Indirect Fluorescent Antibody Technique Research Articles

An indirect fluorescent antibody technique for the diagnosis of Hematodinium sp. infection of the Norway lobster Nephrops norvegicus

An indirect fluorescent antibody technique (IFAT) has been developed to detect Hemato­ dinium sp. in the haemolymph and tissues of the Norway lobster Nepiiiops norvegicus. The IFAT, being more sensitive and reliable than previously used field and laboratory diagnostic techniques, detects both lower-level haemolymph infections as well as previously undiagnosable tissue infections. Low- level haemolymph and organ Hematodinium sp. infections have been found in apparently uninfected lobsters at all times of year, including late summer and autumn, when the parasite was previously thought to be absent from host lobsters. Currently, IFAT is routinely used for laboratory studies and the calibration of field diagnostic techniques.

Monoclonal Antibodies Raised against Coccidia and Malarial Parasites Recognize Antigenic Epitopes Found in Lankesterellid and Adeleorin Parasites

Three murine monoclonal antibodies (MAbs) raised against poultry coccidia or murine malarial parasites were tested for cross-reactivity with 2 sporozoan parasites with different life histories and hosts: Lankesterella minima (Eimeriorina), an intraerythrocytic parasite of frogs that is transmitted by leeches; and Heptazoon catesbianae (Adeleinorina) that infects the red blood cells of frogs and is transmitted by mosquitoes. MAb 1209 recognized both refractile bodies of sporozoites of L. minima, using the indirect fluorescent antibody (IFA) technique and immunoelectron microscopy, and recognized antigens with relative rates of migration (M(r)) of 17, 23, 26, 43, and 48 kDa on a chemiluminescent western blot of L. minima sporozoite antigens. MAbs C(3)4F1 and E12 demonstrated spotty cytoplasmic staining and labeling of the anterior pellicle of L. minima sporozoites, respectively. Gamonts of H. catesbianae labeled with only MAb E12, using IFA. These gamonts exhibited staining similar to that observed with the L. minima sporozoites. The presence of the cross-reactive epitopes recognized by these MAbs in the same conserved locations suggests that these antigens are homologous.

Serologic evidence of respiratory and rickettsial infections among Somali refugees.

Somali refugees living in a camp located in Djibouti were studied in October 1991 and May 1992. The refugees had been living at the camp for about two years. The median age of volunteers was 25 years, of whom 69% were female. Paired sera obtained seven months apart were evaluated by complement fixation, microimmunofluorescence, indirect fluorescent antibody, streptococcal antibody, and enzyme-linked immunosorbent assay techniques for evidence of pathogen infection. Fifty-two percent, 31.3%, 8.0%, 5.9%, and 25.4% of the volunteers had serologic evidence for pre-enrollment infection with Chlamydia pneumoniae, Mycoplasma pneumoniae, Rickettsia typhi, R. conorii, and Coxiella burnetti, respectively. Similarly, 43.5%, 5.2%, 6.1%, 10.7%, 15.8%, and 11.9% of the volunteers studied had serologic evidence for new infection with Streptococcus pyogenes, C. pneumoniae, M. pneumoniae, R. typhi, R. conorii, and Cox. burnetii, respectively. These data suggest that the studied pathogens may be endemic in displaced populations living in the Horn of Africa.

Pattern of polar extension of the cell wall in the fission yeast Schizosaccharomyces pombe

The indirect fluorescent-antibody technique has been used to establish the pattern of polar extension in the fission yeast Schizosaccharomyces pombe 160 over a complete cell cycle in liquid medium, thus avoiding the possibility of perturbations being introduced by growth on an agar pad, which is the technique used in most other investigations. Nearly all of the cells (about 98%) showed more growth at the old end than at the new end that was formed by cleavage of the septum at the previous division. Importantly, there was no evidence of the abnormal growth pattern (i.e., the significant contribution of new ends to extension) in cells of S. pombe growing on agar pads reported by Miyata et al. (H. Miyata, M. Miyata, and B.F. Johnson. 1986. Can. J. Microbiol. 32: 528-530 and 1990. Can. J. Microbiol. 36: 390-394). In addition, extension over the cycle was inversely related to birth length (cells shorter than the mean at birth tended to produce daughter cells longer than themselves and vice versa), there was a small but significant asymmetry in the position of the septum, and the time of initiation of extension at the new end was estimated at about 0.24 of the cycle.

