Fluorescent Antibody Procedure Research Articles

Constructed wetlands – Are they safe in reducing protozoan parasites?

Constructed wetlands have been promoted in recent literature for use in rural communities in developed as well as in developing countries as an appropriate technology to be handled with low operational maintenance costs. Within a joint project supported by BMBF (Project No O2WA0107 and No 02WA0108) research was done concerning the sanitation effect of constructed wetlands on wastewater effluents. This article will focus on the detection and the removal of cysts of Cryptosporidium parvum and Giarda lamblia, those being the most frequently identified pathogenic protozoan parasites worldwide with increasing medical and economical consequences. Two plants, one installed in 2000 as a pilot plant at Langenreichenbach near Leipzig (Saxony, Germany), the other one in routine operation since 1993 in a training center at the town of Belzig (Brandenburg, Germany) were tested for three years. Detection methods from the US EPA (ICR Protozoan Method for Detecting Giardia Cysts and Cryptosporidium Oocysts in Water by a Fluorescent Antibody Procedure (EPA/814-B-95-003;US EPA 1995) were employed in order to assess protozoal and bacterial reduction in the wastewater passing through different combinations of filter beds and fillings. Removal of cysts of Cryptosporidium and Giardia spp. turned out to be a 2 log reduction in all plants. The most effective structural element was a two-stage combination of filter beds leading to the highest removal efficiency both for the protozoan and the bacterial indicator organisms. Also, washed sand (0-2mm grain size) in the filter bed proved to be most effective filter material; the planted reed (phragmites spp.) or willow (salix spp.), however, turned out to be of minor importance for the filtering activity.

Infection and Seroconversion of Susceptible Animals Introduced into a Babesiosis Endemic Area

The study aimed to determine the incubation period of Babesia sp. infection in naive cattle and to monitor the serological response once exposed to natural Boophilus microplus (Rhipicephalus microplus)-infested paddocks. The study was carried out on a farm located in Veracruz, Mexico. Five groups of five steers were relocated every 3 months from a tick-free area to a tick-infested paddock. Animals were introduced in October, January, April, July, and October. Blood samples were taken daily until day 21 to determine packed cell volume (PCV), percentage of parasitized erythrocytes (PPE), and antibody titers to Babesia bigemina and B. bovis by the indirect fluorescent antibody procedure. Detection of Babesia in blood was also performed by species-specific PCR. The estimated incubation period was 6-14 days post introduction to paddocks (PIP), with fever (41 degrees C) for at least 3 days. PCV decreased by >25% and Babesia parasites were observed during the clinical phase of the disease. The highest individual PPEs (0.44% and 0.22% for B. bovis and B. bigemina, respectively) were observed from animals introduced in October. The four other groups showed a mean PPE ranging from 0.002-0.146% at day 14 PIP. All animals were detected as PCR positive between 8-14 days PIP. The highest antibody titers were 1:3328. The environmental conditions were favorable throughout the year for tick reproduction as the farm showed enzootic stability and hyperendemic conditions for bovine babesiosis. In this type of farm, strategic tick control could be accompanied by babesiosis vaccination, particularly for cattle relocated from tick-free areas.

Triple-Label Fluorescent Antibody Staining of Imaginal Discs in Drosophila

INTRODUCTIONThis protocol describes a triple-label, fluorescent-antibody staining procedure that uses three different primary antisera generated in three different species. Biotin and rhodamine-streptavidin are used for the most critical of these antibodies, as they have the best signal-to-noise ratio. Fluorescein is used to visualize the second antibody, while Cy5 is used for the third. Cy5 is only poorly visible using a standard fluorescence microscope, but is used by the third channel on many laser-scanning confocal microscopes. Obviously, other fluorescent tags, as well as double and single fluorescent stains, are possible. Although these methods are useful for visualizing genetic mosaic clones in wandering larval stage imaginal discs and pupal tissues from Drosophila, the immunohistological techniques are generally applicable to the description of protein expression.

Antinuclear antibody reduces the pregnancy rate in the first IVF-ET treatment cycle but not the cumulative pregnancy rate without specific medication.

