Effect of passive antibodies derived from rotavirus-like particles on neonatal calf diarrhea caused by rotavirus in an oral challenge model.

Our objective was to evaluate the efficacy of bovine rotavirus antigen-specific passive antibody for reducing the duration of diarrhea induced by oral challenge with bovine rotavirus in a neonatal calf model. The bovine rotavirus-specific passive antibodies were produced before the study by hyperimmunization of pregnant cows during the dry period with an adjuvanted vaccine containing recombinantly-expressed rotavirus virus-like particles. Eighty-three calves were cleanly collected at birth and randomly assigned to 1 of 3 groups as follows: (1) control group that was colostrum deprived and fed milk replacer for first feeding, (2) group that was colostrum deprived and fed milk replacer mixed with antirotavirus antibodies for first feeding, or (3) group that was fed colostrum replacer mixed with antirotavirus antibodies and a product approved by the US Department of Agriculture containing antibodies against Escherichia coli K99 and bovine coronavirus for first feeding. One of the 3 treatments was administered within 6 h of birth to each calf, followed by oral challenge with bovine rotavirus 3 h later. Calves were observed through 7 d of age and scored according to a standardized scale for clinical signs of diarrhea, change in appetite, depression, and dehydration. Twice daily, measurements of rectal temperature and collection of feces were performed. Fecal samples were assessed for infectious agents commonly associated with diarrhea, and bovine rotavirus shedding was quantified. There were 24 of 28 (86%) calves in the control group that received no antibodies that had signs of severe diarrhea, whereas 57% of the calves that received antirotavirus in milk replacer experienced severe diarrhea, and 7% of calves that received colostrum replacer mixed with antigen-specific bovine rotavirus antibodies showed signs of severe diarrhea. Calves that received colostrum replacer mixed with antigen-specific bovine rotavirus antibodies had a mean duration of 0.9 d of diarrhea compared with 2.7 d in the control group. Calves in the group that was colostrum deprived and fed milk replacer with antirotavirus antibodies had a mean duration of diarrhea of 1.7 d. Rotavirus peak fecal shedding was 3.5 d in the group with milk replacer only, 5.5 d in the milk replacer with antibody group, and 6.5 d in calves in the colostrum replacer group. When bovine rotavirus antigen-specific antibody was fed in milk replacer to colostrum-deprived calves or in conjunction with colostrum replacer that also contained supplemental antibodies against Escherichia coli K99 and bovine coronavirus, those calves were observed to have reduced the onset, duration, and severity of diarrhea when compared with milk replacer placebo.

Immunodeterminant specificity of human immunity to type III group B streptococcus.

The type III polysaccharides of group B Streptococcus in its native state chemically consists of glucose, galactose, glucosamine, and sialic acid. The core of this polysaccharide lacks sialic acid and precipitates with type III antiserum to give a partial identity with the precipitate between the native antigen and this serum. The core determinant is immunochemically similar to the capsular polysaccharide of type XIV Streptococcus pneumoniae, while the native type III group B streptococcal polysaccharide does not cross-react with type XIV pneumococcal antiserum. In human sera, it is antibody directed to the native antigen which correlates very highly with opsonic immunity (r = 0.94) while a poorer correlation exists between antibody to the core antigen and opsonins (r = 0.51 P less than 0.001). In natural infections, as association exists between low levels of maternal antibody to the native antigen and risk of disease in the infant. This association is not true for antibody to the core structure, where both infected infants and their mothers have much higher levels of antibody to the core than the native antigens. Infected infants are also more likely to respond to infection by developing antibody to the native antigen. Immunization of 12 adults with multivalent pneumococcal polysaccharide induced significantly better antibody response to the core antigen than to the native, and this vaccine induced opsonic activity in only one recipient. Immunization of adults with type III group B streptococcal antigens induced antibody to the native determinant which correlated with opsonic activity. Therefore, it would appear that native group B streptococcal polysaccharides will provide the best candidate antigens for immunization.

In vitro reconstitution of exocytosis from plasma membrane and isolated secretory vesicles.