Immunological detection of the fungal parasite, Pythium sp.; the causative organism of red rot disease in Porphyra yezoensis

The red rot disease of Porphyra yezoensis Ueda (Rhodophyta) is caused by a parasitic fungus, Pythium sp. To facilitate the detection of this pathogen in infected thalli of P. yezoensis, polyclonal and monoclonal antibodies were prepared. Antibodies were raised against antigen prepared from an isolate of fungal hyphae obtained from red-rot infected thallus of P. yezoensis from Aichi Prefecture. Polyclonal antibody was obtained from the antisera of immunized rabbits. Monoclonal antibody was obtained from the culture supernatant of a hybridoma which had been established by cell fusion between a myeloma cell line and spleen cells of immunized mice. Hyphae were detected by means of indirect fluorescent antibody technique. Titers of polyclonal antibodies obtained were too low to recognize fungal hyphae that had penetrated the thalli of P. yezoensis; however, monoclonal antibody was useful for the detection of fungi that had penetrated algal thalli. The monoclonal antibody was specific for the Pythium sp. from red-rot infected thalli of P. yezoensis from Saga (western Japan) and from Aichi Prefectures (central Japan), but was ineffective for infections from Miyagi Prefecture (northern Japan). It is evident, therefore, that Pythium sp. can give rise to immunologically distinct groups of red rot disease. Based on chemical and enzymatic treatments, the antigenic determinant appeared to localize on the sugar chains of glycoconjugates or the polysaccharides of the hyphal cell wall.

Development of monoclonal antibodies to iron-regulated outer membrane proteins and lipopolysaccharide of Aeromonas salmonicida

DAO Diseases of Aquatic Organisms Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials DAO 21:201-208 (1995) - doi:10.3354/dao021201 Development of monoclonal antibodies to iron-regulated outer membrane proteins and lipopolysaccharide of Aeromonas salmonicida Neelam B, Thompson KD, Price NC, Tatner MF, Adams A, Ellis AE, Stevens L Two monoclonal antibodies (MAbs) were obtained against the iron-regulated outer membrane proteins (IROMPs) and 4 MAbs were obtained against the lipopolysaccharide (LPS) of Aeromonas salmonicida, as demonstrated by immunoblotting. MAbs directed against LPS reacted with whole bacteria, both in the enzyme linked immunosorbent assay (ELISA) and by the indirect fluorescent antibody technique (IFAT). Of the 2 MAbs directed against the IROMPs, one did not react with whole bacteria either by IFAT or by ELISA, whereas the other reacted weakly with whole bacteria in both the tests. Some potential applications of the MAbs, including screening of bacterial isolates, are also discussed. Aeromonas salmonicida . Iron-regulated outer membrane proteins . Lipopolysaccharide . Monoclonal antibodies Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in DAO Vol. 21, No. 3. Publication date: March 30, 1995 Print ISSN:0177-5103; Online ISSN:1616-1580 Copyright © 1995 Inter-Research.

An outbreak of Pontiac fever due to Legionella pneumophila serogroup 7. II. Epidemiological aspects

From August 20 to 22, 1994, an outbreak of acute febrile illness occurred in a Training Center building of a company in Shibuya-ku, Tokyo. All 43 trainees attended in two groups and 2 Center staffs were attacked. Illness was self- limiting, generally lasting three days. Though strains of legionellae, isolated from the water of the cooling tower located at the top of the building, were identified as Legionella pneumophila by microplate DNA-DNA hybridization, they failed to agglutinate with antisera against L. pneumophila serogroups 1 through 6. Two strains were sent to the Centers for Disease Control, Atlanta, Georgia, USA, and determined as serogroup 7 of the species. Since the clinical courses agreed with the definition of Pontiac fever by Glick et al. and seroconversion in a patient against the cooling tower strain (EY3698)from 1:16 to 1:256 was determined by indirect fluorescent antibody technique, the epidemic of acute febrile illness was concluded as an outbreak of Pontiac fever due to L. pneumophila serogroup 7. The cooling tower was a cylindrical open style, with volumetric flow rate of 130 liter/min, and was used for air- conditioning exclusively to the third floor of the building. The building equipped no air-inlet, and indoor-air of the training room exchanged at every break time through windows of 168 cm in height and 72 cm in width. The cooling tower was not operated for five days before the Group A trainees checked in the Center on 18 August followed by Group B trainees on 19 August. It was speculated that high atmospheric temperature and stagnation of cooling water during this period would lead L. pneumophila to overly multiply, which could be a source of infection by flowing in through opened windows to the training rooms.

Possibility of Lyme disease as an occupational disease-- seroepidemiological study of regional residents and forestry workers.

