Experimental tracheal and conjunctival infections with Cryptosporidium sp. in pigs.

Tracheal and conjunctival infections with Cryptosporidium were established in pigs by inoculation of oocysts into the trachea and onto the conjunctival sacs. The protozoa were found attached to epithelial cells by an electron-dense band and a folded, vacuolated feeder organelle. They were situated in a parasitophorous vacuole surrounded by a double-layered membrane covered by glycocalyx. Trophozoites, schizonts, merozoites, macrogametes and oocysts could be demonstrated. In addition to normal stages of the life-cycle, degenerate stages were found. The infections with Cryptosporidium were characterized by focal destruction and loss of epithelial cells. Numerous intraepithelial lymphocytes were associated with affected foci, as were infiltrations with lymphocytes, monocytes, and macrophages.

Subclinical cryptosporidiosis of turkeys in Iowa.

Cryptosporidium oocysts were found in the feces of 16 of 20 (80%) 17-day-old and 38 of 100 (38%) 24-day-old turkeys in a healthy commercial flock in central Iowa. Mean dimensions of 50 oocysts were 4.5 x 5.3 micron. In experimentally inoculated turkeys and chickens, the parasites frequently infected the ceca, colon, and cloaca, but rarely infected the small intestine or bursa of Fabricius. These data differ from published descriptions of Cryptosporidium baileyi in chickens and turkeys. The results indicate that the cryptosporidia of poultry are not a homogeneous group and demonstrate that Cryptosporidium infections can occur in apparently healthy poultry flocks.

Experimental cryptosporidiosis in fetal lambs.

Fetal lambs were infected in utero with purified sporulated oocysts of Cryptosporidium parvum in order to study pathogenesis and host cellular response to the enteropathogen. Ileal loops (IL) of fetuses, 124-130 days of gestation, were inoculated with 1-4 x 10(6) oocysts usually via cannulae in the abdominal wall of the ewe. Oocysts, both free and phagocytosed, were observed in the IL content as early as day 1 post-inoculation (PI). The percentage of oocysts phagocytosed by the host's polymorphonuclear neutrophils (PMN's) and mononuclear cells remained high up to day 13, the last day of examination. Numerous parasites were observed at days 6, 7, and 12 PI in the microvilli of the ileum with hypercellularity of the lamina propria, which consisted of a mixed infiltration of PMN's, mononuclear cells, including lymphoid cells, and a few eosinophils. Cytolysis and extrusion of epithelial cells, often heavily parasitized by various stages of the parasite, as well as inflammatory cells, were prominent in luminal contents. Germinal centers were prominent in mesenteric lymph nodes draining the infected loops by day 12 PI. Depletion of lymphoid cells was already present in Peyer's patches by day 4 PI.

Effects of colostral antibody on susceptibility of calves to Cryptosporidium parvum infection.

Effects of colostral antibody on susceptibility of calves to Cryptosporidium parvum infection were examined. Six calves were fed pooled colostrum that contained C parvum antibody, 6 times daily (at 4-hour intervals) for 7 days and then milk replacer for 7 days. Colostrum was obtained from healthy cows or cows inoculated parenterally with C parvum oocysts before parturition. Antibody content was determined in serum and colostrum whey, using an ELISA for anticryptosporidia immunoglobulin. Six calves were fed colostrum from healthy cows 1 time, and then milk replacer 6 times daily for 14 days. On day 1, all calves were challenge exposed with C parvum, PO, and were monitored daily for diarrhea and oocyst shedding. Bovine colostrum containing specific antibody to C parvum, at ELISA titers up to 10,240, was not effective in protecting calves against challenge exposure to C parvum.

Synthetic retinal analogues modify the spectral and kinetic characteristics of microbial rhodopsin optogenetic tools.

Optogenetic tools have become indispensable in neuroscience to stimulate or inhibit excitable cells by light. Channelrhodopsin-2 (ChR2) variants have been established by mutating the opsin backbone or by mining related algal genomes. As an alternative strategy, we surveyed synthetic retinal analogues combined with microbial rhodopsins for functional and spectral properties, capitalizing on assays in C. elegans, HEK cells and larval Drosophila. Compared with all-trans retinal (ATR), Dimethylamino-retinal (DMAR) shifts the action spectra maxima of ChR2 variants H134R and H134R/T159C from 480 to 520 nm. Moreover, DMAR decelerates the photocycle of ChR2(H134R) and (H134R/T159C), thereby reducing the light intensity required for persistent channel activation. In hyperpolarizing archaerhodopsin-3 and Mac, naphthyl-retinal and thiophene-retinal support activity alike ATR, yet at altered peak wavelengths. Our experiments enable applications of retinal analogues in colour tuning and altering photocycle characteristics of optogenetic tools, thereby increasing the operational light sensitivity of existing cell lines or transgenic animals.

