Collaborative problem-solving in telemedicine and evidence interpretation in a complex clinical case

The objective of this study was to evaluate the effect of different infection levels of Ostertagia ostertagi and Cooperia oncophora in a simulated 'first grazing season' on the resistance of calves to an artificial challenge infection. The infection levels were determined by the infection schedules and the chemoprophylaxis used. Thirty six 7-11-month old Holstein-Friesian bull calves were randomly divided into four groups. The animals of group B received an ivermectin sustained release bolus (ISRB) on day 0. The calves of group D were treated on days 0 and 56 with a subcutaneous injection of doramectin (0.2 mg kg(-1) BW). Group C was the untreated control group. The calves of group N were used as helminth-naive controls, while the animals of groups B, C and D were trickle infected for 24 weeks. The infection schedules were designed to simulate the expected infection pattern for each treatment group under set-stocked conditions in temperate climate areas. After the last infection, all animals were treated with oxfendazole. One week later, all animals received a challenge infection of 50,000 O. ostertagi L3 and 100,000 C. oncophora L3, spread over 10 consecutive days. During the primary infection period the faecal egg output and the serum pepsinogen and antibody levels reflected the different levels of host-parasite contact between the groups (group C > group D > group B > group N). After the challenge infection, faecal egg counts, total Ostertagia burden, size of the adult worms and abomasal globule leucocyte counts all indicated a positive relationship between the level of Ostertagia infection during the primary infection period and the level of acquired resistance. A reduction of host-parasite contact during the primary infection period, as a consequence of the infection schedule and the chemoprophylaxis used, resulted in a diminished level of resistance to the artificial challenge infection with O. ostertagi. Faecal cultures and small intestine worm counts indicated that all previously infected groups had acquired a high degree of resistance to the Cooperia challenge infection.     

Acquired immunity against Cooperia spp. and Ostertagia spp. in calves: effect of level of exposure and timing of the midsummer increase

In two experiments, groups of calves were exposed to different levels and patterns of infection with Ostertagia spp. and Cooperia spp. The experimental design simulated the stereotypic pattern of herbage infestation, including both as normal and a delayed midsummer increase, under conditions of set-stocking. After this simulated 'first grazing season', calves were followed during the subsequent winter housing. At the end of that housing period some calves were challenged with 100,000 L3 Cooperia spp. and 40,000 L3 Ostertagia spp. and slaughtered 23 days later. All previously infected calves were protected against the establishment of the challenge infection with Cooperia spp., but not against Ostertagia spp. For the latter a significant negative correlation was found between worm count and previous level of exposure to infection. During the simulated first grazing season, changes in the ratio of Cooperia to Ostertagia eggs in the faecal egg output and the genus-specific egg count were influenced by both the level of exposure and the timing of the midsummer increase. It is concluded that acquired immunity against both parasite genera develops depending on the level of exposure to infection during a first grazing season, and that delaying the midsummer increase results in a delay of the acquisition of an effective immunity as measured by faecal egg counts and the ratio of Cooperia to Ostertagia egg output.     

Relationship of impairment of schistosome 28-kilodalton glutathione S-transferase (GST) activity to expression of immunity to Schistosoma mattheei in calves vaccinated with recombinant Schistosoma bovis 28-kilodalton GST

Sera from calves vaccinated with the recombinant Schistosoma bovis-derived 28-kDa glutathione S-transferase (28GST) and subsequently naturally or experimentally exposed to Schistosoma mattheei were studied for their content of specific immunoglobulin G (IgG) and IgA antibodies to recombinant S. bovis 28GST as well as for their capacity to inhibit the enzymatic activity of the antigen. The results were analyzed in regard to the presence (natural infection) or absence (experimental infection) of a protective effect(s) (reductions in worm burden, egg load, fecal egg counts, and excretion of viable eggs) toward S. mattheei challenge. Under such conditions, no differences in the IgG- and IgA-specific antibodies to recombinant S. bovis 28GST or in the ability to block the catalytic function of the antigen between the two groups were recorded. Nevertheless, correlation analysis between the specific antibody responses to recombinant S. bovis 28GST and the inhibition of GST activity suggested an association with IgG in experimentally infected vaccinated animals, while in naturally infected vaccinated calves, the inhibitory activity appeared to be linked to a greater degree with IgA. These results suggest that in contrast to schistosomiasis in humans, IgG antibodies in calves with schistosomiasis may exhibit inhibitory functions toward GST enzymatic activity or have a modulatory effect on IgA antibody properties. Furthermore, sera from animals immunized with recombinant S. bovis 28GST recognized the native S. mattheei 28GST and achieved comparable levels of inhibition of activity of recombinant S. bovis 28GST and S. matthei 28GST, indicating the presence of cross-reactive epitopes on these two molecules.     

