The labelling of pulmonary surfactant phosphatidylcholine in 19-31-days-old lambs

The labelling of surfactant phosphatidylcholine and disaturated phosphatidylcholine was studied in 19-31-days-old lambs. Following the placement of small bore tracheal catheters, the animals were given radioactively labelled palmitic acid and/or choline by intravenous injection and multiple samples were recovered from the distal airways of each animal via a small catheter. The specific activities of the phosphatidylcholine and/or disaturated phosphatidylcholine were measured in these samples of surfactant. The labelled phospholipids accumulated in the samples of surfactant in a linear fashion; the mean time required to reach maximal specific activities in phosphatidylcholine and saturated phosphatidylcholine with either palmitic acid or choline as precursor was 28 h. Subsequently the specific activities of the labelled phospholipids from the surfactant samples decreased semi-logarithmically. The mean t1/2 for phosphatidylcholine and disaturated phosphatidylcholine labelled with radioactive palmitic acid was 35 h. The saturated phosphatidylcholine labelled with radioactive choline had a t1/2 of 251 h. The results demonstrate that surfactant labelling studies can be done by multiple sampling of single large animals.     

Modification of the fatty acid composition of phospholipid in measles virus-persistently infected cells

The composition of phospholipid was studied in BGM cells uninfected, persistently infected or lytically infected with measles virus, strain Hallé. In persistently infected cells, phosphatidylcholine palmitic acid content, and phosphatidylethanolamine palmitic acid and arachidonic acid contents were significantly increased. Lytically infected cells had a similar phospholipid fatty acid composition to the uninfected. Phosphatide composition showed minor modifications, but the content of total choline-derivative phospholipids was unchanged in either type of infection.     

Identification of foot-and-mouth disease virus-specific linear B-cell epitopes to differentiate between infected and vaccinated cattle

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals. For several years, vaccination of animals, which had proven to be successful for the eradication of the disease, has been forbidden in the United States and the European Community because of the difficulty of differentiating between vaccinated and infected animals. In this study, detailed investigations of the bovine humoral immune response against FMD virus (FMDV) were performed with the aim of identifying viral epitopes recognized specifically by sera derived from FMDV-infected animals. The use of overlapping 15-mer synthetic peptides, covering the whole open reading frame of FMDV strain O(1)K in a peptide enzyme-linked immunosorbent assay, allowed the identification of 12 FMDV strain O(1)K-specific linear B-cell epitopes. Six of these linear B-cell epitopes, located in the nonstructural proteins, were used in further assays to compare the reactivities of sera from vaccinated and infected cattle. Antibodies recognizing these peptides could be detected only in sera derived from infected cattle. In further experiments, the reactivity of the six peptides with sera from animals infected with different strains of FMDV was tested, and strain-independent infection-specific epitopes were identified. Thus, these results clearly demonstrate the ability of a simple peptide-based assay to discriminate between infected and conventionally FMD-vaccinated animals.     

Immune responses to Mycoplasma conjunctivae in alpine ibex, alpine chamois, and domestic sheep in Switzerland

The humoral immune response of three alpine chamois (Rupicapra rupicapra rupicapra), two alpine ibex (Capra ibex ibex) and three domestic sheep naturally affected with infectious keratoconjunctivitis (IKC), and four ibex and two sheep experimentally infected with Mycoplasma conjunctivae was analysed. In addition, the local immune response to M. conjunctivae was analysed using conjunctival washes from chamois and sheep. Immunoblot analysis of sera using whole cell antigens of M. conjunctivae revealed the major immunogenic proteins which had molecular masses of 175, 83, 68, 60, 50, 42, 36, and 33 kDa. Major antigens were found at 83, 68, 60, and 42 kDa in both sera and conjunctival washes from naturally infected animals of all three Caprinae species. In experimentally infected animals, antibodies to the 68 and 60 kDa antigens were dominant. Naturally infected animals showed much stronger immune reactions than those experimentally infected, and specific antibodies appeared 2 to 4 wk after experimental infection. To evaluate possible cross-reactions, whole cell antigen of M. conjunctivae was analysed by immunoblot against hyperimmune sera of closely related Mycoplasma spp. Antibodies to the 175, 73, 68, 60, and 33 kDa antigens appeared to be specific to M. conjunctivae. Cross-reactions mainly with 83, 50, and 42 kDa antigens were detected, in particular with M. ovipneumoniae and M. bovoculi hyperimmune sera, but also with antisera against M. capricolum capricolum and M. putrefaciens.     