We describe the reconstitution of exocytotic function through recombination of purified cortical secretory vesicles (CVs) and plasma membrane from sea urchin eggs. CVs were dislodged from a cell surface complex preparation by gentle homogenization in an isotonic dissociation buffer, and purified by differential centrifugation. CV-free plasma membrane fragments were obtained by mechanically dislodging CVs from cortical lawn (CL) preparations with a jet of CL isolation buffer. This procedure produced a "plasma membrane lawn" preparation, consisting of plasma membrane fragments attached via their vitelline layer (an extracellular glycocalyx) to a polylysine-coated microscope slide. When freshly prepared CVs were incubated with plasma membrane lawns, CVs reassociated with the cytoplasmic face of the plasma membrane, forming an exocytotically competent, reconstituted cortical lawn (RL). Exocytosis in RLs was monitored by phase-contrast microscopy, and quantitated with a sensitive microphotometric assay. Half-maximal exocytosis in RLs occurred at 18.5 microM free Ca2+; half-maximal exocytosis in control lawns occurred at 5.7 microM free Ca2+. Greater than 90% of the purified CVs that were not attached to a plasma membrane lawn remained intact when bathed in a buffer containing millimolar Ca2+. This result excluded the possibility that Ca2+-triggered CV lysis was responsible for our observations, and confirmed that the association of CVs with the plasma membrane was required for exocytosis in RLs. Evidence that the Ca2+-stimulated release of CV contents in CLs and RLs is the in vitro equivalent of exocytosis was obtained with an immunofluorescence-based vectorial transport assay, using an antiserum directed against a CV content protein: stimulation of RLs or partially CV-depleted CLs with Ca2+ resulted in fusion of the CV and plasma membranes, and the vectorial transport of CV contents from the cytoplasmic to the extracytoplasmic face of the egg plasma membrane.

Endoglycosidase f cleaves the oligosaccharides from the glucose transporter of the human erythrocyte.

The glucose transporter from human erythrocytes is a heterogeneously glycosylated protein that runs as a very broad band of average apparent Mr 55 000 upon sodium dodecyl sulfate polyacrylamide gel electrophoresis. When the purified preparation of transporter, solubilized in Triton X-100, was treated with endoglycosidase F, much of it ran as a sharp band of Mr 46 000 upon electrophoresis. Moreover, endoglycosidase F released 80% of the radioactivity in a preparation of the transporter labeled in its oligosaccharides with galactose oxidase and tritiated borohydride, and almost none of the remaining radioactivity was located in the Mr 46 000 band. These results suggest that endoglycosidase F can release virtually all of the carbohydrate linked to the transporter polypeptide. A quantitative analysis of the gels was complicated by partial aggregation of polypeptides that occurs due to prolonged incubation in Triton X-100, but at least 65% of the protein in the preparation of purified transporter is the 46 kDa polypeptide. The extracellular domain of the transporter is very resistant to proteolysis; no cleavage occurred upon treatment of intact erythrocytes with seven different proteases at high concentration.

Antibody-based immunotherapy of cryptosporidiosis.

Passive antibody immunotherapy (PAI) for cryptosporidiosis is a treatment strategy that has been actively pursued in laboratory studies and early-stage clinical studies for the last decade. Several experimental approaches have been initiated, including use of bovine colostrum and colostral antibodies (hyperimmune and natural), monoclonal antibodies, chicken egg yolk antibodies, and even orally administered human plasma antibodies. Most studies have employed oral administration to treat or prevent this intestinal infection. The interest in this treatment strategy has been sparked by the lack of an effective or approved therapy, increased awareness of the widespread nature of this parasite, epidemiological evidence that humoral immunity plays an important role in host resistance, and several early case reports of antibody therapy in which remarkable resolution of the disease was observed. Most studies using a variety of preparations of antibodies administered to animals and humans have shown some degree of efficacy, though the responses have been, for the most part, partial rather than complete resolution of the disease. This chapter examines critically the scientific rationale and the evidence for PAI for cryptosporidiosis, including practical considerations and future approaches.

Water-borne cryptosporidiosis: detection methods and treatment options.