We conducted a study to investigate the state of human infection by the Lyme disease pathogen by determining the prevalence of positivity for serum anti-body to Borrelia burgdorferi among regional residents and forestry workers in Nagano Prefecture. Assay of the antibody titer was performed by indirect fluorescent antibody technique (IFA) using Borrelia burgdorferi strain B-31 as the antigen. The study was based on the results of an earlier investigation to test our prediction of high infection rates for adult ticks of I.persulcatus and I.ovatus in Nagano Prefecture. The study results showed infection to regional residents and forestry workers by the Lyme disease pathogen and a significantly higher positive rate in forestry workers than in regional residents. These findings indicate the need to consider this disorder as an occupational disease of forestry workers.

Survival of the fish pathogen Aeromonas salmonicida in the marine environment

Abstract. The fate of the fish pathogen Aeromonas salmonicida in the marine environment was examined by culture, epifluorcscence microscopy, the indirect fluorescent antibody technique (1FAT), direct viable counts (= Kogure technique) and the assessment of respiratory activity by the reduction of tetrazolium compounds to coloured formazans. In laboratory‐ based systems containing sediment, water, algae and invertebrates, cells of A. salmonicida were observed microscopically after plate counts on tryptone soya agar declined to zero. Survival was maximal at a salinity of 25%o, and in wood and sediment, notably mud, rather than the water column.

An indirect fluorescent antibody technique for detection of anti-Dermatophilus congolensis antibodies in sheep.

An indirect fluorescent antibody (IFA) technique has been developed for detection of anti-Dermatophilus antibodies in sheep. Sera from 25 bacteriologically confirmed clinically affected sheep and from 10 negative non affected lambs were used. Whole cell antigen from brain heart infusion cultures of D. congolensis was used and all sera were tested in the same way for cross-reactivity against antigens obtained from cultures of Actinomyces viscosus, Micrococcus luteus, Nocardia asteroides, and Corynebacterium pseudotuberculosis. Sera from Dermatophilus-infected sheep gave positive results with D. congolensis antigen and negative results with the antigens from other bacteria. The whole cell antigens employed were simple to prepare and easy to recognise by microscopy. Cross-reactivity was further tested using the D. congolensis culture whole cell antigen and 3 sera from sheep with bacteriologically confirmed natural infections due to Corynebacterium pseudotuberculosis, Actinomyces pyogenes and Nocardia asteroides. None of these sera showed positive reactions. The authors recommend this technique for serological surveys and research on dermatophilosis.

Chlamydia trachomatis antibody testing is more accurate than hysterosalpingography in predicting tubal factor infertility

To compare the likelihood of abnormal Chlamydia trachomatis antibody test results with that of abnormal hysterosalpingography (HSG) test results in patients with tubal factor infertility. Anti-C. trachomatis immunoglobulin G antibodies were determined prospectively in 211 consecutive infertility patients by means of an indirect fluorescent antibody technique. The results were compared with the results of HSG with respect to their predictive value of tubal factor infertility. Likelihood ratio calculations were used. University hospital-based, tertiary care infertility clinic. A series of 211 consecutive infertility patients. C. trachomatis antibody testing, HSG, laparoscopy. Likelihood ratios for abnormal C. trachomatis antibody test results and abnormal HSG results in infertility patients, as assessed by laparoscopy. The positive likelihood ratio for C. trachomatis antibody testing was 9.1, indicating a patient with tubal factor infertility to be 9.1 times more likely to have abnormal serology results than a patient without tubal factor infertility. This was superior to HSG, which had a positive likelihood ratio of 2.6 in our study and of 1.6 to 6.1 in the literature. The odds ratio of C. trachomatis antibody testing was 31.5 in our study. Its 90% confidence interval (8.3 to 138.5) did not overlap that of HSG as calculated from a meta-analysis of literature reports (5.3 to 7.9). C. trachomatis antibody testing is simple, inexpensive, and causes minimal inconvenience to the patient. It is more likely than HSG to be abnormal in patients with tubal factor infertility. C. trachomatis antibody testing deserves to become an integral component of the initial fertility work-up.

Geographical variation of seropositivity to Ehrlichia risticii (equine monocytic ehrlichiosis) of horses in New York state.

