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PubMed1997

Efficacy of different methods for detection of low Cryptosporidium parvum oocyst numbers or antigen concentrations in stool specimens.

The detection of Cryptosporidium parvum oocysts in stool specimens by acid-fast (AF) stains or immunofluorescence assays (IFA) requires the presence of large numbers of oocysts. To determine whether new commercially available enzyme immunoassays (EIAs) are more sensitive alternatives, three EIAs, a direct IFA, and the modified cold Kinyoun AF stain were compared, particularly with respect to detection of low oocyst numbers or antigen concentrations. Thirty-one negative and 31 calf stool-enriched human stool specimens were tested. One EIA method detected only nine positive specimens, demonstrating a sensitivity significantly less (p < 0.0001) than that of the IFA, the AF stain, and the other two EIAs. No differences could be found with respect to specificity. In addition, serial dilutions of 28 patients' stool samples containing cryptosporidian oocysts were prepared and examined using two EIAs, IFA, and the AF stain. One EIA yielded significantly inferior results (p < 0.0001), whereas the other one and the two microscopic methods did not differ significantly in either part of the study. The results indicate that the new EIAs do not exhibit higher sensitivities for detection of Cryptosporidium parvum than the two routinely used microscopic methods. Thus, for most laboratories, the IFA or AF stain may still represent the preferred method for the diagnosis of cryptosporidiosis.

AIDS-Related Opportunistic Infections↗
PubMed1981

Serological prevalence of bovine babesiosis in Guyana.

The serological prevalence of bovine babesiosis in Guyana was determined utilising the indirect fluorescent antibody (IFA) and the complement fixation (CF) tests. Babesia bigemina IFA and CF antibody activity was detected in 80% and 40% respectively of the sera from native cattle while IFA and CF antibody activity to Babesia bovis was observed in 61% and 16% of the sera. The prevalence of antibody was compared between calves less than 6 months of age and mature cattle. There was little difference between the CF and IFA tests for B. bigemina in the calves but the prevalence of IFA antibody activity to B. bovis in calves and to B. bovis and B. bigemina was greater in adult cattle. Examination of sequential serum samples from imported calves revealed that CF antibody activity appeared earlier but by day 51 following exposure to natural infection the prevalence of antibody as determined by both tests was the same.

Animals↗
PubMed1994

Monoclonal antibody-based immunohistochemical demonstration of Entamoeba histolytica in liver tissues of experimentally infected hamster (Mesocricetus auratus).

Histopathological changes and the presence of Entamoeba histolytica trophozoites was sequentially followed after intrahepatic inoculation of the parasites in 42 hamsters, 35 of which received no treatment whereas the remaining seven were treated with metronidazole. The liver tissues were examined for amoebic trophozoites by a monoclonal antibody (mAb)-based immunofluorescence assay (IFA), a mAb-based immunoperoxidase (IPx) and H & E staining. The number of hamsters developing abscesses was increased with time and was highest on day 30. Cellular infiltration with inflammatory cells and glycogen depletion were observed as early as day 5, followed thereafter by more intense inflammation of portal canals, periportal fibrosis, bile duct proliferation and hepatocyte degeneration. In 7 metronidazole-treated hamsters, no obvious pathological damage was seen. In a group of seven hamsters each, both IPx and IFA were positive in 3, 3, 4, 5 and 4 hamsters and in 3, 4, 3, 3 and 5 hamsters on days 5, 10, 15, 20 and 30, respectively. In 18 control hamsters, IPx, IFA and H & E were all negative. If the result from H & E was used as a gold standard, agreement between H & E and IFA and H & E and IPx were 91.4%, and 88.6%, respectively. Sensitivity and specificity were 93.8% and 89.5%, respectively for IFA, and 93.8% and 84.2%, respectively for IPx.

Animals↗
PubMed1984

Dermal hypersensitivity reactions to imported fire ants.