一种实用的筛选病毒抗原表位方法的建立(英)

采用基因分段克隆、表达结合蛋白质印迹, 筛选到了口蹄疫病毒非结构蛋白3ABC上高结合力、保守的感染相关线性表位,分别位于3ABC蛋白上第106～155和156～190位氨基酸.这两个表位可与感染不同血清型口蹄疫病毒动物康复血清反应,但不与来自健康免疫动物和未接触病毒动物的血清发生反应.实验表明,用基因工程表达的多肽筛选抗原表位的方法是可行的.     

Detection of the antibody to lymphocytic choriomeningitis virus in sera of laboratory rodents infected with viruses of laboratory and newly isolated strains by ELISA using purified recombinant nucleoprotein

An enzyme-linked immunosorbent assay (ELISA) was developed to detect the antibody against lymphocytic choriomeningitis virus (LCMV) in sera of laboratory animals. In this ELISA system, LCMV-nucleoprotein (NP) expressed by recombinant baculovirus and purified with high molar urea was used as the antigen. Sera from laboratory animals experimentally infected with the Armstrong strain or the newly isolated M1 strain of LCMV were examined to detect anti-LCMV antibody by the ELISA system, and the reactivity was compared with that of IFA test. Regardless of LCMV strain, all the sera of adult mice infected with LCMV were positive with very high optical density (OD). Also, the sera from mice neonatally infected with LCMV M1 strain were positive with slightly lower OD than adult mice. In contrast, all the sera of uninfected mice were negative to LCMV-NP antigen. Similarly, anti-LCMV antibodies were detected in all the sera of hamsters, mastomyses, and gerbils infected with the LCMV Armstrong strain. The results of the ELISA were in complete agreement with those of IFA, and indicate the high sensitivity and specificity of the ELISA system in the detection of anti-LCMV antibody. Because this ELISA system does not require handling infectious LCMV in the course of the antigen preparation and serological assay, there is no risk of contamination in the laboratory or nearby animal facility. In addition, by using negative control antigen in parallel with positive antigen in ELISA, we can exactly check the LCMV contamination in laboratory animals.     

Brucella abortus in wildlife on selected cattle farms in Alabama

Two studies of brucellosis in wildlife on farms where the brucellosis infection prevalence in cattle was known are reported. On a research farm, 233 feral animals of 22 mammalian species and 12 of seven avian species were trapped during three time periods. Sixty were studied before cattle were introduced, 128 were studied while 501 cattle infected with Brucella abortus were calving and aborting, and 60 specimens were collected 20 mo after the last infected cow calved. Selected tissues from 229 wild animals were cultured and sera from 138 were examined using the brucellosis card, standard tube agglutination (STA), 2-mercaptoethanol (2-ME) and rivanol (RIV) tests. Brucella abortus was not recovered from any animals sampled prior to cattle being introduced and all sera collected were negative. Brucella abortus was isolated from four opossums (Didelphis virginiana) and one raccoon (Procyon lotor) in the group of animals trapped during the calving period. Three serums were tested and had STA titers ranging from 1:100 to 1:200. Of 68 sera only one had antibodies. Brucella were not isolated from 59 animals trapped after the calving period and only one of 42 serums had antibodies. On regional cattle farms, 243 wild animals were trapped. Brucellae were not isolated from 223 animals which were cultured. No serums had significant titers. The data from this study suggest opossums and raccoons can be infected from cattle but are unlikely to maintain the infection.     