A total of 2,579 serum samples from horses in New York state during 1985-1986 were examined for seropositivity to Ehrlichia risticii using the indirect fluorescent antibody technique. Cluster analysis statistical technique was used to group counties according to their estimated EME-disease rate (seropositive proportion of sampled horses). Counties were clustered into 4 groups of different EME-disease rates, representing high (86% seropositive), medium (66% seropositive), medium-low (47% seropositive) and low (6% seropositive) risk regions. The logistic regression statistical technique was used to examine whether the geographic variation of E. risticii seropositivity is due to other EME-risk factors. Risk factors known to be associated with EME (age, breed, sex, month of sample submission) have not contributed to the apparent geographical variation of the disease in New York state. This lack of an association between equine host factors or temporal factors with the geographical variation of EME suggests that conditions necessary for establishment of EME endemic foci involves other factors. The county equine population density and presence of a major equine racetrack were not associated with the geographical variation of the disease in New York state. The high risk regions for EME were located predominately along river valleys, but an explanation of why EME activity varies geographically remains speculative.

Observations on the Occurrence of Bacterial Gill Disease and Amoeba Gill Infestation in Rainbow Trout Cultured in a Water Recirculation System

Abstract Spontaneous outbreaks of bacterial gill disease (BGD) occurred in 17-44-g rainbow trout Oncorhynchus mykiss after they were placed in a water recirculation system. In each of five groups stocked from September 1991 through July 1992, BGD occurred within 6–8 d after stocking. In each instance, BGD was followed by a secondary amoeba infestation. The spontaneous BGD outbreaks did not occur among previously stocked groups that had recovered from earlier BGD disease outbreaks. Examination of gill tissue by Gram stain and indirect fluorescent antibody technique showed increased numbers of filamentous bacteria associated with BGD after the rainbow trout were stocked into the system. Bacterial numbers decreased after a 1-h treatment with chloramine-T at concentrations of 9–15 mg/L but increased within 2 d after treatment. Although the chloramine-T treatments controlled mortality related to BGD, the amoeba infestation persisted. Histological examination of gills showed some focal hyperplasia before the ra...

Isolation, initial characterization and serological studies of glycolipid antigens from Babesia bovis-infected erythrocytes

Serological analysis of Babesia bovis-derived glycolipids by ELISA and the indirect fluorescent antibody technique demonstrated the existence of their antigenic and immunogenic activities not only in B. bovis but also in B. bigemina infections. This indicates that serological cross-reactivity of B. bovis and B. bigemina relates to glycolipids. The negative ELISA reaction obtained with Anaplasma marginale antisera suggested the specificity of the reaction to the genus Babesia. Fractionation of these glycolipids by Florisil Sep-Pak column chromatography with subsequent HPTLC immunostaining and Orcinol staining suggested the presence of carbohydrate antigenic determinants in B. bovis glycolipids.

Humoral immune response of carp Cyprinus carpio to Myxobolus artus (Myxozoa: Myxobolidae) infection

The circulating antibody which reacted with sonicated spores of Myxobolus artus was detected in some naturally infected carp. However, some other fish had no detectable sign of infection, though they had the antibody. When carp were injected either with intact or sonicated spores, the antibody was not produced, while fish injected either with developing stages (presporogonic and sporoblast stages) of the parasite or sonicated spores with bovine serum albumin elicited the antibody production. The results of the injection experiments suggest that (1) developing stages have antigenicity to carp, and (2) spores have lost the antigenicity; sonicated spores are haptens, with which the antibody can react. In an indirect fluorescent antibody technique, sera positive for the antigen reacted with developing stages of the parasite, but not with the spore.The mechanism of the host immune response against M. artus is discussed in relation to a previous observation that the parasite sometimes underwent abnormal development, in which host encapsulation was imperfect or even lacking, probably leading to degeneration of pseudocysts before the completion of spore formation. It is plausible that the antibody was produced when pseudocysts which showed abnormal growth ruptured during their developing stages, resulting in exposure of the young parasite to the host immune system.

Sero-Prevalence ofToxoplasmainfection in human and dog population in Shiraz, Iran

Abstract Shad-Del, F., Sarvestani, R. G. and Milani, M. S. 1993. Sero-prevalence of Toxoplasma infection in human and dog population in Shiraz, Iran. J. Appl. Anim. Res., 3: 83–89. Sero-prevalence of Toxoplasma gondii infection in human beings by indirect fluorescent antibody technique and dogs by Sabin-Feldman dye test, of Shiraz and its suburbs was studied Out of 225 human serum samples examined, 21.3% were found positive showing the serum titers 1:64 to 1:1024. Persons with close contacts with animals i.e., farmers, butchers and cat owners showed maximum infection. Agewise, those in the age group of 18–27 years showed higher infection (35.7%). Sex differences were not significant. Out of 100 dogs examined, 31(31.0%) were positive for T. gondii. Of these 41.8% strary and 9.1% pet dogs were positive. The dogs above 3years of age showed highest (57.14%) injection rate and like human beings, sex of dogs did not affect infection rate. The high infection rate in stray dogs might pose serious public health pr...