It has been shown that the presence of antinuclear antibody (ANA) might reduce pregnancy rates after in vitro fertilization-embryo transfer (IVF-ET). However, the mechanism of implantation failure by ANA has not yet been clarified. This study was performed to investigate the impact of ANA on pregnancy rates after IVF-ET, and the necessity of specific medication for infertile women who have ANA in their sera. A total of 108 infertile women were treated by IVF-ET or intracytoplasmic sperm injection (ICSI)-ET. ANA was examined by an indirect fluorescent antibody procedure. Data from women under 40 years old were analyzed retrospectively. The implantation rates per embryo transferred in the first treatment cycles were 14.8% (eight of 54) and 32.4% (33 of 102), in women with and without ANA, respectively. There was a significant difference in the implantation rates between the two groups (P < 0.05). The pregnancy rates per ET in the first treatment cycles were 28% (seven of 25) and 54.2% (26 of 48), respectively. There was also a significant difference in the pregnancy rates between the two groups (P < 0.05). Afterwards, treatments with IVF-ET or ICSI-ET were repeatedly performed for unsuccessful patients, without any specific medication for ANA. The average ET cycles were 1.80 +/- 1.13 and 1.27 +/- 0.54, in women with and without ANA, respectively. The cumulative pregnancy rates per patient were 68% (17 of 25) and 55.6% (35 of 63), respectively. There was no significant difference in the overall pregnancy rates between the two groups. These findings suggest that ANA might have an impact on implantation failure in women treated by IVF-ET or ICSI-ET. ANA reduced the pregnancy rates in the first IVF-ET or ICSI-ET cycles but not the cumulative pregnancy rates without medication. This indicates that the mechanisms of implantation failure by ANA could be solved, and effective and safe medication should be developed for better implantation rates, especially in the first treatment cycle.

Serologic survey for canine coronavirus in wolves from Alaska.

Wolves (Canis lupus) were captured in three areas of Interior Alaska (USA). Four hundred twenty-five sera were tested for evidence of exposure to canine coronavirus by means of an indirect fluorescent antibody procedure. Serum antibody prevalence averaged 70% (167/240) during the spring collection period and 25% (46/185) during the autumn collection period. Prevalence was 0% (0/42) in the autumn pup cohort (age 4-5 mo), and 60% (58/97) in the spring pup cohort (age 9-10 mo). Prevalence was lowest in the Eastern Interior study area. A statistical model indicates that prevalence increased slightly each year in all three study areas. These results indicate that transmission occurs primarily during the winter months, antibody decay is quite rapid, and reexposure during the summer is rare.

Evaluation of two concentration methods for detecting Giardia and Cryptosporidium in water

The cartridge filtration method and membrane filtration method based on the fluorescent antibody procedure were evaluated for their recovery efficiencies and detection limits of Giardia and Cryptosporidium. We assessed the performances of the two concentration methods for water samples collected from Taiwan water treatment plants. The membrane filtration method was characterized by higher recovery rate and detection limit comparing with the cartridge filtration method. The occurrences of both parasites, and the relationships of parasite concentrations with indicator microorganisms show inconsistency between the two methods. It was discovered that water turbidity reduced the recovery efficiencies, and raised the detection limits for both parasites regardless of the method used.

Isolation ofAcholeplasma laidlawiifrom Centrarchids in a Central Florida Lake

Abstract In 1991, the poor physical condition of largemouth bass Micropterus salmoides from Lake Harris, Florida, was associated with the decline of the lake's fishery. The swim bladders of emaciated bass had mild inflammation and ecchymotic hemorrhages. A mycoplasma-like organism isolated from swim bladders was initially believed to be the causative agent. The organism was later identified as Acholeplasma laidlawii by using a fluorescent antibody procedure and was demonstrated to be nonpathogenic. Parenteral injection of the organism into healthy largemouth bass fingerlings produced no signs of disease or difference in growth rate compared with control fish during a 16-month period. Field studies resulted in isolation of A. laidlawii from black crappies Pomoxis nigromaculatus, bluegills Lepomis macrochirus, and redear sunfish L. microlophus, but not from noncentrarchids in Lake Harris or from any fish species in a control fishery (Lake Holly, Florida). The absence of organisms in all emaciated bass, our ...