Since the infamous outbreak in Milwaukee, WI, USA, of water-borne cryptosporidiosis affecting over 400,000 people, there have been at least 20 smaller outbreaks associated with this parasite in the UK and North America. These events have led to an explosion of interest in and research on the nature of cryptosporidiosis as a dangerous water-borne pathogen, particularly patients with acquired immune deficiency syndrome (AIDS). In addition, several major environmental laws and proposed regulations specifically address the control of this parasite. The possible ramifications of these laws include billions of dollars of modifications to water-treatment facilities in the USA. Unfortunately, the methods used to gather the information on which these laws are based have serious deficiencies that could lead to gross underestimation of the magnitude of this problem. The present review considers gaps in our understanding of water-borne cryptosporidiosis, new methods under investigation that could improve our ability to monitor water for the presence of this organism, and treatment and control strategies to limit the threat to our water supplies.

In vitro reconstitution of exocytosis from sea urchin egg plasma membrane and isolated cortical vesicles.

We have succeeded in reconstituting an exocytotically active egg cortex fraction by recombining purified cortical vesicles (CVs) with egg plasma membrane (PM). CVs were dislodged from a suspension of egg cortex by gentle homogenization in a dissociative buffer with a pH of 9.1, and purified by two rounds of differential centrifugation. Egg PM was prepared by shearing the cortical vesicles from a cortical lawn preparation with a jet of isotonic buffer. PM lawns produced by this procedure consist of an array of CV-free PM fragments attached via their extracellular surface to a polylysine coated glass slide. When a neutralized suspension of CVs was recombined with a PM lawn, CVs reassociated with the cytoplasmic face of the plasma membrane to form a reconstituted lawn (RL). RLs undergo a morphological change in response to Ca2+-containing buffers that is similar to the exocytotic release of CV contents from cortical lawns. In both reactions CV contents are vectorially transferred from the cytoplasmic to the extracytoplasmic face of the egg PM. A quantitative binding assay was developed and used to show that adherence of CVs to a heterologous PM lawn prepared from human red blood cells is minimal.

Polycation inhibition of exocytosis from sea urchin egg cortex.

The Ca2+-stimulated release of vesicle contents from cortical fragments prepared from sea urchin eggs is an in vitro model for exocytosis. Cortical fragments have been isolated either in suspension (cell surface complex, CSC preparation), or attached to polycation-coated surfaces (cortical lawn, CL preparation). CL, but not CSC, have been reported to undergo a rapid "aging" process whereby they fail to respond to micromolar free Ca2+. Since, in principle, the only difference between the two preparations is the use of polycations in the CL preparation, polycations were suspected of being inhibitory. This hypothesis was tested by evaluating the effects of polycation-containing buffers on the Ca2+ threshold, rate, and extent of exocytosis in CL prepared from the eggs of Strongylocentrotus purpuratus. A sensitive microphotometric assay, based on light scattering by the individual cortical vesicles in the CL, was used to quantitate the exocytotic response. Buffers containing polylysine were found to be potent inhibitors of cortical exocytosis. The Ca2+ threshold of CL that had been treated for 15 min at room temperature with 50 micrograms/ml of polylysine was more than three orders of magnitude greater than that of freshly prepared CL. The other polycations tested (protamine, spermine and neomycin) were also found to be inhibitory, but to a lesser degree than polylysine. Two lines of evidence suggested that the polycations used in the preparation of CL are responsible for the rapid "aging" phenomenon: CSC fragments that had been affixed to polylysine-coated coverslips were shown to acquire "aging" characteristics similar to the CL preparations; control CSC that had been maintained in suspension did not. Radiolabeled poly-L-lysine was shown to dissociate from coated coverslips and redistribute onto CL.

Purification of a major membrane protein of Toxoplasma gondii by immunoabsorption with a monoclonal antibody.

The principal iodinatable surface protein (P30) of our cloned RH strain of Toxoplasma gondii has an apparent molecular weight of 30,000, as measured by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate under reducing conditions. Monoclonal antibody B specifically immunoprecipitated protein P30 from a detergent extract of surface radioiodinated T. gondii. Monoclonal antibody B in the presence of complement was also parasiticidal for T. gondii, and this parasiticidal effect could be blocked by protein P30. Monoclonal antibody B was purified from mouse ascitic fluid and linked to cyanogen bromide-activated Sepharose. The resulting immunoabsorbent was used to purify 1.7 mg of protein P30 from a large number of parasites. The efficiency of recovery of protein P30 was measured by assays of radioactivity and of parasiticidal blocking activity. Protein P30 represented 3 to 5% of the total protein. It is also present in a recently isolated strain of T. gondii. A convalescent human antitoxoplasma serum immunoprecipitated radiolabeled protein P30. Three convalescent antisera when quantitated by an ELISA test had a high anti-protein P30 titer. Charge shift electrophoresis showed that protein P30 has an extensive hydrophobic region and thus is probably an integral membrane protein. Electrophoresis under nonreducing conditions showed no evidence that protein P30 exists as a disulfide linked homo- or heterodimer, although it probably has intramolecular disulfide bonds.