Optimized serodiagnosis of sheep fascioliasis by Fast-D protein liquid chromatography fractionation of Fasciola hepatica excretory-secretory antigens

Current methods for the serodiagnosis of sheep fascioliasis show suboptimal sensitivity, specificity, or both. With the aim of developing an improved method, we fractionated native Fasciola hepatica excretory-secretory antigens (ESAs) by size-exclusion FPLC (fast protein liquid chromatography) on a Superdex 75 HR 10/30 column and then tested the serodiagnostic value of the antigens contained in each one of the 4 peaks obtained (peaks I-IV). Serodiagnostic value was assessed using sera from sheep naturally infected with F. hepatica (group A); sera from the individuals of a fluke-free herd (most of which also had other intestinal nematodes, lung nematodes, Moniezia spp., and/or Cysticercus tenuicollis) sera from a fluke-free herd (group B); sera from lambs experimentally infected with 10-40 F. hepatica metacercariae (group C); and sera from uninfected control lambs (group D). Enzyme-linked immunosorbent assay (ELISA) with peak I or II as target antigens (and to a lesser extent with peak III as target) showed reactivity with negative sera, so that it was not possible to establish cutoff values discriminating infected and uninfected animals. In contrast, when peak IV was used as target, a low cutoff value of 0.235 optical density units (mean + 4 SD) discriminated infected and uninfected animals, with 100% sensitivity and 100% specificity. ELISA with peak IV as a target identified infected animals (even animals that had received only 10 metacercariae) within 3-5 wk of infection and subsequently throughout the rest of the 14-wk monitoring period. In Western blotting analysis, again only the antigens contained in peak IV (range 7-40 kDa, under reducing conditions) were specific for diagnosis of infected animals. These results indicate that molecular sieving of F. hepatica ESAs by this procedure is a fast, simple, reproducible way of obtaining antigens useful for serodiagnosis of sheep fascioliasis.     

H and 13C n.m.r. studies of serum from normal and Echinococcus multilocularis infected jirds

The major components of the 13C and high field region of the 1H nuclear magnetic resonance (n.m.r.) spectra of normal and Echinococcus multilocularis infected jirds were identified and compared. Substantial depletion of the glucose and fatty acid chains from lower density lipoproteins was detected in sera from infected animals. In addition, this proliferating metacestode markedly changed the appearance of the spectral region recently assigned to N-acetyl protons of carbohydrate side chains of N-acetylated glycoproteins.     

Identification of antigens with potential for immunodiagnosis of Parelaphostrongylus tenuis and Elaphostrongylus cervi infections in red deer (Cervus elaphus elaphus)

Red deer (Cervus elaphus elaphus) were infected experimentally with Parelaphostrongylus tenuis in New Brunswick, Canada, and with Elaphostrongylus cervi in New Zealand. Excretory-secretory (E-S) antigens from adult P. tenuis were evaluated for their serodiagnostic potential in identifying P. tenuis and heterologous E. cervi infections in a Western blot. The antigen recognition profile of sera from animals infected with P. tenuis varied between individuals and with duration of infections, whereas that of pooled sera from animals infected with E. cervi showed less variation. A single molecule of 42-43 kDa was recognized consistently by sera from all animals infected with either P. tenuis or E. cervi. Sera from unexposed control deer and from those with other heterologous nematode infections did not consistently identify this antigen. Serorecognition of the 42-43-kDa antigen by deer infected with P. tenuis resulted in a sensitivity of 99% and a specificity of 85% (> or =1 mo postinfection). Although antibody to this antigen waned with time, the persistence of recognition up to 34 mo postinfection with P. tenuis exemplifies its diagnostic value. The sensitivity and specificity of diagnosis using this molecule were each 100% for identifying deer infected with E. cervi (> or =3 mo postinfection). Two other molecules from E-S of adult P. tenuis, 26-28 and 10-12 kDa, were also diagnostic, although their recognition was not persistent throughout infections. These 2 molecules may prove useful in combination with the 42-43-kDa antigen to help identify all infected animals during all phases of infections. This research represents the first conclusive identification of antigens with real potential for reliable antemortem immunodiagnosis of both P. tenuis infections and heterologous E. cervi infections.     

