Breast-milk IgA and lactoferrin survival in the gastrointestinal tract--a study in rural Gambian children.

The survival of breast-milk secretory-IgA and lactoferrin has been investigated in 23 Gambian children aged 1.5, 3 and 17 months. Endogenous excretion of these immune proteins was measured in 7 weaned 34-month-old children. Defaecation rate was the prime determinant of faecal secretory-IgA and lactoferrin outputs, indicating that partial degradation occurs in the large intestine. Calculations showed that at least 30% of IgA and 2% of lactoferrin ingested from breast-milk must survive in the small intestine. Variations in faecal immune protein outputs were related to differences in intake and defaecation rate and were not affected by age or solid food consumption. The raised faecal outputs of 5 children with diarrhoea were a consequence of their high stool frequencies. IgA disappearance in the large intestine proceeded twice as fast in Gambian breast-fed children as in comparable Cambridge infants, suggesting that differences in gut flora may influence IgA survival. Thus breast-feeding, irrespective of age or additional food, can deliver significant quantities of these antimicrobial proteins to the small intestine but differences in defaecation rate and gut flora may affect their protective potential in the large intestine.

Natural transmission of group A rotavirus within a pig population.

Five litters of piglets born within two days of each other, together with their dams, were investigated for faecal excretion of group A rotavirus antigen from birth to two months old. All the 50 piglets in these litters became infected with the virus between 19 and 35 days old. Rotavirus excretion was first seen in one litter which was housed with other litters not included in this study. Two days later, piglets of the second litter in another farrowing room began to excrete rotavirus, and then infection spread to the other three litters in the same room. Within each of these litters, one or two piglets were infected early and thereafter infection spread to other piglets. It took four to 10 days for rotavirus to infect every piglet within a litter, and 16 days in total before all piglets in the five litters were infected. No rotavirus antigen was demonstrated in faeces from sows during the investigation period.

Sensitive fluorogenic enzyme immunoassay on nitrocellulose membranes for quantitation of virus.

A highly sensitive fluorogenic enzyme-linked immunosorbent assay (FELISA), which utilizes nitrocellulose membranes as solid phase support, has been developed for the detection and identification of virus in clinical samples. Reagents were standardized and, using purified Newcastle disease virus (NDV) as a model, the theoretical lower limits of test sensitivity of the FELISA were compared, in both "sandwich" and "indirect" formats, to those of a comparable chromogenic enzyme-linked immunosorbent assay (CELISA). Of the systems evaluated, the "sandwich" FELISA exhibited maximum sensitivity and detected 10 fg of purified virus protein per milliliter of test sample (500 ag per test volume). Specificity of the "sandwich" FELISA was evaluated by challenging the system with heterologous strains of NDV and with other serologically related and unrelated viruses. In a clinical trial in which fecal materials from chickens undergoing vaccination with NDV were assayed directly by FELISA, the virus was detected from the first to approximately the tenth day post-vaccination. The test is simple to perform and results can be obtained in approximately 4 h.

Immunochemical properties of the antigens involved in the pigeon breeder's disease.

The pigeon breeder's disease is a form of extrinsic allergic alveolitis caused by the exposure to pigeon droppings. Chemical analysis of this antigen was carried out employing column fractionation techniques. Sephadex G-50 and DEAE cellulose were performed and several proteins and hexoses peaks were recorded. Molecular weights were determined by comparison with standardized marker proteins passed through a drop counting fraction collector. The whole extract revealed a molecular weight of 100 Kd while fraction 1 showed 66 Kd and fraction 2 only 30 Kd. A guinea pig experimental model was developed with the whole extract injected by the intradermal route, administered by an intragastric catheter or aerosolized in a glass chamber. Histopathological studies were carried out with the lungs, kidneys, liver and spleen obtained by the necropsy of the animals. The lungs and the kidneys showed the paramount changes in their structures with lymphomononuclear infiltrates and an Arthus-like phenomenon surrounding the vessels. Immunological techniques were applied to the sera and the lymphocytes obtained from the animals. Precipitin and hemagglutinating IgG antibodies were detected against the whole extract and the fractions obtained by column fractionation. Sensitized lymphocytes were also detected. This experimental model represents a guide in the approach to the human allergic alveolitis whose immunological findings will be presented in a forth-coming report.

Measurement in vitro of a larval migration inhibitory factor in gastrointestinal mucus of sheep made resistant to the roundworm Trichostrongylus colubriformis.