The cell-mediated response to schistosomal antigens at the clonal level. III. Identification of soluble egg antigens recognized by cloned specific granulomagenic murine CD4+ Th1-type lymphocytes

Granulomatous inflammation in schistosomiasis is a consequence of T cell-mediated hypersensitivity to parasite egg Ag. In the present study we used three consecutive independent chromatographic procedures to fractionate and identify the soluble egg Ag recognized by schistosome-specific, cloned, murine, CD4+ Th1-type lymphocytes, which had been shown previously to be capable of mediating granuloma formation in vivo when adoptively transferred to normal syngeneic hosts challenged with an i.v. injection of eggs. The stimulatory activity resided in two acidic egg molecules, with apparent molecular masses of 64 to 68 kDa and 38 to 42 kDa, each of which ran as a single band on SDS-PAGE after purification. Fast performance liquid chromatography and SDS-PAGE performed under reducing conditions suggested that the two molecules are related and that the 38- to 42-kDa molecule is a subunit of the 64- to 68-kDa molecule. Polyclonal lymphoid cells from schistosome-infected mice were similarly stimulated by the purified 64- to 68-kDa and 38- to 42-kDa molecules, implying that these are sensitizing Ag in the natural disease.     

Serological response to Chlamydia pneumoniae in adults with coronary arterial fatty streaks and fibrolipid plaques

The antigen-specific serological response to Chlamydia pneumoniae was studied in 45 adults with coronary artery atherosclerosis and compared with that in 40 adults with acute respiratory infection. C. pneumoniae antigen and DNA were detected in lesions more frequently in patients with low immunoglobulin G titers against C. pneumoniae than in those with high immunoglobulin G titers. Reactivities with the 42-kDa (46%) and 52-kDa (31%) proteins were observed more frequently in sera from seropositive individuals with atherosclerosis than in sera from patients with acute respiratory infection. Antibodies against the C. pneumoniae-specific 42- and/or 52-kDa protein may be a marker for chronic C. pneumoniae infection.     

Decline in serum follicle-stimulating hormone concentrations alters key intrafollicular growth factors involved in selection of the dominant follicle in heifers