Enzyme-linked immunosorbent assay of antigens from Candida albicans circulating in infected mice and rabbits: The role of mannan

Various antisera raised either to antigens ofCandida albicans or to sub-lethal infections of blastospores (convalescent sera) were tested for their efficacy in diagnosing systemic disease in artifically infected animals. Globulin from convalescent serum, when conjugated with alkaline phosphatase and used in enzyme-linked immunosorbent assays (ELISA), was the only antiserum type which detected circulatingCandida-related antigen in the serum of infected animals. Conjugates made from anti-mannan, anti-blastospore or antimycelial globulin did not detect antigen. Mannan did not appear to be related to an antigen produced in sera of experimentally infected mice. The significance of these results in the diagnosis of systemic candidosis is discussed.     

Effect of Mengovirus Replication on Choline Metabolism and Membrane Formation in Novikoff Hepatoma Cells

Infection of Novikoff rat hepatoma cells (subline NlSL-67) with mengovirus resulted in a two- to threefold increase in the rate of choline incorporation into membrane phosphatidylcholine at about 3 hr after infection, without affecting the rate of transport of choline into the cell or its phosphorylation. The time course of virus-stimulated phosphatidylcholine synthesis was compared with the time courses of other virus-induced processes during a single cycle of replication. The formation of viral ribonucleic acid (RNA) polymerase and of viral RNA commenced about 1 hr earlier than the virus-stimulated choline incorporation. Further, isopycnic centrifugation of cytoplasmic extracts indicated that the excess of phosphatidylcholine synthesized by infected cells is not located in the membrane structures associated with the viral RNA replication complex, but with structures of a lower density (1.08 to 1.14 g/cc). These membrane structures probably represent the smooth vesicles which accumulate in the cytoplasm of infected cells during the period of increased phosphatidylcholine synthesis between 3 and 5 hr after infection. They are formed with both newly synthesized phosphatidylcholine and phosphatidylcholine present prior to infection. However, concomitant protein synthesis is not required for the stimulated synthesis of membranes; the effect was not inhibited by treating the cells with inhibitors of protein synthesis at 3 hr after infection, although virus production was inhibited about 90% and virus-induced cell degeneration was markedly reduced and delayed. Production of mature virus began normally at about the same time as the stimulation of phosphatidylcholine synthesis. Treatment of infected cells with puromycin at 2 hr, on the other hand, completely inhibited the stimulation of phosphatidylcholine synthesis.     

Serum glycoproteins in rats with experimental coccidioidomycosis

Summary The changes in the serum glycoproteins and the development of specific antibodies were examined during the course of experimental coccidioidomycosis in rats. The total glycoprotein, seromucoid hexose, seromucoid protein, and haptoglobin levels were signigicantly higher in sera from infected animals compared with sera from non-infected controls. These changes were evident at three days but not at two weeks following inoculation withC. immitis. The non-seromucoid hexose and total protein concentrations were not significantly different between infected animals and controls. The highest percentages of animals exhibiting positive tests for complement fixing and precipitin antibodies and positive cultures forC. immitis in organs at autopsy were found one week after infection.This study was supported in part by USPHS Grants T1 A1 52-07, A106048-02 and the Dermatologic Research Foundation of California, Inc.     