Identification of Mycoplasma Isolates by ELISA

An antigen-based enzyme-linked immunosorbent assay (ELISA) was developed for the rapid identification of Mycoplasma gallisepticum and Mycoplasma synoviae. Two trials were conducted to compare the ELISA with the conventional fluorescent antibody technique. In trial 1, broilers were inoculated with M. gallisepticum F, M. gallisepticum S6, and M. synoviae. In trial 2, hens were inoculated with M. gallisepticum F, M. synoviae, and a combination of the two. Swab isolates were obtained from the choanal cleft and cultures were grown in broth media with a pH indicator. The organisms were bound to 96-well plates for the ELISA or cultured on agar for the fluorescent antibody test. Monoclonal antibodies directed to M. gallisepticum F, M. gallisepticum S6, and M. synoviae were reacted with the ELISA aliquots. The ELISA test identified the mycoplasmas in these samples and was completed within 8 hr but did not identify as many isolates as the fluorescent antibody procedure. The principal advantage of this ELISA is the ability to identify the strain of mycoplasma.

Detection of microsporidial spores in fecal specimens from patients diagnosed with cryptosporidiosis.

Patients infected with Cryptosporidium parvum may have concurrent infections with microsporidia. Two modified trichrome stains and a polyclonal indirect fluorescent-antibody procedure were used for the detection of microsporidia; the Merifluor Cryptosporidium-Giardia monoclonal direct immunofluorescence detection kit was used for the detection of C. parvum. Formalinized stool specimens from 60 immunocompromised patients strongly suspected of having or previously diagnosed with cryptosporidiosis or microsporidiosis were examined. All patients were positive for one or both parasites, 18 (30%) with C. parvum only, 25 (42%) with microsporidia only, and 17 (28%) with both C. parvum and microsporidia. These findings emphasize the importance of considering both organisms as potential causative agents of diarrhea in compromised patients.

Characterization of Monoclonal Antibodies and Corresponding Epitopes of Vibrio vulnificus.

Monoclonal antibody (Mab) P3C3 raised against V. vulnificus reacted with nine of thirteen V. vulnificus strains. In comparison, Mab P3F10 reacted with only three V. vulnificus strains. No cross-reactions were observed among fourteen non-V. vulnificus strains. P3C3 and P3F10 isotypes were characterized as IgG1k. When examined via an indirect fluorescent antibody procedure, epitopes that were recognized by the P3C3 and P3F10 Mabs were found to be on the cell surface. Results of an additivity test suggest that these two epitopes are at different positions on the cell surface. The number of P3F10 epitopes on the TG617 strain was larger than the number of P3C3 epitopes. In addition, the former. was probably physically more accessible than the latter. The resultant additive index suggested that the binding of Mab P3F10 to its corresponding epitopes interferes in three dimensions with the binding of Mab P3C3 to its epitopes; however, the reverse is not true. Epitope P3C3 was shown to be a protein with a molecular weight of 40 kDa, but epitope P3F10 was shown to be a glycopeptide with a molecular weight ranging from 10 to 21 kDa.

Measles virus replication in lungs of hispid cotton rats after intranasal inoculation.

Hispid cotton rats were inoculated intranasally with either measles virus (MV) Edmonston, a multipassaged, tissue culture-adapted strain of MV, or with one of three clinical MV isolates that had limited passages (three to five times) in tissue culture cells. MV Edmonston was recovered from the lungs of every (n = 37) hispid cotton rat inoculated with this virus for at least 7 days after virus inoculation. Peak pulmonary titers occurred on Day +4 (3.3-4.4 log10/g lung). Scattered areas of inflammation were observed interstitially in lung sections from infected animals stained with hematoxylin and eosin, and a similar pattern of diffuse fluorescence was seen in cryostat sections stained with an indirect fluorescent antibody procedure specific for virus antigens. Fluorescent antibody and virus isolation studies on lung lavage cells both suggested that lung leukocytes were a primary target of the virus. In contrast to these findings, virus was isolated only sporadically from hispid cotton rats inoculated with any of the clinical measles virus isolates. Despite the restricted growth of MV in these animals, cotton rats may be useful for studying certain aspects of measles virus pathogenesis and for screening potential antiviral compounds in vivo.