Lambs were challenged by dosing with 2500 T. colubriformis larvae per day for 34 weeks, rested for 4 weeks and then re-challenged with the infective larvae for a further 10 weeks. A technique for the measurement of inhibition of larval migration from agar gels was used to investigate the antiparasitic activity of mucus, obtained from the small intestine and abomasum of the lambs, at the end of the re-challenge period. Measurements were also made on ileal digesta samples collected at different times during the development of the initial resistance, the rest period and the re-challenge period, and on faeces collected during the re-challenge infection. The mucus from the small intestine and abomasum paralysed and inhibited larval migration from agar gels significantly more (P less than 0.01 and P less than 0.05, respectively) than corresponding mucus from parasite-free control animals. This inhibitory activity was also detected (P less than 0.01) in the ileal digesta of infected animals from Week 6 of primary dosing. The magnitude of the inhibition in the ileal digesta increased with time during the infection period and was again detected during re-infection (P less than 0.01). It was not detectable in resistant sheep towards the end of the rest period. Slight inhibitory activity was detected in faeces after 2 weeks of re-infection. These observations suggest that substances secreted into the lumen of the small intestine of infected animals are responsible for resistance against ingested larvae.

A method of obtaining, processing, and analyzing human intestinal secretions for antibody content.

Human intestinal secretions can be readily obtained using a commercially available intestinal lavage solution. Although such secretions contained abundant protease activity, significant loss of immunoglobulins was prevented by the addition of a mixture of protease inhibitors. The total content of IgA, IgM, and IgG antibody in secretions was measured using sandwich ELISA. In the secretions of ten normal volunteers IgA was most abundant (197 micrograms/ml +/- 103 SD) followed by IgM (12.5 micrograms/ml +/- 6.8 SD) and IgG (0.24 micrograms/ml +/- 0.04 SD). The IgA in secretions was predominantly secretory IgA as shown by sucrose density centrifugation. The effect of intestinal secretions on the sensitivity of the antigen-specific ELISA was tested by adding murine myeloma IgA anti-TNP added to samples of human secretions. IgA anti-TNP activity could be detected as low as 1 ng/ml, and there was no evidence of interference with the ELISA by other constituents in the secretions. Using these methods an antigen-specific secretory IgA anti-cholera toxin B subunit response in the secretions of volunteers given an oral B subunit vaccine was readily demonstrated.

An enzyme-linked immunoassay for the possible detection of non-A, non-B viral antigen in patients with epidemic viral hepatitis.

An enzyme-linked immunosorbent assay has been developed by using IgM antibodies from the acute stage as a source to capture the antigen in stools of patients with epidemic non-A, non-B (NANB) viral hepatitis. 29/69 (42.3%) of the patients and 3/9 (33.3%) contacts were positive for a suspected NANB viral antigen. However, only 1/27 (3.7%) of the negative controls drawn from amongst the patients with amoebiasis, giardiasis, hepatitis due to virus A and healthy individuals was positive for NANB antigen in the stool. The suspected NANB viral antigen was more frequently detected in stools collected between the 14th and 18th day of icteric hepatitis. The study suggests that IgM antibodies from patients with acute viral NANB hepatitis react with an antigen present in the stools of a high proportion of patients with epidemic NANB viral hepatitis. This serological test may be useful to establish the etiological diagnosis of non-A, non-B (fecal-oral) viral hepatitis. ELISA-positive stools contained 27 nm viral particles.

The major allergen of the house dust mite, Dermatophagoides pteronyssinus, is synthesized and secreted into its alimentary canal.

The natural production of Der pl, the major faecal allergen from the house dust mite (Dermatophagoides pteronyssinus), was investigated. Mite cultures grown on radiolabelled substrate were examined at intervals over a period of 21 days and incorporation of radiolabel into the mite proteins measured over the time course. Mite proteins were extracted, purified by chromatofocussing and characterized by immunodiffusion, SDS-PAGE, electroblotting and autoradiography. The faecal allergen, Der pl, did not incorporate a significant level of label until the 14th day (p less than 0.05). The results suggest that Der pl is a protein synthesized and excreted by the house dust mite.

Development of nasal, fecal and serum isotype-specific antibodies in calves challenged with bovine coronavirus or rotavirus.