Declining FSH after a transient rise coincides with selection of a dominant follicle (DF) and atresia of the remaining cohort follicles (subordinates) in cattle. The objectives of this study were to determine 1) whether intrafollicular amounts of inhibins, activin-A, insulin-like growth factor I (IGF-I), and IGF-I-binding proteins (IGFBP) are altered during selection of the first-wave dominant follicle (DF1) and 2) whether these biochemical markers are FSH dependent. Beef heifers received six or eight 6-h injections of saline (controls) or eight 6-h injections of recombinant bovine FSH (1 mg/injection) at 38 to 42 h after estrus (Day 0). Daily ultrasound scanning was used to define selection of DF1. Controls (n = 6 per group) were ovariectomized 1) on Day 3 of the estrous cycle before DF1 selection (preselection follicles) and 2) after DF1 selection on Day 4.8 +/- 0.5. In controls, FSH declined between Days 2 and 3 and selection of DF1 occurred between Days 3 and 5. During this interval, intrafollicular estradiol concentrations increased > 5-fold in DF1, yet declined 4-fold in subordinates (p < 0.05). In DF1, total IGF-I increased 1.3-fold (p < 0.05), whereas the amounts of the 40- to 47-kDa and the 35-kDa IGFBP (ligand hybridization) decreased 2.4- and 2.5-fold, respectively (p < 0.05), compared to values in preselection follicles on Day 3; total dimeric inhibin-A decreased 1.8-fold (p < 0.05). In contrast, amounts of the 30- to 32-kDa IGFBP increased 12.4-fold (p < 0.05) in subordinates on Day 4.8 compared with preselection follicles on Day 3, while the amount of inhibins > 34 kDa decreased 4- to 9-fold (p < 0.05). In FSH-treated heifers, both selection of DF1 and atresia of subordinates were delayed by 2.2 days. Preselection follicles recovered on Day 4.9 +/- 0.1 from FSH-treated heifers were similar (p > 0.05) in almost all biochemical parameters to preselection follicles from control heifers; however, they differed markedly from both DF1 and subordinate follicles recovered from control heifers on Day 4.8 +/- 0.5. In conclusion, the decline in FSH beginning after Day 2 of the heifer estrous cycle causes differential alterations in FSH-dependent growth factors and hormones within the cohort of preselection follicles, simultaneously inducing growth and enhanced estradiol-producing capacity of the DF and atresia of subordinate follicles.     

The value of quantitative gallium-67 single-photon emission tomography in the clinical management of malignant external otitis

Crude and partially purified somatic (S) and excretory-secretory (ES) antigens of Fasciola hepatica were subjected to Western blot analysis in order to identify polypeptides that would enable specific and sensitive immunodiagnosis of horse and pig fasciolosis to be undertaken. Sera from 20 horses and 20 pigs with natural infections of F. hepatica and the same number of uninfected hosts of each species were tested, together with sera from 2 pigs with Cysticercus cellulosae infections. Using crude S antigens, sera from infected horses and pigs reacted specifically with a wide range of polypeptides of 14-19, 22-30, 35-37 and 42 kDa. Likewise, specific reactivity between polypeptides of 14-17, 22-30 and 40-42 kDa in crude ES antigens and sera from infected horses and pigs was obtained. Against the criteria of high sensitivity and specificity, the 22-30-kDa polypeptides would appear to be the most suitable candidate antigens for use in the immunodiagnosis of fasciolosis in horses and pigs.     

Differential pattern of c-fos mRNA in rat brain following central and systemic administration of interleukin-1-beta: implications for mechanism of action

The epidemiology of gastrointestinal nematodes was studied in a spring calving herd in northeast Mississippi. Pregnant, mixed breed beef cows (n = 15) were placed on a 10 ha fescue/bermuda grass pasture from January 1990-February 1992. In both years, calves were born from February-April and were weaned and removed from the pasture in mid-October. Fecal egg counts (EPG) and generic composition of nematodes in fecal cultures were determined monthly for cows and calves. Estimation of numbers of third-stage larvae on herbage also was determined monthly from March 1990-February 1992. Worm-free tracer calves (2-3 per month) were allowed to graze for 1 month periods and slaughtered for counting and identification of gastrointestinal nematodes. The mean monthly EPG of cows was consistently low (0.23-3.41); EPG of calves increased from spring through fall of both years. Five nematode genera were identified from fecal cultures of cows and calves. Ostertagia and Trichostrongylus spp. were the predominant nematodes in cows, while Ostertagia and Cooperia spp. were predominant in calves. Numbers of third-stage larvae on herbage declined from spring through summer and remained at low levels until late fall/winter, when numbers increased markedly. Eleven nematode species were identified from tracers, but O. ostertagi and Cooperia spp. predominated in most months. Seasonal changes in tracer worm counts coincided with similar changes in counts of third-stage larvae on herbage. Inhibition of O. ostertagi occurred in tracer calves during spring, but did not give rise to a marked increase in egg production in cows during fall.     