Isolation and characterization of a monoclonal antibody-resistant antigenic mutant of Toxoplasma gondii.

Several monoclonal antibodies against Toxoplasma gondii were parasiticidal in the presence of normal human serum as measured by reduction in plaque titer or in assays that detected lysis. One monoclonal antibody, G, was used to select a resistant mutant from a large population of chemically mutagenized wild-type parasites. This mutant retained the wild-type sensitivity to other monoclonal antibodies and to polyclonal antisera. A comparison of surface radioiodinated wild-type and mutant parasites by using one and two-dimensional electrophoresis showed that the mutant lacked a protein (or proteins) of approximate m.w. 22,000. An immunoprecipitation procedure using monoclonal antibody G yielded a protein(s) of this m.w. from surface radioiodinated wild-type T. gondii but not from surface radioiodinated mutant parasites.

Animal model system for studying virulence of and host response to Bacteroides fragilis.

Experimental animal model systems have been used by many investigators to explore the pathogenicity of obligate anaerobes. During the last 15 years, research in our laboratory has utilized an experimental model for intraabdominal sepsis to define the contribution of obligate anaerobes to the infectious process. These studies have shown that obligate anaerobes are important components of the polymicrobic flora present during such infection. Moreover, certain anaerobes, such as Bacteroides fragilis, possess specific virulence factors, such as the capsular polysaccharide, that appear to be important to the infectious process. More recent research has used modifications of the original model system to evaluate the host immune response to B. fragilis. These studies indicate that immunization with the capsular polysaccharide provides a T cell-dependent immunity to abscess development when animals are challenged with B. fragilis. It has also been shown that the killing of B. fragilis is T cell dependent. The observations made with regard to B. fragilis in this animal model system are discussed.

T cell regulation of Bacteroides fragilis-induced intraabdominal abscesses.

Intraabdominal abscesses (IAA) caused by Bacteroides fragilis are a major sequela to colonic spillage into the peritoneum. The development of an animal model that closely reproduces the disease observed in humans permitted careful inspection of the cellular and/or humoral contributions to the development and control of this disease. The results obtained thus far describe an immunoregulatory T cell circuit that governs both the development of and the immunity against these abscesses. T cells of CD4+8+ phenotype induce the development of IAA in response to B. fragilis. CD8+ T cells generated in response to immunization with the B. fragilis capsular polysaccharide confer protection against the development of IAA. These cells elaborate an antigen-specific factor that mediates the observed protection by these cells. Moreover, a third type of T cell, a CD8+ cell that is also present in nonimmune individuals, is required for the immune T cell or its factor to confer protection. Thus, in the specific disease process of IAA induced by the encapsulated microorganism B. fragilis, immunity proceeds by cellular and not humoral mechanisms.

Characterization of a > 900,000-M(r) Cryptosporidium parvum sporozoite glycoprotein recognized by protective hyperimmune bovine colostral immunoglobulin.

Cryptosporidium parvum, a zoonotic Apicomplexan pathogen, causes profound diarrhea, malnutrition, and dehydration in patients with AIDS. A less severe, self-limited disease occurs in immunocompetent individuals, particularly children, animal handlers, and residents of the developing world. Very little is known about the biology of the organism, the pathophysiology of the disease process, or the mechanism of protective immunity. There is no effective therapy for cryptosporidiosis, but hyperimmune bovine colostrum raised against Cryptosporidium oocysts and sporozoites has ameliorated infection and disease in some patients with AIDS, and a variety of monoclonal antibodies, as well as hyperimmune bovine colostrum, have significantly reduced cryptosporidial infection of mice and calves. We report here the identification and initial characterization of a > 900,000-M(r) Cryptosporodium sporozoite glycoprotein (GP900) that is a prominent antigen recognized by protective hyperimmune bovine colostral immunoglobulin. Three of six murine anticryptosporidial monoclonal antibodies reacted with GP900, indicating that the molecule is highly immunogenic in mice as well as in cows. GP900 is Triton X-100 soluble and N glycosylated. Western blotting of the N-deglycosylated protein, detected with antibodies eluted from recombinant clones expressing a partial GP900 fusion protein, suggested that the polypeptide backbone of the glycoprotein has an M(r) of < 190,000. GP900 is encoded by a single-copy gene that resides on the largest Cryptosporidium chromosome.