Rapid isolation of Escherichia coli O157:H7 from enrichment cultures of foods using an immunomagnetic separation method

A method employing immunomagnetic beads was developed for the isolation of Escherichia coli O157:H7. Magnetic beads precoated with sheep anti-rabbit IgG were coated with rabbit antiserum against E. coli O157. The level of bacteria, amount of beads, amount of antibody coating the beads and incubation time were varied to determine their effects on recovery of the organism. The sensitivity of the test for recovery of E. coli O157:H7 was 1 × 101 cfu ml−1. The organism was selectively recovered from 24 h enrichment cultures of meat products in which the initial inoculum level was 1 bacterium g−1. Sorbitol negative colonies were selected from Sorbitol Mac-Conkey agar and were positively identified as E. coli using a direct fluorescent antibody procedure. The results indicate that immunomagnetic separation is a convenient and rapid method for the isolation of E. coli O157:H7 from foods.

A comparison of the sensitivity and specificity of sialodacryoadenitis virus, Parker's rat coronavirus, and mouse hepatitis virus-infected cells as a source of antigen for the detection of antibody to rat coronaviruses

SummarySialodacryoadenitis virus (SDAV) and Parker's rat coronavirus (PRC) are two recognized viral strains which cause spontaneous disease in the laboratory rat. Currently there is no recognized practical procedure which will accurately differentiate infections with these strains. Using SDAV- and PRC-infected L-2 cells as the source of antigen, and sera from rats collected post inoculation with either of these viral strains, the indirect fluorescent antibody (IFA) procedure was used to determine whether antibody titers could be used to differentiate infections from the homologous and heterologous virus. There was no detectable difference in the sensitivity or specificity of these systems in detecting antibody to the homologous or heterologous virus. Thus there was no evidence that SDAV- and PRC-infected cells would serve to differentiate antibody to the homologous virus using the IFA technique. In addition, antibody titers were similar when mouse hepatitis virus (MHV)-infected cells were used as the source of antigen for the IFA technique. However, using MHV or SDAV-infected cells as the source of antigen, there was a significant difference in antibody titers to the homologous virus detected using the immunoenzyme technique.

Diagnosis of Bovine Viral Diarrhea Virus Infection using Monoclonal Antibodies

The monoclonal antibody (MAb) D89 against bovine viral diarrhea virus (BVDV) was used in conjunction with fluorescein-conjugated anti-mouse immunoglobulin in an indirect fluorescent antibody (IFA) procedure on frozen tissue sections and cell culture. During the 2-year study, BVDV was isolated from specimens submitted in 460 cases. The D89 Mab detected all but 2 BVDV isolates, both cytopathic. In 316 of the cases in which BVD virus was detected by IFA, specimens were inoculated on bovine turbinate cells and examined for BVDV antigens at 3-5, 10, and 20 days postinoculation. The BVDV was detected in 238/316 cases (75%) after 3-5 days incubation. The remainder were not detected until 10 or 20 days postinoculation. Virus isolation was enhanced in the early test if plates were centrifuged at the time of inoculation. Results suggest that D89 monoclonal antibody is a suitable diagnostic reagent for the detection of BVDV isolated from diagnostic specimens. The D89 MAb can be used for the detection of BVDV in both cell culture and tissues. Combination of D89 with another BVDV MAb (C17) did not improve the ability to detect BVDV in tissues compared to using D89 only, and the combined Mab's resulted in an increase in nonspecific fluorescence when used on tissues. Although pooling of different BVDV monoclonal antibodies may be necessary to detect all strains of BVDV in cell culture, pooling should be used with caution on tissues. Early detection of BVDV in cell culture by this IFA procedure permits faster confirmation of BVDV diagnosis when compared to the usual routine testing for noncytopathic BVDV at termination of first passage in cell culture.

