. Mice receiving mAb 17.41 shed significantly fewer (P < 0.005) C. parvum oocysts than scid mice receiving isotype control mAb. Intestinal (but not gastric) infectivity scores were also reduced for scid mice treated with mAb 17.41 (P < 0.01).     

Effects of Escherichia coli heat-stable enterotoxin STb on intestines of mice, rats, rabbits, and piglets

There are at least two classes of Escherichia coli heat-stable enterotoxins, STa and STb. Unlike STa, which is active in suckling mice and piglets, STb is inactive in suckling mice but active in piglets and older, weaned pigs. This study examined the activity of STb in several animals and its effect on intestinal histology and cyclic GMP levels in intestinal mucosal cells. STb did not cause fluid secretion in suckling mice up to 12 days old or in rat or rabbit intestinal-loop preparations. STb-induced fluid secretion in weaned-pig intestinal loops occurred by 30 min and became maximal by 3 to 6 h. STb did not disrupt intestinal histology and did not alter cyclic GMP levels in intestinal mucosal cells from piglet intestinal loops after 0.5- and 6-h incubations. Our studies support the concept that STb is a second heat-stable E. coli enterotoxin with properties and a mechanism of action unlike those of STa.     

Neutrophils do not mediate the pathophysiological sequelae of Cryptosporidium parvum infection in neonatal piglets

Cryptosporidium parvum is a minimally invasive protozoal pathogen of intestinal epithelium that results in villus atrophy, mucosal lipid peroxidation, diarrhea, and diminished barrier function. Influx of neutrophils is a consistent feature of human and animal cryptosporidiosis, and yet their contribution to the pathological sequelae of infection has not been investigated. Accordingly, we used an established neonatal piglet model of C. parvum infection to examine the role of neutrophils in disease pathogenesis by inhibiting their recruitment and activation in vivo using a monoclonal anti-CD18 antibody. Infected piglets were treated daily with anti-CD18 or isotype control immunoglobulin G and euthanized at peak infection, at which time neutrophil infiltrates, lipid peroxidation, severity of infection, and intestinal barrier function were quantified. C. parvum infection resulted in a significant increase in mucosal neutrophil myeloperoxidase activity that was prevented by treatment of piglets with anti-CD18 antibody. Neutrophil recruitment was dependent on mucosal superoxide formation (prevented by treatment of infected piglets with superoxide dismutase). Neutrophils did not contribute to peroxynitrite formation or peroxidative injury of C. parvum-infected mucosa and had no impact on the severity of epithelial infection, villus atrophy, or diarrhea. The presence of neutrophils in C. parvum-infected mucosa was associated with enhanced barrier function that could not be attributed to mucosal elaboration of prostaglandins or stimulation of their synthesis. These studies are the first to demonstrate that neutrophilic inflammation arising in response to infection by a noninvasive epithelial pathogen results in physiologic rather than pathological effects in vivo.     

Present and future control of cryptosporidiosis in humans and animals

Although water treatment processes to remove Cryptosporidium are improving and detection methods for identifying the parasite in water are becoming more sensitive, outbreaks of cryptosporidiosis continue in the human population. Animals, especially dairy calves, often become infected as C. parvum oocysts are present in high numbers, remain viable for long periods of time after excretion and no disinfectants, prophylactic or therapeutic reagents exist. Vaccination against C. parvum is being attempted in ruminants for the purpose of generating hyperimmune colostrum containing antibodies that may be effective in passive immunotherapy against cryptosporidiosis in the young. A number of recombinant C. parvum surface or internal antigens have been expressed by DNA-cloning technology. Immune colostrum specific for several recombinant C. parvum proteins have demonstrated efficacy in murine and ruminant models against cryptosporidiosis. The P23 and CP15 antigens appear to be the most promising candidates for vaccine development. Recent studies have demonstrated efficacy of the drug nitazoxanide against C. parvum infection in humans. In the near future, control of this parasitic disease in humans and animals will rely on a combination of passive immunotherapy and selective drug treatment.     