Antibody response to Chlamydia pneumoniae infection in children with respiratory illness

Serologic diagnosis of Chlamydia pneumoniae infection has been based on the microimmunofluorescence test (MIF). However, recent prospective studies in children have found that >50% infected with C. pneumoniae failed to develop any antibodies detectable by MIF. In this study, single sera from 46 culture-positive and 42 culture-negative children with respiratory infection and known MIF status were examined by immunoblotting. Forty-one (89.1%) of the single sera from culture-positive and 27 (64.3%) from culture-negative children reacted to C. pneumoniae antigens in immunoblot. C. pneumoniae proteins most frequently recognized by sera from culture-positive patients were at 101-102, 72-76, 50-52, 48-49, 43-44, 41-42, and 30-31 kDa. However, there did not appear to be a correlation of specific band patterns and culture status.     

Efficacy of albendazole against gastrointestinal nematodes of cattle

Forty-five calves with artificial and pasture-acquired nematode infections were medicated with albendazole at dose levels of 0, 2.5, 5.0, or 10 mg/kg of body weight. A dose level of 2.5 mg/kg removed at least 99% of adult Trichostrongylus axei, Trichostrongylus colubriformis, Cooperia oncophora, and Bunostomum phlebotomum. Burdens of Haemonchus contortus, Strongyloides papillosus, and Ostertagia ostertagi were reduced 79, 88, and 97%, respectively. At a dose level of 5.0 mg/kg, at least 95% of all adult nematodes were removed; at 10 mg/kg, at least 97% were removed. At least 99% of 4th-stage larvae of O ostertagi, T axei, C oncophora and T colubriformis and 96% of H contortus were expelled at a dose level of 2.5 mg/kg. At 5.0 and 10 mg/kg, 99 to 100% of all species of larvae were removed. Trichuris spp adults were slightly susceptible at all dose levels; larvae were susceptible (83%) only at 10 mg/kg.     

Generation and regulation of beta-amyloid peptide variants by neurons

Studies of processing of the Alzheimer beta-amyloid precursor protein (betaAPP) have been performed to date mostly in continuous cell lines and indicate the existence of two principal metabolic pathways: the "beta-secretase" pathway, which generates beta-amyloid (A beta(1-40/42); approximately 4 kDa), and the "alpha-secretase" pathway, which generates a smaller fragment, the "p3" peptide (A beta(17-40/42); approximately 3 kDa). To determine whether similar processing events underlie betaAPP metabolism in neurons, media were examined following conditioning by primary neuronal cultures derived from embryonic day 17 rats. Immunoprecipitates of conditioned media derived from [35S]methionine pulse-labeled primary neuronal cultures contained 4- and 3-kDa A beta-related species. Radiosequencing analysis revealed that the 4-kDa band corresponded to conventional A beta beginning at position A beta(Asp1), whereas both radiosequencing and immunoprecipitation-mass spectrometry analyses indicated that the 3-kDa species in these conditioned media began with A beta(Glu11) at the N terminus, rather than A beta(Leu17) as does the conventional p3 peptide. Either activation of protein kinase C or inhibition of protein phosphatase 1/2A increased soluble betaAPP(alpha) release and decreased generation of both the 4-kDa A beta and the 3-kDa N-truncated A beta. Unlike results obtained with continuously cultured cells, protein phosphatase 1/2A inhibitors were more potent at reducing A beta secretion by neurons than were protein kinase C activators. These data indicate that rodent neurons generate abundant A beta variant peptides and emphasize the role of protein phosphatases in modulating neuronal A beta generation.     

Sperm autoantigens recognized by autoantibodies in developing rats following prepubertal obstruction of the vas deferens