Intraperitoneal host cellular responses and in vivo killing of Bacteroides fragilis in a bacterial containment chamber.

A bacterial containment chamber was used to evaluate the peritoneal cellular response to Bacteroides fragilis during intraperitoneal challenge. This containment system was also used to determine the fate of bacteria within the peritoneal cavities of animals immunized, either actively or through adoptive transfer of cells or cell lysates, with the capsular polysaccharide of B. fragilis. This system demonstrated that the dominant cell types in the peritoneal cavities within 48 h of implantation of the containment chambers containing B. fragilis were neutrophils and macrophages. However, the early cellular response in immunized animals included an increase in the lymphocyte population within 4 h of challenge which was not detected in naive animals. In immunized animals, a later dramatic increase in the lymphocyte population at approximately 4 to 6 days following implantation of the containment chambers occurred. This increase in the lymphocyte population in immunized animals coincided with a decline in the viable bacterial counts within the chambers from 10(8) to 10(9) CFU/ml to less than 10(2) CFU/ml. A similar decline was not seen in naive animals challenged in the same manner. Killing of B. fragilis within containment chambers occurred when spleen cells, T cells, or lysates of T cells from actively immunized animals were passively transferred to naive recipient animals. It was shown that the factor responsible for bacterial killing was not antibody mediated, since bacteria contained within dialysis sacs with an exclusion of 50 kilodaltons were still killed in this model. Moreover, removal of T cells from adoptively transferred cell populations before transfer abrogated the decline in viable bacterial populations. The postulated mechanisms by which this bacterial killing occurred are discussed.

Milk immunoglobulin with specific activity against purified colonization factor antigens can protect against oral challenge with enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is the most commonly isolated pathogen responsible for travelers' diarrhea and the cause of up to 650 million cases of pediatric diarrhea per year in the developing world. As a safe alternative to the prophylactic use of antibiotics, a hyperimmune bovine milk antibody product with specific activity against purified colonization factor antigens (CFAs) was developed and evaluated in a human challenge study. Twenty-five healthy adult volunteers were challenged orally with 10(9) cfu of a virulent CFA/I-bearing ETEC. In the randomized double-blind trial, 7 of 10 volunteers receiving a lactose-free placebo developed clinical diarrhea after challenge, compared with only 1 of 15 cases in volunteers receiving active product (Fisher's exact test, P < .0017). It is concluded that antibodies against CFAs alone are sufficient for protection and that prophylaxis with milk-derived immunoglobulin is a feasible alternative to existing drug interventions.

Virulence of three distinct Cryptosporidium parvum isolates for healthy adults.

The infectivity of three Cryptosporidium parvum isolates (Iowa [calf], UCP [calf], and TAMU [horse]) of the C genotype was investigated in healthy adults. After exposure, volunteers recorded the number and form of stools passed and symptoms experienced. Oocyst excretion was assessed by immunofluorescence. The ID50 differed among isolates: Iowa, 87 (SE, 19; 95% confidence interval [CI], 48.67-126); UCP, 1042 (SE, 1000; 95% CI, 0-3004); and TAMU, 9 oocysts (SE, 2.34; 95% CI, 4.46-13.65); TAMU versus Iowa, P=.002 or UCP, P=.019. Isolates also differed significantly (P=.045) in attack rate between TAMU (86%) and Iowa (52%) or UCP (59%). A trend toward a longer duration of diarrhea was seen for the TAMU (94.5 h) versus UCP (81.6 h) and Iowa (64.2 h) isolates. C. parvum isolates of the C genotype differ in their infectivity for humans.