Local peroxynitrite formation contributes to early control of Cryptosporidium parvum infection

In intestinal inflammation, mucosal injury is often exacerbated by the reaction of NO with neutrophil-derived superoxide to form the potent oxidant peroxynitrite. Peroxynitrite also has antimicrobial properties that aid in the killing mechanism of macrophages and neutrophils. Cryptosporidium parvum parasitizes intestinal epithelium, resulting in loss of epithelial cells and mucosal inflammation. Synthesis of NO is significantly increased and arises from the induced expression of inducible nitric oxide synthase (iNOS) by the infected epithelium. Inhibition of iNOS results in intensified epithelial parasitism and oocyst excretion. We hypothesized that formation of peroxynitrite is restricted to sites of iNOS expression by the epithelium and contributes to host defense in C. parvum infection. Accordingly, the location and biological effects of peroxynitrite formation were examined in neonatal piglets infected with C. parvum. Infected piglets were treated daily with a selective iNOS inhibitor [L-N6-(1-iminoethyl)-lysine] or one of two peroxynitrite scavengers [5,10,15,20-tetrakis(N-methyl-4'-pyridyl)porphyrinato iron(III) or uric acid] or received vehicle. At peak infection, peroxynitrite formation was restricted to sites of iNOS expression by parasitized epithelium and lamina propria of the apical villi. Peroxynitrite formation was dependent on iNOS activity and was inhibited by treatment with peroxynitrite scavengers. Scavengers increased the number of intracellular parasites and the number of infected epithelial cells present per villus and significantly exacerbated oocyst excretion. Recovery from infection was not delayed by ongoing treatment with scavenger. The present results are the first to demonstrate an in vivo role for peroxynitrite formation in acute mucosal defense against a noninvasive intestinal epithelial pathogen.     

Biological evaluation of a methanol-soluble, heat-stable Escherichia coli enterotoxin in infant mice, pigs, rabbits, and calves.

Escherichia coli P16 was shown to produce two heat-stable toxins (ST) with differing biological activity. The toxins were separated by methanol extraction, and the first, STa, was methanol soluble, partially heat stable, active in neonatal piglets (1 to 3 days old) and infant mice, but inactive in weaned pigs (7 to 9 weeks old); the second, STb, was methanol insoluble, active in weaned pigs and rabbit ligated loops, but inactive in infant mice. It is therefore suggested that use of suckling mice as indicators of ST production will fail to identify certain ST-producing strains.     

Complete development of Cryptosporidium parvum in bovine fallopian tube epithelial cells.

Cryptosporidium parvum is a coccidian parasite responsible for causing protracted and life-threatening diarrheal illness in immunocompromised humans, especially patients with AIDS. The lack of medications effective in treating people suffering from cryptosporidiosis has prompted the development of in vivo and in vitro models for this disease. This study is the first to demonstrate that C. parvum can complete its entire life cycle (from sporozoite to infective oocyst) in a primary culture of bovine fallopian tube epithelial (BFTE) cells. Scanning and transmission electron photomicrographs were used to detail the ultrastructure of individual parasitic stages. Successful infections were produced by inoculating cell cultures with either oocysts or purified sporozoites. Infection of BFTE cells with C. parvum close paralleled in vivo infections with regard to host cell location and chronology of parasite development. Infecting BFTE cells with sporulated oocysts provided a reproducible and quantitative cultivation system with significantly (P < or = 0.001) higher infection rates than in Madin-Darby canine kidney cells. Oocysts produced in BFTE cells were infective for immunosuppressed adult C57BL/6N mice. Cultivation of C. parvum in BFTE cells will facilitate the study of interactions between parasites and host cells as well as provide a reliable system for evaluating anticryptosporidial compound efficacy.     

Resistance of severe combined immunodeficient mice to infection with Cryptosporidium parvum: the importance of intestinal microflora.

Cryptosporidium parvum is a protozoan parasite which colonizes intestinal epithelium, causing transient diarrheal illness in immunocompetent hosts and severe chronic disease in immunocompromised hosts. We examined the resistance of severe combined immunodeficient mice, either bearing intestinal flora or germfree, to intestinal infection with C. parvum. Infection was not readily detected in flora-bearing adult severe combined immunodeficient mice until 5 to 7 weeks following oral challenge with C. parvum. In contrast, germfree adult severe combined immunodeficient mice were heavily infected 3 weeks following challenge. These data support the hypothesis that resistance of adult mice to C. parvum infection does not require a specific immune response but can be mediated by nonspecific mechanisms associated with the presence of intestinal flora.     