Development of an enzyme-linked immunosorbent assay for detection of IgM antibodies to Babesia bigemina in cattle

A crude antigenic preparation of Babesia bigemina was used to develop an ELISA for the detection of IgM antibodies. Optimal dilutions of the antigen, using positive and negative reference sera, were determined by checkerboard titrations. Negative sera from cattle imported from tick-free areas, serum samples collected from infected B. bigemina cattle were used to validate the test. The specificity was 94% and sensitivity of the Elisa 87.5%. Sera from 385 cattle deriving from areas free from tick-borne diseases, which were submitted to a preimmunization process, were screened by this technique. The Elisa detected seroconversion on the 14th day post-inoculation in animals either infested with Boophilus microplus ticks (infected with B. bigemina), or inoculated with B. bigemina infected blood. Antibody titers decreased after day 33; however, all animals remained positive until the end of the experiment (124 days). The ELISA described may prove to be an appropriate serological test for the detection of IgM antibodies against B. bigemina.     

Measles-virus-persistent infection in BGM cells. Modification of the incorporation of [3H]arachidonic acid and [14C]stearic acid into lipids.

In BGM cells chronically infected with measles virus, although the composition of the phospholipids is unaltered, the fatty acid composition is modified. Uninfected, lytic and persistently infected cells were labelled with [3H]arachidonic acid and [14C]stearic acid and their metabolic fate analysed. No difference in the total incorporation was observed in the different systems. However, the [14C]stearic acid and [3H]arachidonic acid were incorporated up to 2-fold and 13-fold respectively greater into the neutral lipid of persistently infected compared with that of uninfected cells. Both radioactive fatty acids were specifically accumulated in the triacylglycerol and non-esterified fatty acids fractions. Lytically infected cells were similar to uninfected cells. Although there was no significant difference in the incorporation of radioactivity into the total phospholipid in either system, there was a large decrease in [3H]arachidonic acid incorporated into phosphatidylethanolamine and to a lesser extent phosphatidylcholine and phosphatidylinositol in persistently infected cells. [14C]Stearic acid incorporation was also reduced in phosphatidylcholine and phosphatidylethanolamine fractions of persistently infected cells.     

Changes in the lipid peroxidation activity and intensity of tissue respiration during healing of aseptic and infected wounds in animal experiments

The variance of lipid peroxidation (LPO) was studied by the concentrations of malonic dialdehyde (MDA) in the tissue of wound bed and blood serum on the model of surface musculocutaneous aseptic and infected wounds simulated in 250 rats. The speed of oxygen consumption by isolated wound tissue was determined simultaneously. It was stated that the time course of MDA concentration in wounds and sera as well as tissue respiration in animals with infected wounds differed from those in animals with aseptic wounds. In a whole, MDA levels were found to be higher in cases with infected wounds and of changeable character. The latter animals demonstrated less intensive respiration of granulation tissue. Correlation between the variance of tissue respiration and MDA levels was established as was that of LPO and respiration with the phases of wound process. The findings could be used for the development of pathogenetic therapy and evaluation of its efficacy.     

口蹄疫病毒NSP 3ABC基因在昆虫细胞中的分泌表达及其活性检测

用设计的特异引物,扩增得到了N-端带有6×His编码序列的口蹄疫病毒完整3ABC基因序列,并将其亚克隆入带有蜂毒溶血肽序列的穿梭质粒pMelBac-B中,构建了重组质粒pMel-3ABC。将该重组质粒与杆状病毒骨架DNABac-N-BlueTM共转染Sf9昆虫细胞,通过噬斑筛选和PCR鉴定,获得了含有目的基因的重组杆状病毒。重组病毒感染Sf9昆虫细胞,采用通过SDS-PAGE和Western blot检测,证明目的基因在昆虫细胞中得到了正确的表达,表达产物分泌至细胞培养上清中,并具有良好的生物活性。表达的目的蛋白经过镍柱亲和层析法纯化后,用间接ELISA方法检测与口蹄疫病毒感染动物血清的反应性,证明表达目的蛋白与感染动物血清有很好的反应性而与正常动物以及免疫动物血清不发生反应。该研究为建立一种更加敏感和特异的口蹄疫病毒感染动物与疫苗免疫动物的鉴别诊断方法奠定了基础。     