Studies of in vitro excystation of Cryptosporidium parvum from calves.

Studies of in vitro excystation of Cryptosporidium parvum from calves showed that sporozoite yields were optimum when oocysts were treated with sodium hypochlorite, then incubated at 37 degrees C for 60 min in the presence of taurocholic acid solutions at pH about 7.0. Trypsin was not required for excystation and high concentrations were inhibitory. Studies using protease inhibitors and direct assays for proteolysis failed to implicate proteolytic enzymes as effectors of excystation. The results suggest that Cryptosporidium uses excystation mechanisms that are different from those used by Eimeria spp.

IgG subtype abnormalities with normal total IgG in a clinical allergy practice.

Thirty-one of 75 patients with recurrent upper respiratory infections were found to have immunoglobulin G subclass deficiencies with normal levels of total immunoglobulin G in a clinical allergy and asthma practice. Sixteen were IgG3 deficient, thirteen IgG2 deficient, and two were IgG1 deficient. Only one patient had an IgA deficiency. Two patients have normal IgG with decreased PRP titers. Serum antibody titers to the capsular polysaccharide of Haemophilus influenzae type B (HibCP) were found to be low in seven patients. Other investigators have established relationships between these deficiencies and recurrent infections. When investigating patients with recurrent infections, it seems prudent to look beyond simple quantitative immunoglobulins.

A pilot study examining the role of zileuton in atopic dermatitis.

BACKGROUND: Atopic dermatitis is a common chronic inflammatory skin condition. Current therapies are often insufficient to control the degree of inflammation and pruritus seen in this disease. OBJECTIVE: To examine the efficacy of zileuton in treating atopic dermatitis by measuring both subjective and objective outcome parameters. METHODS: This was an open-label pilot study using zileuton to treat atopic dermatitis. Following a 1-week run-in period off all corticosteroids and antihistamines, six adult patients received 6 weeks of zileuton 600 mg po QID. Subjects were not allowed to use their usual atopic dermatitis medications during the study. At baseline, and at 2, 4, and 6 weeks into the zileuton course, patients were asked to report on their disease dissatisfaction and their pruritus. Objective skin scoring was also performed by an examiner at these visits. RESULTS: Disease dissatisfaction score decreased from a mean of 8 (out of a possible 10) to 4.4 over the 6-week period (P = .03). Pruritus scores showed a trend toward improvement during the study, decreasing from a mean of 7.3 to 4.3 (out of a possible 10, P = .06). Objective skin erythema scores decreased from a baseline mean of 24 (out of a possible 60) to 14 following zileuton treatment (P = .03). CONCLUSIONS: In this pilot study, zileuton showed efficacy in significantly improving the symptoms and objective skin findings seen in atopic dermatitis. The encouraging results seen in this trial should encourage larger, placebo-controlled studies looking at this novel approach toward treating this condition.

Characterization of cyclophosphamide-rat model of cryptosporidiosis.

A cyclophosphamide-treated-rat model for cryptosporidiosis is described. The optimal conditions for inducing a cryptosporidial infection in rats were a 50 mg/kg dose of cyclophosphamide per day in the drinking water for a minimum of 14 days before inoculation with 10(4) or more infectious oocysts. Eighteen days after inoculation, 80% or more of the animals were infected. Cryptosporidia were attached to the terminal 24 cm of the small intestine, and the immunosuppressed animals remained infected as long as they received cyclophosphamide. However, the intestinal infection cleared within 7 to 10 days after cyclophosphamide was withdrawn. Oocysts continued to be detected in the feces for an additional 5 to 7 days after cryptosporidia were no longer visibly attached to the ideal enterocytes. This study characterized an animal model that can be used to investigate the pathogenesis of and evaluate therapeutic regimens for cryptosporidiosis in the immunocompromised host.

Experimental intrauterine infection of adult BALB/c mice with Cryptosporidium sp.