The activity of aminoglycoside antibiotics against Trypanosoma brucei

The trypanocidal activity of four aminoglycosides was determined against Trypanosoma brucei in vitro. The drug activity in descending order, was as follows; paromomycin kanamycin>gentamycin > neomycin. Paromomycin bad the highest activity and the concentration that inhibited 50% of trypanosome growth (IC50) was 11.4microM. The effect of paromomycin on the causative agents of the East African form of sleeping sickness - T.b. rhodesiense KETRI 265, 2285, 2545, 2562 and EATRO 110,112, 1152 was subsequently assessed. Variations sensitivities between the trypanosome populations were observed and IC50 values ranging from 13.01 to 43.06 microM recorded. However, when paromomycin was administered intraperitoneally (i.p) at 500 mg/kg, it was not effective in curing mice infected with T. b. rhodesienseKETRI 2545 the most drug-sensitive isolate in vitro. Lack of in vivo activity may be because the trypanosome is an extracellular parasite. The pharmacokinetics of paromomycin in the mouse model need to be determined.     

Inhibition of Cryptosporidium infection in mice treated with a cyclodextrin inclusion complex with diloxanide furoate

The efficacies of diloxanide furoate, beta-cyclodextrin and a cyclodextrin inclusion complex against Cryptosporidium parvum were evaluated in a suckling murine model. Efficacy was established by numbers of oocysts recovered from the intestinal tract of mice on day 7 postinfection. The level of infection in treated mice was significantly lower than in control mice and, surprisingly, the most efficacious treatment was beta-cyclodextrin, an excipient used in pharmaceutical technology.     

Infectivity and neutralization of Cryptosporidium parvum sporozoites.

Cryptosporidiosis, a diarrheal disease of calves and humans caused by the coccidian parasite Cryptosporidium parvum, is terminated in hosts with normal immune systems. To assess the mechanisms of immunity in cryptosporidiosis, it is necessary to isolate and quantitate sporozoites, the infective stage of Cryptosporidium spp. Here we report the (i) separation of infective C. parvum oocysts from calf feces by ether extraction, sieving, and hypochlorite treatment; (ii) separation of viable C. parvum sporozoites from intact and excysted oocysts by anion-exchange chromatography; and (iii) quantitation of sporozoite infectivity in vivo by direct intraintestinal injection of isolated sporozoites in 7-day-old BALB/c mice. When isolated sporozoites were incubated with heat-inactivated immune bovine serum, 25 times the 50% infective dose for 7-day-old mice was completely neutralized. Sporozoites incubated with preimmune bovine serum were infectious for 7-day-old mice.     

Single and combined humoral and cell-mediated immunotherapy of Pneumocystis carinii pneumonia in immunodeficient scid mice.

Homozygous mutant scid/scid (severe combined immunodeficiency) mice (referred to as scid mice) lack both specific humoral and cell-mediated immune functions and are exemplary in vivo models for analysis of host-parasite relationships. In our colony, scid mice routinely and predictably develop spontaneous Pneumocystis carinii pneumonia (PCP) with high morbidity. Previous studies have identified both T cells (specifically, CD4+ cells) and antibody as independent mechanisms of effective anti-P. carinii resistance; however, CD4+ T cells also cause an often fatal hyperinflammatory reaction. The current study has explored the optimal application of these immune components for conferring protection against P. carinii. Anti-P. carinii hyperimmune serum was highly effective at reducing the number of P. carinii organisms in early, intermediate, and advanced stages of PCP and was capable of increasing the mean life expectancy of P. carinii-infected scid mice by more than threefold if provided on a continuing basis. When a short course of hyperimmune-serum therapy was provided prior to transfer of P. carinii-sensitized normal lymphocytes, scid mice were rendered permanently free of P. carinii without the pathological sequelae of the hyperinflammatory reaction. These findings are discussed in the contexts of mechanism and clinical relevance.     

Gamma interferon functions in resistance to Cryptosporidium parvum infection in severe combined immunodeficient mice.

Severe combined immunodeficient (SCID) adult mice are relatively resistant to Cryptosporidium parvum infection, even though they are deficient in both T- and B-cell function. The requirement for gamma interferon (IFN-gamma) in this resistance was examined by treatment of these mice with monoclonal antibody to IFN-gamma. SCID mice injected intraperitoneally with monoclonal anti-IFN-gamma 4 h before and three times weekly after challenge with C. parvum had heavy intestinal infections 3 weeks postchallenge. SCID mice similarly injected with irrelevant antibody were not infected. Furthermore, SCID mice receiving a single injection of anti-IFN-gamma either 2 h before or 18 h after challenge were also susceptible to infection. Although IFN-gamma was not detected in SCID mouse intestinal samples, it was found in the supernatant of SCID mouse splenocyte cultures after stimulation with C. parvum antigens. On the other hand, SCID mice receiving multiple injections of antibodies against tumor necrosis factor remained resistant to infection. These data indicate that the resistance of SCID mice to C. parvum infection is IFN-gamma dependent, whereas tumor necrosis factor appears not to play a significant role.     