Unsuckled specific pathogen free calves were inoculated at 3-4 weeks of age, either intranasally (IN) or orally (O) with bovine coronavirus or O plus IN (O/IN) or O with bovine rotavirus. Shedding of virus in nasal or fecal samples, and virus-infected nasal epithelial cells were detected using immunofluorescent staining (IF), ELISA or immune electron microscopy (IEM). Isotype-specific antibody titers in sera, nasal and fecal samples were determined by ELISA. Calves inoculated with coronavirus shed virus in feces and virus was detected in nasal epithelial cells. Nasal shedding persisted longer in IN-inoculated calves than in O-inoculated calves and longer than fecal shedding in both IN and O-inoculated calves. Diarrhea occurred in all calves, but there were no signs of respiratory disease. Calves inoculated with rotavirus had similar patterns of diarrhea and fecal shedding, but generally of shorter duration than in coronavirus-inoculated calves. No nasal shedding of rotavirus was detected. Peak IgM antibody responses, in most calves, were detected in fecal and nasal speciments at 7-10 days post-exposure (DPE), preceeding peak IgA responses which occurred at 10-14 DPE. The nasal antibody responses occurred in all virus-inoculated calves even in the absence of nasal shedding of virus in rotavirus-inoculated calves. Calves inoculated with coronavirus had higher titers of IgM and IgA antibodies in fecal and nasal samples than rotavirus-inoculated calves. In most inoculated calves, maximal titers of IgM or IgA antibodies correlated with the cessation of fecal or nasal virus shedding. A similar sequence of appearance of IgM and IgA antibodies occurred in serum, but IgA antibodies persisted for a shorter period than in fecal or nasal samples. Serum IgG1 antibody responses generally preceeded IgG2 responses and were predominant in most calves after 14-21 DPE.

The transfer of serum IgG1 antibody into the gastrointestinal tract in newborn calves.

Transfer of functional blood IgG1 to the gastrointestinal tract was measured in neonatal calves. Radiolabelled immunoglobulin G1 (IgG1) anti-DNP antibody was administered to 2 day old calves by intravenous injection. The serum clearance rate was measured and was compared to the rate of protein-bound 125I excretion in the feces over a 10 day period to determine the importance of transfer to the gastrointestinal tract as a mechanism of serum IgG1 clearance. The amount of protein-bound and DNP-binding 125I present in the gastrointestinal tract of 10 day old calves at necropsy was also measured. Fecal excretion of protein-bound 125I accounted for 32% of the serum 125I-IgG1 clearance. Protein-bound 125I was present in the gastrointestinal tract at necropsy in amounts estimated to account for 68% of the total 125I-IgG1 clearance, and retained 65% of the DNP-binding ability of the original antibody. The discrepancy between the fecal excretion (32% of total IgG1 clearance) and the GI clearance estimated from protein-bound 125I in the gut (68% of total IgG1 clearance) is explained in part by IgG1 proteolysis occurring after transfer to the gastrointestinal tract but before fecal excretion. These results indicate that transfer to the calf gastrointestinal tract accounts for most IgG1 clearance in young calves, and that the intestinal antibody retains antigen binding function and may contribute to intestinal immunity.

Candidate rotavirus vaccine (rhesus rotavirus strain) in children: an evaluation.

Fifty children, 3 months to 12 years of age, were given an experimental, orally administered, live attenuated rotavirus vaccine. Overall evidence of vaccine effectiveness as judged by vaccine virus shedding or a serologic response was seen in 82% of vaccinees. No clinical illness was seen in the rotavirus vaccinees when compared with 40 concurrently studied control children. No transmission to control children was observed even with close daily contact in a day-care setting. Young infants, generally less than 1 year of age, who had not previously experienced wild-type rotavirus infection shed significantly more vaccine virus. Limitation of virus shedding in those already exposed may be related to a prompt copro-IgA response which was significantly elevated by three days after vaccination. In summary, the development of this rotavirus vaccine, rhesus rotavirus-MMU-18006, is a promising step in the development of immunoprophylaxis against this major enteric pathogen.

A combined dot nitrocellulose-enzyme immunoassay for rotavirus and adenovirus.

A combined enzyme immunoassay using nitrocellulose membrane as solid support is described for the detection of rotavirus and adenovirus in faeces from children with gastroenteritis. Its sensitivity and specificity are comparable to those of a previously described assay performed on plastic microplates (Pereira et al. (1985) J. Virol. Methods 10, 21-28). The introduction of nitrocellulose membrane as support for the immune reactions greatly simplifies the multiple washing steps and precludes the need for disposable plastic plates.

Measurement of class specific antibody against cryptosporidium in serum and faeces from experimentally infected calves.

Anti-cryptosporidium antibody levels were measured in serum and faeces of experimentally infected calves. In serum, IgG was detectable six days after infection and remained elevated throughout infection. IgA and IgM in serum showed little change. IgG, IgA and IgM levels all rose in the faeces five or six days after infection and reached a peak between days 8 and 14 after infection and then declined.

A double-antibody sandwich ELISA for the detection of Entamoeba histolytica antigen in stool samples of humans.