Sperm protein autoantigens recognized by serum antisperm autoantibodies during development following vas deferens obstruction were studied using western blot analysis. At age 10 days, rats in an obstructed group underwent bilateral ligation and division of the vas deferens, whereas animals assigned to a sham group received a sham operation. At ages 14, 21, 35, 56, 91, and 128 days, rats were sacrificed and blood samples were obtained. Sperm antigens were recognized infrequently and with low intensity by most sera obtained at ages 14 through 56 days. Subsequently, the incidence as well as the intensity of staining of antigens in blots increased at 91- and 128-day intervals in obstructed animals. The increase in antisperm antibodies correlated with the appearance of sperm throughout the epididymis at approximately 56 days postnatally. A set of autoantigens including proteins migrating at 82-78, 76-73, 68, 57, 54, 48, 44, 42, 38-42, 36, and 22 kDa were recognized with the highest frequency and intensity after puberty. A 42-kDa protein appeared to be one of the first autoantigens recognized when obstructed animals underwent sexual maturation. Although individual animals recognized different patterns of sperm autoantigens, a repertoire of 10-12 autoantigens dominated the antisperm antibody response when obstructed animals underwent sexual maturation.     

Mechanisms of insulin resistance in experimental hyperinsulinemic dogs

The aim of this work was to identify which proteins in horse dander extracts are allergens and to characterise them. Two-dimensional PAGE showed that horse dander preparations are composed of up to 50 proteins, all having acidic isoelectric points in the pH range 3-4.5. Immunoblots of two-dimensional PAGE were used to compare the reactivity of the proteins with IgE from 23 allergic patients. Patient sera were divided into two main groups recognising either allergens of 18.5 kDa or proteins of 27-29 and 31 kDa. The proteins of 27-29 kDa and 31 kDa were all N-glycosylated and their glycan chains seem to play a role in the binding of IgE from allergic patients. The sugar composition of their carbohydrate moiety was determined and lectin-binding experiments indicated presence of terminal sialic acid linked alpha-(2-->6) to galactose, galactose linked beta-(1-->4) to N-acetylglucosamine, and possibly presence of sialic acid linked alpha-(2-->3) to galactose. The 27-29-kDa glycoproteins had heterogeneous isoelectric points, most probably due to different degrees of sialylation in their oligosaccharide chains. The two 18.5-kDa allergens exhibited slightly different isoelectric points and their N-terminal sequences were identical, showing that they most likely were isoforms of the same protein. Sequence analyses revealed that their N-terminal sequences are similar to proteins belonging to the lipocalin family. We named the two 18.5-kDa proteins Equ c 2.0101 and Equ c 2.0102, according to International Allergen Nomenclature recommendations [King, T. P., Hoffman, D., Lowenstein, H., Marsh, D. G., Platts-Mills, T. A. E. & Thomas, W. (1995) J. Allergy Clin. Immunol. 96, 5-14]. The N-terminal of the allergens of 27-29 kDa were blocked and their sequences were not determined. Their amino acid compositions were determined and comparison with acidic mammalian proteins in the Swiss-Prot database revealed high scores with lipocalin proteins. This suggests that the glycosylated horse dander allergens belong to the lipocalin family, like Equ c 2.0101 and Equ c 2.0102.     

Serological detection of heat shock protein hsp27 in normal and breast cancer patients

Heat shock protein Mr 27,000 (hsp27) is found in many human breast cancer cells and tissues; its expression is associated with the presence of estrogen receptors, lower cell proliferation, and resistance to certain chemotherapies. The purpose of this study was to assess whether hsp27 may be present in sera from women with primary breast cancer and to know whether autoantibodies to hsp27 may be found in these patients. The study was performed by Western blot analyzing sera from 42 normal premenopausal women, 20 normal postmenopausal women, and 36 breast cancer patients. hsp27 was clearly detected in sera by immunoblotting but only after immunoprecipitation. The mean hsp27 levels in cancer patients were higher than in the control patients; however, 66% of the breast cancer patients showed hsp27 within the normal range, indicating low sensitivity. Moreover, cancer patients with metastatic disease did not show significantly higher hsp27 levels than cancer patients without metastases. Serum hsp27 levels did not correlate with the hsp27 levels in tumor tissues detected by immunohistochemistry. Elevated CA 15-3 levels were not associated with high hsp27 values. Autoantibodies against hsp27 were not detected by immunoblotting in normal sera and in sera from breast cancer patients. As a consequence, serological determination of this biomarker is unlikely to be of utility in the detection and follow-up of breast cancer patients.     