A quick procedure for identifying doubly homozygous immunodeficient scid beige mice

This article describes a rapid and reliable procedure for identifying mice which are doubly homozygous at the scid and beige (bg) loci starting from CB17 scid (no T and B cells) and B6 bg mice (no NK activity). The [scid, bg] mice are directly identified in the F2 progeny by monitoring (1) the hypogammaglobulinemia for the scid gene and (2) the prolonged bleeding associated with the bg gene. Like CB17 scid mice, the [scid, bg] mice show a high susceptibility to infections and die early in life unless they are protected against potential infections. This is achieved by a graft of splenocytes plus bone marrow cells from (B6 bg x CB17 scid) F1 mice. These [scid, bg] mice combine the bg and scid immunodeficiencies and should be better recipients for xenografts than classical scid mice.     

Protection of piglets against Edema disease by maternal immunization with Stx2e toxoid

Edema disease (ED) in piglets is caused by Shiga toxin Stx2e-producing Escherichia coli. We show that a genetically disarmed Stx2e toxoid is a safe antigen that generates antiserum protecting piglets against the Stx2e toxin. Immunization of suckling piglets with the Stx2e toxoid was safe, had no adverse effects on growth of the piglets, and resulted in effective prevention of edema disease clinical symptoms after challenge with the Stx2e toxin. Our data showed that maternal immunity against the Stx2e toxoid can be transmitted from the vaccinated sows to the piglets via the colostrum. Very high levels of Stx2e-specific serum antibodies persisted in these piglets until 1 month postweaning, bridging the critical period in which the weaned piglets are most susceptible to edema infection. Challenge with Stx2e toxin resulted in clinical signs of edema disease and death of all control piglets from nonimmunized sows, whereas none of the piglets from immunized sows developed clinical signs of ED.     

Comparison of oocyst shedding and the serum immune response to Cryptosporidium parvum in cattle and pigs

 A comparison was made between oocyst shedding and the presence of specific serum IgG antibodies to Cryptosporidium parvum in 108 bovines and 90 pigs. Oocysts were detected by a commercial immunofluorescence assay in feces from 26.8% of bovines and 34.4% of pigs, whereas positive titers as determined by an indirect fluorescent antibody method were found in sera from 12.9% and 48.9% of the respective animals. Infection was significantly most frequent in suckling calves (82.7%) and weaned piglets (87.5%). By contrast, the numbers of seropositives were highest in weaned calves (17.1%) and fattening pigs (76.6%). The results of coprological and serological analysis corresponded in 65.7% of bovines and 56.7% of pigs. When used to diagnose the shedding of cryptosporidial oocysts, the detection of specific IgG antibodies had a sensitivity ranging from 10.3% (cattle) to 58.1% (pigs) and a specificity of 86.1% (cattle) and 55.9% (pigs). Received: 07 December 1995 / Accepted: 16 January 1996     

Anti-Cryptosporidium parvum antibodies inhibit infectivity in vitro and in vivo.

Cryptosporidium parvum causes acute diarrhea in immunocompetent individuals and a severe life-threatening disease in immunocompromised individuals, including AIDs patients. No efficacious therapy for cryptosporidiosis has yet been reported. However, treatment of some patients with cryptosporidiosis with hyperimmune bovine colostrum has ameliorated or eliminated clinical symptoms. Consequently, it is important to identify and characterize C. parvum antigens which are the targets of protective antibodies to facilitate the development of more efficacious therapy. We report that hyperimmune bovine colostral immunoglobulin inhibits C. parvum infectivity in a reproducible in vitro assay, and we correlate this inhibition with the protective capacity of the bovine colostrum in vivo. We have also identified the major C. parvum sporozoite antigens recognized on Western blots (immunoblots) by this colostral immunoglobulin preparation. Antibodies that recognize some surface molecules of other Apicomplexan parasites are protective in vivo. Consequently, we radioiodinated membrane proteins of sporozoites and immunoprecipitated 19 molecules which are the target of immunoglobulin that is protective in vivo and in vitro.