モノクローナル抗体を用いた蛍光抗体法の鶏伝染性気管支炎診断への応用

Indirect fluorescent antibody (IFA) technique using a monoclonal antibody (M-Ab) specific to nucleocapsid protein of avian infectious bronchitis (IB) virus was applied to diagnosis of IB of chickens. Since affected chickens of 69 days of age showed respiratory signs and severe diarrhea, they were suspected of IB clinically. The IB virus specific antigens were clearly demonstrated in thin sections of the swollen kidneys collected from chickens. The IB virus was isolated from the kidneys and virus neutralizing antibody titers against the isolates rose significantly in convalescent sera of chickens. From these results, the disease was diagnosed as IB, and the IFA technique using M-Ab was judged to be usuful for diagnosis of IB.

Immunological detection of cytoskeletal proteins in the exoerythrocytic stages of malaria by fluorescence and confocal laser scanning microscopy.

Using monospecific antibodies, the presence and distribution of tubulin, actin, myosin, intermediate filaments, and lamins were examined in the exoerythrocytic liver schizont of Plasmodium berghei by conventional indirect fluorescent antibody methods and confocal laser scanning microscopy. The binding reactivity of the antibodies to parasite proteins was determined by Western blot analysis. The localisation of all antibodies in control host hepatocytes followed expected distributions in both uninfected and infected hepatocytes; by contrast, reactivity to the exoerythrocytic schizont was variable. The parasite reacted positively with selected anti-tubulin, -actin, and -myosin antibodies in both fluorescence and Western blot analysis. Anti-lamin antibodies were positive by confocal indirect fluorescent antibody labelling, but no labelling was detected with anti-intermediate filament antibody. Within the technical limits of resolution of the methods as applied to asynchronous parasite infections, not one of the antibodies reacting positively with the parasite by the indirect fluorescent antibody technique could be shown to identify unequivocally the classic architectural features associated with their respective target organelles, i.e. microtubules, stress-fibres or the nuclear envelope.

Retrospective evaluation of factors associated with the risk of seropositivity to Ehrlichia risticii in horses in New York state

SUMMARY A retrospective study was designed to determine the distribution of equine monocytic ehrlichiosis among the equine population in New York state, and to identify factors associated with risk of disease. Serum samples submitted to the diagnostic laboratory of the university during the period from January 1985 through December 1986 were examined for antibodies to Ehrlichia risticii, using the indirect fluorescent antibody technique. Factors evaluated included geographic origin and date of submission of the sample, and age, breed, and sex of the horse. Logistic regression analysis was used to identify which factors were significantly associated with the risk of seropositivity to E risticii, while simultaneously controlling for other factors. Of the 2,579 tested samples, 1,950 (76%) had positive results. Factors significantly associated with risk of seropositivity to E risticii were: breed of the horse (Thoroughbreds were 3 times more likely to have been exposed to E risticii, compared with non-Standardbred, non-Thoroughbred breeds); sex (female horses were 2.7 times more likely to have been exposed, compared with male horses); age of the horse (the risk of being exposed to E risticii increased with age, peaked at around 12 years, and decreased thereafter); and month of submission (horses tested during November and December had the highest odds of being seropositive [odds ratio = 2.1], and horses tested during March through April were least likely to be seropositive [odds ratio = 0.5], compared with horses tested during January and February).

Demonstration of rabies virus-specific antibody in the sera of free-ranging Iowa raccoons (Procyon lotor).

Between 1984 and 1988, a study was conducted to evaluate the frequency of rabies virus neutralizing antibodies in raccoons (Procyon lotor) in two counties in Iowa. Nine hundred eighty five raccoons were trapped and tagged in Guthrie and Cerro Gordo counties during the spring, summer and fall of each year. Sex, age and weight were recorded for each animal and a blood sample was collected. Serum samples were tested for the presence of serum neutralizing antibodies (SNA) by the rapid fluorescent focus inhibition test (RFFIT), mouse serum neutralization test (MSN), and an indirect fluorescent antibody (IFA) technique for detecting immunoglobulin G. Fifty-one raccoons (5%) were found to have SNA by the RFFIT. Thirty-six serum samples (24 with RFFIT antibody titer greater than 3.0, and 12 less than 3.0) were also tested by the MSN, with results correlating well with the RFFIT results (r = 0.86, P less than 0.01, Kappa = 0.93). In 35 raccoons with SNA by the RFFIT, six individuals had immunoglobulin G binding activity by the IFA test. These results provided serologic evidence of exposure of raccoons to rabies virus in an area free of enzootic raccoon rabies.