Factors affecting the bacteriological contamination of commercial washing machines

Mycoplasma gallisepticum- and Mycoplasma synoviae-free chickens were infected with 0.2 ml broth culture of M. gallisepticum strain 1226 intra air sac at 3, 14, 18, 28, 42, 49 and 65 days of age. Blood samples were taken 0-5 weeks before infection and 1-6 weeks after infection (depending on age of infection). The antibody response was examined by Western blot. As a control of infection, serum plate agglutination test (SPA), pathological lesions, and presence of Mycoplasma in air sacs were used. Antibodies to p64-67 kDa appeared in all groups of birds on the first week post-infection. Antibodies to p56 were detected from the second week post-challenge if infection was performed at 3 or 14 days of age, while on first week if challenge was done at 18, 28, 42, 49 or 65 days of age. Antibodies to p200, p120, p98, p80, p75, p72, p60, p50, p45, p40, p35, p33, p31, p28, p26, p24 and p22 were also detected.     

Dynamic study on relationship between serum SEAIC level and hepatic pathological changes in mice infected with Schistosoma japonicum

Using purified rabbit polyclonal antibodies to SEA, avidin-biotin system and capture ELISA technique, we observed the dynamic changes in the level of the circulating soluble egg antigen-antibody complex (SEAIC) in murine sera at various weeks post infection. Simultaneously, the diameter and area of liver egg granuloma were measured by using profile analytical technique. Serum SEAIC was first detected 4 weeks post infection (p.i.), reaching peak level at 6-7th week, and then gradually dropped, and maintained at moderately high level till the end of the observation (12 weeks p.i.). Schistosome eggs appeared in liver tissue at 4 weeks p.i. No egg granuloma could be found until 6 weeks p.i. The peak of the average diameter and area of liver egg granuloma was noted at 7 weeks p.i., then dropped gradually. Its dynamic changes were consistent with that of the serum SEAIC level. It is therefore suggested that the serum SEAIC level could be a reference index reflecting the extent of the pathological changes of the liver. Moreover, SEAIC might play an important role in the pathogenesis of schistosomiasis japonica.     

Kinetics of duck hepatitis B virus infection following low dose virus inoculation: one virus DNA genome is infectious in neonatal ducks

Using pooled serum from congenitally duck hepatitis B virus (DHBV)-infected ducks as inoculum, we examined the effect of virus dose on the incubation period of infection and on the patterns of spread of virus infection in the liver. The pooled serum inoculum contained 9.5 x 10(9) DHBV genomes per milliliter and had an infectivity titre (ID50) in newly hatched ducks of 1.5 x 10(10) per milliliter with a 95% confidence interval of 3.0 x 10(9) to 6.3 x 10(10) ID50/ml, indicating the equivalence between one DHBV genome and one infectious unit within the limits of the assays. The incubation period of infection was inversely related to the dose of inoculum and the onset of viraemia ranged from Day 6 with the highest dose to Day 14 or 29 with the lowest dose inoculum. To study the spread of virus infection from a low percentage of initially infected cells we inoculated newly hatched ducks intravenously with sufficient DHBV (1.5 x 10(3) ID50) to infect only approximately 0.0001% of total liver cells. DHBV infection first reached detectable levels on Day 4 postinoculation (p.i.) and was detected in approximately 0.035% of hepatocytes, most of which occurred as single cells or pairs of cells, indicating that a number of rounds of infection had occurred with the spread of virus both to adjoining cells, i.e., by cell-to-cell spread, and to cells located in other parts of the liver lobule. Despite some bird-to-bird variation in timing, the percentage of infected hepatocytes increased exponentially with a mean doubling time of 16 hr from Day 4 to Day 14 p.i., by which time replication was seen in > 95% of hepatocytes. This rapid dissemination from a small number of infected hepatocytes suggests that, in neonatal ducks, there are no major delays in virus replication within the liver, that any innate and adaptive defence mechanisms operating during the first 10 to 14 days of infection are insufficient to contain virus spread, and that even a small number of infected hepatocytes produce enough progeny to rapidly infect the remaining hepatocytes.