Inoculation of adult, female BALB/c mice with 2 X 10(5) bleach-treated Cryptosporidium sp. oocysts isolated from calf feces resulted in infection of the uterine mucosa in more than 50% of the animals. Cryptosporidium sp. completed the entire life cycle in the uterus, and infectious oocysts were passed into the vagina. Two methods of application were used to establish intrauterine infection. The inoculum was either injected into the uterus after abdominal surgery or intracervically instilled. Mice were susceptible at all phases of the sexual cycle, but the highest infection rates were obtained during estrus and diestrus. Parasites were demonstrated as early as 5 days postinfection. Phagocytic cells in the uterine lumen and in the vagina contained Cryptosporidium sp. Phagocytosis may be an important immune response and a mechanism of parasitic clearance. These results suggest that Cryptosporidium sp. is a potential pathogen of the reproductive tract.

Resistance of calves to Cryptosporidium parvum: effects of age and previous exposure.

Cryptosporidium parvum is a coccidian parasite that causes diarrheal disease in many vertebrate species, including young (less than or equal to 1 month old) calves. Older calves and adult cattle are resistant to infection. In this study, newborn calves were raised in isolation from C. parvum for 1 week to 3 months before experimental challenge with the parasite. Calves orally challenged with C. parvum at 1 week of age shed oocysts in their feces and had diarrhea after challenge exposure. When these calves were rechallenged at 1 and 3 months of age, they neither shed oocysts nor had diarrhea. There was no significant increase in the mean anticryptosporidium enzyme-linked immunosorbent assay serum antibody titer in these calves following any of the challenge exposures. Calves orally inoculated with C. parvum for the first time at 1 month of age shed oocysts, had diarrhea after challenge exposure, and were resistant to rechallenge at 3 months of age. These calves had a twofold increase in serum antibody titer after the first challenge and no increase after the second challenge. Calves orally inoculated with C. parvum for the first time at 3 months of age shed oocysts, and two of seven animals had diarrhea. These calves had a 10-fold increase in serum antibody to C. parvum after exposure. This study demonstrates that calves raised in isolation from C. parvum remain susceptible to challenge until at least 3 months of age. Furthermore, within this time period, initial exposure and recovery renders calves resistant to further challenge with the parasite. The data also suggest that exposure of young calves to C. parvum may inhibit the development of a serum antibody response to the parasite.

Persistent Cryptosporidium infection in congenitally athymic (nude) mice.

Nude (nu/nu) BALB/c mice and their white (nu/+) littermates were experimentally infected with Cryptosporidium sp. at 6 days of age. In white mice, the infection was transient, but in nude mice a persistent infection developed that was characterized by diarrhea and, occasionally, death. There were villus atrophy and crypt hyperplasia in the small intestine of infected nude and white mice necropsied at 11 days of age. Persistently infected nude mice had, in addition to the above small intestinal lesions, diffuse cystic mucosal hyperplasia and crypt abscesses in the large intestine at 56 days of age. These results suggest that T cells are required for recovery from the Cryptosporidium infection but are not required for epithelial cell loss in cryptosporidiosis. Both nude and white mice appeared to be relatively more resistant to Cryptosporidium infection at 42 days of age than at 6 days of age.

Factors affecting motility and morphology of Cryptosporidium sporozoites in vitro.

In vitro motility and morphology of Cryptosporidium sporozoites were examined in the presence of various solutions. Crude preparations of the bile salt, taurocholic acid, maintained both motility and morphology in a dose-dependent manner. These effects appeared to be due to the taurocholic acid itself, and not simply due to pH variations, osmotic factors, or contaminants. Lysis of sporozoites was also observed and was found to be dependent on pH, with acidic conditions (pH less than 6.2) triggering the lysis.

Susceptibility of germfree or antibiotic-treated adult mice to Cryptosporidium parvum.

Adult mice are more resistant than neonatal mice to intestinal colonization with the protozoan parasite Cryptosporidium parvum. Development of a mature intestinal flora may play a role in this resistance. We compared susceptibilities to colonization with C. parvum in adult conventional mice, adult germfree mice, and adult conventional mice treated with oral antibiotics to deplete the intestinal flora. Germfree mice of both CD1 and BALB/c strains were colonized at day 7 following inoculation with C. parvum oocysts isolated from the feces of an infected, diarrheic calf. Age-matched conventional mice of the same strains were comparatively resistant to colonization. Conventional mice treated with antibiotics remained resistant to colonization. These results suggest that the microflora in the intestine was not the sole determinant of resistance or susceptibility to colonization. The germfree adult mouse as an experimental model of cryptosporidiosis is discussed.

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.