Time courses of antibody levels in Mastomys natalensis after infections with Litomosoides carinii, Dipetalonema viteae, Brugia malayi or B. pahangi, Determined by ELISA

Using a Litomosoides carinii adult antigen, time courses of antibody levels were followed by an ELISA in L. carinii, Dipetalonema viteae, Brugia malayi and B. pahangi infected Mastomys natalensis. Using various groups of infected animals, periods up to 400 days after infection were covered. In L. carinii infected Mastomys, antibodies were first detected 11 days p.i. and levels increased rapidly until day 40. Temporarily reduced levels about the beginning of patency were followed by increasing values until about 100 days p.i. Then the antibody content of the sera remained more or less constant until about 250 days p.i. although maximum levels were found at day 170. Thereafter, the antibody concentration in the sera declined slowly but high levels were still observed 390 days p.i. The antibody content was usually higher in animals with high microfilariae densities than in those with low microfilariae counts but relations could not be proven statistically. In D. viteae infected Mastomys, maximum antibody values were reached within the beginning of patency. Levels were not altered markedly until about 110 days p.i. Thereafter they decreased slightly but then remained constant until the end of the investigation period 350 days p.i. B. malayi infected animals showed a rapid increase of the antibody content in the sera; a maximum was reached by 20 days after the infection. Thereafter, somewhat constant levels were found for 4--5 months. After 300 days p.i. the antibody levels declined progressively, accompanied with increasing parasitaemia densities; after 380 days the levels reached about 2/3 of the maximum. However, despite this, no relation was found between the levels of parasitaemia and antibody in individual animals. In B. pahangi infections the main prepatent antibody increase occurred during week 5 p.i., when maximum values were observed. The beginning of patency and the early patency were accompanied with slightly declining antibody levels. From 150 days p.i. until the end of the investigation 400 days p.i., the antibody content of the sera was fairly constant.     

Automated, Quantitative Microhemagglutination Assay for Treponema pallidum Antibodies

An automated, quantitative microhemagglutination assay for antibodies to Treponema pallidum was developed by using T. pallidum-sensitized erythrocytes and an automatic serial-dilution instrument. Reactivity was found in sera from 54 rabbits and 6 chimpanzees infected with T. pallidum. Reactivity was also found in sera from animals infected with T. pertenue, T. carateum, and T. cuniculi. No reactivity was found in sera from 75 normal rabbits or from 129 rabbits immunized with cultivatable treponemes or a variety of other bacteria. In approximately 3 min, 13 twofold serial dilutions of each of 8 preabsorbed sera and the addition of sensitized erythrocytes to each dilution were accomplished automatically. The automated assay can serve as a research tool in quantitating antibodies to pathogenic treponemes, and evaluation of its clinical usefulness seems warranted.     

Development of an immunochromatographic strip for the rapid detection of Toxoplasma gondii circulating antigens

We developed a rapid immunochromatographic strip (ICS) procedure that can detect circulating antigens in the blood of animals during the acute stage of toxoplasmosis. The aim of this study was to evaluate this test using sera from field samples and from experimentally infected animals. The sensitivity and specificity of the ICS were compared with those of an enzyme-linked immunosorbent assay (ELISA). Both assays detected circulating antigens in the sera of animals experimentally infected with the Gansu Jingtai strain of Toxoplasma gondii, and the agreement between the two assays was 100%. In the infected animals, circulating antigens could be detected as early as the second day post-infection (PI) and in all animals by the fourth day. In the 381 field serum samples, the positive rates of the ICS and ELISA were 5.2% and 5.8%, respectively. In addition, there was no cross-reactivity of the antigens with Neospora caninum. The results presented here suggest that the ICS is a feasible, convenient, rapid and effective method to detect infection by T. gondii. This test could be a powerful supplement to the current diagnostic methods. Taken together, the results of this study encourage further research toward the production of commercial diagnostic tests for detecting T. gondii in animals.