A double-antibody sandwich ELISA was developed to detect detergent-solubilized antigens of Entamoeba histolytica in stool samples of humans. The test system was evaluated for its methodical and diagnostic sensitivity and specificity. In recovery experiments the lower limit of detection was 400 ng E. histolytica (HK9) protein/ml stool, corresponding to approximately 2000 amoebic trophozoites/ml stool. Samples of 97 patients with suspected intestinal amoebiasis were examined. Specific antigens were detected by ELISA (= positive reaction) in 14 (93%) out of 15 stool samples containing trophozoites of E. histolytica. In contrast, 68 (93%) of 73 samples with other protozoa, including Entamoeba coli, E. hartmanni, Endolimax nana, Iodamoeba buetschlii and Giardia lamblia, did not react in the test system (= negative reaction). The test was shown to detect only trophozoites of E. histolytica and not the cyst stage. This fact could facilitate the differentiation between cyst carriers and persons excreting trophozoites. The results of this preliminary study justify a further large scale evaluation of the test system.

Protection between different serotypes of bovine rotavirus in gnotobiotic calves: specificity of serum antibody and coproantibody responses.

In a previous study, different U.S. isolates of bovine rotavirus were studied for their serotypes and cross-protective properties (G. N. Woode, N. E. Kelso, T. F. Simpson, S. K. Gaul, L. E. Evans, and L. Babiuk, J. Clin. Microbiol. 18:358-364, 1983). Three viruses belonging to two different serotype groups were used as vaccines in gnotobiotic calves, which were subsequently challenged with B641 or B223, representing the two bovine serotypes. In the present work, the experiments were repeated with more calves and the specificity of their antibody responses was measured and compared with the results of the protection studies. Protection between different serotypes occurred under both homologous and heterologous conditions but was not directly serotype dependent. B223 virus showed both homologous and heterologous protection against B223 and B641 challenge viruses. This was a one-way reaction, as B641 did not induce protection against B223. Neonatal calf diarrhea virus vaccine produced neither homologous (against B641) nor heterologous (against B223) protection. The plaque reduction neutralization titers of serum antibody and coproantibody did not predict a state of protection against the challenge virus. Calves vaccinated with neonatal calf diarrhea virus or B641 developed neutralizing antibodies to their respective heterologous challenge viruses but were not protected. After challenge, the boosted coproantibody plaque reduction neutralization response to the original vaccine virus was greater than that to the challenge virus.

Analysis of latex agglutination test for Clostridium difficile toxin A (D-1) and differentiation between C difficile toxins A and B and latex reactive protein.

Virulent toxigenic and avirulent non-toxigenic strains of Clostridium difficile gave a positive result in the latex agglutination test (LAT) for C difficile toxin A (D-1). Similar concentrations of latex agglutinating antigen were produced by these strains in vivo. Positive reactions were also given by C sporogenes, proteolytic C botulinum Types A, B, and A/F, and Bacteroides assaccharolyticus. The latex agglutinating antigen was denatured by boiling for 10 minutes, but not by heating at 56 degrees C for 30 minutes. The reaction was abolished by incubation of test material with crude C difficile antitoxin but not with other clostridial antitoxins or specific antitoxin to C difficile toxin A. The latex agglutinating antigen present in C difficile eluted between 0.39% and 0.47% M sodium chloride, and that produced by the other clostridia, between 0.35% and 0.43% M sodium chloride by fast protein liquid chromatography. The latex agglutinating antigen of C difficile was neither cytotoxic nor mouse lethal and was distinct from toxin A and toxin B. In the analysis of faecal specimens from patients with diarrhoea the latex agglutination test correlated better with the presence of C difficile than with toxin B and detected both toxigenic and non-toxigenic strains. The latex agglutination test should only be used in the laboratory as an alternative to culture for C difficile and not as a method for the detection of C difficile toxins.

Investigations of culture medium-free house dust mites. III. Antigens and allergens of body and fecal extract of Dermatophagoides farinae.

Crossed immunoelectrophoresis (CIE) and crossed radioimmunoelectrophoresis (CRIE) were used to characterize antigens (Ags) and allergens derived from Dermatophagoides farinae (DF) culture media-free mite body and mite fecal matter extracts. CIE of DF body and DF feces extracts revealed the presence of 35 and 20 Ags, respectively. CRIE experiments demonstrated IgE binding by 14 and seven DF body and DF feces Ags, respectively, when CIE gels were incubated with reference sera from clinically mite-sensitive patients. Binding of specific IgE to the various Ags in the two extracts varied significantly both in frequency and in strength from patient to patient and within the same patient's serum. Sera from some patients demonstrated IgE binding predilection for specific DF body Ags, whereas other sera exhibited greater binding preference for DF feces Ags. Homologous, heterologous, and intermediate gel CIE and CRIE clearly demonstrated that DF bodies and DF feces share some common Ags or epitopes, but the two different extracts also were quantitatively different. Some Ags and allergens originate from mite body material and are not present in mite feces. These results indicate that only extracts containing high concentrations of both body and fecal allergens should be used in clinical testing and therapy.