Diagnostic Tests for Lyme Disease-Reply

<h3>In Reply.—</h3> Our article attempted to assess the interlaboratory and intralaboratory reliability of serological tests for Lyme disease. The two most commonly performed methods for these tests are indirect fluorescent antibody procedures and enzyme-linked immunosorbent assays.¹Although one of these test methods reports titers from serial dilutions (eg, 1:128) and the other reports an optical density, the clinician usually is most interested in whether the laboratory is reporting the sample as "positive" or "negative." Thus, we disagree with Dr Gambino's contention that we "mix apples and oranges" in our article, as these two methods both report the presence (positive) or absence (negative) of antibody to<i>Borrelia burgdorferi</i>. We do not disagree that enzymelinked immunosorbent assays are probably a better test method; however, many laboratories, including those in state departments of health, commercial laboratories, and research laboratories, are still performing immunofluorescent antibody tests. Thus, it was important to consider this

A Comparison of Flow Cytometry and Other Techniques Used for Diagnosing Mycoplasma Infections

Mycoplasma isolates from chickens are usually identified by a fluorescent antibody (FA) technique that requires 7 to 10 days from sampling to the completion of identification. The FA procedure uses Mycoplasma-positive antibodies conjugated with fluoroisothiocyanate (FITC) to identify the colonies of the organism on agar. This paper describes a flow cytometric procedure that uses the same antibody-FITC conjugate and can be completed in 3 or 4 days. Broilers and egg-type hens inoculated with Mycoplasma gallisepticum and M. synoviae were swabbed from the choanal cleft and cultures were grown in broth. Broth aliquots were briefly incubated with polyclonal antisera to M. gallisepticum and M. synoviae conjugated with FITC, the same reagents routinely used in the FA technique. The sample was then analyzed by flow cytometry for the association of the fluorescent label and the organism. Samples from inoculated chickens incubated with the homologous antibody-FITC had greater fluorescent intensity than did those incubated with the heterologous antibody-FITC. Diagnosis by flow cytometry was consistent with that by the FA technique except that in one trial one sample was incorrectly identified by flow cytometry as positive for M. gallisepticum, although it was not inoculated with M. gallisepticum. Also, flow cytometry did not correctly identify M. synoviae organisms as readily when broilers were inoculated with both M. gallisepticum and M. synoviae.

Adherence of Streptococcus agalactiae to synchronously growing human cell monolayers without lipoteichoic acid involvement.

Freshly isolated virulent and nonvirulent strains of Streptococcus agalactiae type III were used to study differences in coccal adherence to synchronously dividing, subconfluent human embryonic amnion and fetal lung monolayers in vitro. The adherence frequency by virulent isolates of mid-logarithmically growing cocci to amnion cells varied markedly with host cell age, being highest shortly after eucaryotic cell division. This variation was not observed with lung cell monolayers, suggesting that cyclic production or exposure of coccal receptor sites on the eucaryotic cell surface with age is not a common property of all primary human cells in vitro. However, and regardless of age, not all cells within these synchronously dividing populations bound virulent cocci, indicating that a very small segment of a population may always be unresponsive to host cell interactions with a coccal pathogen. By comparison, adherence of nonvirulent coccal isolates to amnion and lung cells remained constant and of a very low order, regardless of host cell age. Maximal adherence of virulent S. agalactiae to young host cells occurred at early and mid-logarithmic phases of growth. However, at the late stationary growth phase, adherence was reduced to almost that of nonvirulent isolates. Pretreatment of virulent S. agalactiae with anti-lipoteichoic acid (LTA) serum failed to inhibit coccal adherence to these different host cells. Heat negated adherence. Group B coccal LTA was cytotoxic for these host cells. However, pretreatment of amnion and lung cells with nontoxic levels of this amphiphile did not prevent attachment of virulent cocci. Finally, coccal pretreatment with pronase abrogated adherence to either host cell even though surface-exposed LTA was uneffected, as observed by the indirect fluorescent-antibody procedure. Likewise, no observable difference in surface LTA was detected when fresh isolates of virulent and nonvirulent coccal strains were compared by this procedure. These studies suggest that protein involvement, rather than LTA, is primarily responsible for mediating virulent S. agalactiae type III attachment to these synchronously growing, subconfluent eucaryotic monolayers in vitro.

Enumeration of Bradyrhizobium japonicum in soil subjected to high temperature: Comparison of plate count, most probable number and fluorescent antibody techniques

The survival of BradyrMzobium japonicum strains in autoclaved and non-autoclaved soil at various temperatures for up to 18 days was determined by plate count, plant infectivity-most probable number (MPN) and modified fluorescent antibody (FA) techniques. The high-temperature tolerant strains studied were BR587, NC1005 and NC1033, and the high-temperature intolerant strains studied were NCI028, 3Ilb24 and 311b123. Each strain was exposed individually in a sandy loam soil at 28, 33 or 37°C under humidity controlled conditions. Those strains reported to be tolerant to elevated temperature in pure culture decreased by <10 1.5 cells g −1 when enumerated by plate count after high-temperature exposure in autoclaved soil. Based on plate counts, the population of two of the three high-temperature intolerant strains added to autoclaved soil decreased by more than 10 3 cells g −1 soil after exposure to elevated temperatures. In non-autoclaved soil, the population of all inoculated strains decreased after exposure to elevated temperature when enumeration was by the plant infectivity-MPN technique. In autoclaved soil at 28°C, the correlation between plate count and FA enumerations was significant at the 0.05 probability level for four of the six strains ( r 2 = 0.65). After high-temperature incubation, counts based on the FA technique were not correlated with plate counts of intolerant strains or MPN enumeration of tolerant and intolerant strains ( r 2 = 0.004). Differential survival of B. japonicum strains in autoclaved soil at high temperature as enumerated by plate counts confirms the designation of temperature tolerance made by Munevar and Wollum (1981). However. MPN enumeration of bradyrhizobia in the non-autoclaved system did not follow the original temperature tolerance classifications. Enumeration by FA procedures did not show a decrease in population after exposure to high temperature as observed with the plate and MPN counts. Thus, the FA technique may not adequately indicate population dynamics in short-term ecological studies.

Reduced immune responsiveness and lymphoid depletion in mice infected with Ehrlichia risticii.

The histopathology of the thymus and spleen and the response of spleen cells to mitogenic stimuli were evaluated in Sprague-Dawley CF-1 mice infected with Ehrlichia risticii. Intraperitoneal injection of 10(4) or 10(6) E. risticii-infected U-937 cells into mice resulted in 100% morbidity and partial mortality. Thymic atrophy became significant between 1 and 2 weeks postinfection and remained for the duration of the study. The atrophy appeared associated with antecedent destruction and rarefaction of lymphocytes, resulting in the loss of corticomedullary demarcation. Splenomegaly was prominent; significantly increased weights were detected 7 days postinfection. Histopathologic examination revealed rarefaction of lymphocytes around central arteries, the presence of necrotic debris in histiocytes, and replacement of erythropoiesis by granulopoiesis in the red pulp. Marked and acute reduction of in vitro proliferative responses of spleen cells to concanavalin A (ConA) and phytohemagglutinin were observed in mice infected with 10(4) or 10(6) E. risticii-infected U-937 cells. Interleukin-2 activity in the supernatant of ConA-stimulated spleen cells was also severely reduced. Both changes were time- and dose-dependent and were not associated with decreased spleen cell viability. Neither morbidity nor mortality occurred in mice infected with 10(2) E. risticii-infected U-937 cells. Although there was temporal reduction in phytohemagglutinin-driven lymphocyte proliferation, reduction in neither ConA-driven lymphocyte proliferation nor interleukin-2 activity was observed with this dosage. All E. risticii-inoculated mice seroconverted between days 18 and 25, as detected by the indirect fluorescent-antibody procedure. The findings indicate for the first time the hypoimmune responsiveness and histopathologic changes in lymphoid organs associated with E. risticii infection.

Immunofluorescence detection of Cryptosporidium oocysts in fecal smears

An indirect fluorescent antibody (IFA) procedure was developed for the detection of Cryptosporidium sp. oocysts in human, nonhuman primate, and bovine fecal smears. The procedure, which takes about 90 min to perform, involves the use of a rabbit antiserum against Cryptosporidium oocysts isolated from dairy cattle. Cross-specificity testing of the IFA method revealed no reactivity with yeasts, various amoebae, Giardia lamblia, Chilomastix sp., or Blastocystis sp. and only very weak cross-reactivity with coccidian oocysts of other genera. IFA detection of oocysts in human and nonhuman primate fecal smears was far more sensitive than was dimethyl sulfoxide-carbolfuchsin staining. Moreover, IFA detection was comparable in sensitivity to auramine O staining with samples of high oocyst concentration and somewhat more sensitive than auramine O with samples containing relatively few oocysts. The IFA procedure may be useful in the clinical diagnosis of human and animal cryptosporidiosis and also in the detection of oocysts in environmental samples.