A survey of suburban residents of New Orleans, La., revealed that 58% of the individuals who responded had been stung by imported fire ants (IFA) within the previous year. More than half of the patients stung had dermal reactions that were distinct from the previously reported reactions to IFA in that immediate wheal-and-flare reactions evolved into pruritic, edematous lesions that persisted about the developing pustule for 24 hr or more. Twenty-one volunteers were stung with live IFA, and the course of the reactions was observed. Nine developed persistent reactions after stings. These reactions could be reproduced by the intradermal injection of IFA--whole body extract in only four of these nine subjects. Biopsy specimens of sting reactions at 6 hr demonstrated the reactions to be "late phase reactions" characterized by dense fibrin deposits like those previously noted in dermal reactions to ragweed and insulin. Eosinophils were present in the sting-associated pustules only in individuals who developed late-phase reactions. These data demonstrate that late-phase reactions occur commonly to IFA stings and that this form of insect hypersensitivity may not always be diagnosed by skin testing with whole body extract.

Ants↗
PubMed1989

Survey of whole body-extract immunotherapy for imported fire ant- and other hymenoptera-sting allergy. Report of the Fire Ant Subcommittee of the American Academy of Allergy and Immunology.

A survey of 5300 allergists was conducted to determine the number and geographic distribution of patients receiving immunotherapy for imported fire ant (IFA) allergy in the United States and Canada. Responses were received from 1293 physicians who reported a total of 2573 patients being treated in 28 states. Most patients were from the southeast, but some patients were reported to be receiving IFA immunotherapy in areas outside the boundaries of known IFA infestation. Although IFA is a widespread health hazard in the southeast, it appears to exert an uneven impact on allergy practice in this region. Of the 1293 physicians responding to the survey, 117 (9%) reported the use of whole body extract (WBE) in the treatment of 1746 patients with winged Hymenoptera-sting allergy. Continued use of WBE immunotherapy was reported by physicians from all regions of the United States and from Canada. The largest numbers of patients receiving winged WBE immunotherapy were reported from Texas (641), Pennsylvania (246), Florida (129), and Canada (127). The results of this survey suggest a need for continuing medical education regarding (1) the magnitude of the health hazard posed by the IFA and (2) the efficacy of venom versus WBE in the treatment of winged Hymenoptera-sting allergy.

Animals↗
PubMed1986

Analysis of anti-HTLV-I antibody by strip radioimmunoassay--comparison with indirect immunofluorescence assay, enzyme-linked immunosorbent assay and membrane immunofluorescence assay.

Antibodies against human T-cell lymphotropic virus type I (HTLV-I) in the sera from 60 patients with adult T-cell leukemia and 21 carriers who were suspected of having HTLV-I infection were investigated by indirect immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA), membrane immunofluorescence assay (MIA) and strip radio immunoassay based on the western blotting technique (SRIA). The sera of 2 of the carriers who were seropositive in IFA and ELISA were negative in MIA and did not react with virus-specific proteins by SRIA. Two sera were negative in IFA and ELISA. These sera were positive in MIA and reacted with only the envelope-related glycoprotein (gp46) and not with gag-related proteins (p28, p24, p19) by SRIA. These findings suggest that the main antigens defined by IFA and ELISA are gag-related proteins and some sera which do not contain anti-HTLV-I antibodies give false-positive results because of the reaction to unknown cellular components. Also some sera may have antibodies against only envelope glycoproteins, and these sera may give false-negative results in IFA and ELISA.

Antibodies, Viral↗
PubMed1989

Rapid diagnosis of varicella-zoster virus infection with a monoclonal antibody based direct immunofluorescence technique.

Diagnosis of varicella-zoster virus (VZV) infection in immunocompromised patients is difficult because of the frequent atypical appearance. Accurate and early diagnosis is important to allow rapid commencement of antiviral chemotherapy, with consequent improvement in antiviral efficacy. A monoclonal based direct immunofluorescence antibody technique (VZV IFA) was assessed in parallel with viral culture in 56 patients with suspected VZV infection. A subgroup of 17 patients from this group with classical dermatomal herpes zoster all had positive VZV IFA tests. Only 6 patients (35%) were positive on viral culture. None of the 15 patients with proven herpes simplex virus infection had a positive VZV IFA, nor did any patient with positive VZV viral culture have a negative VZV IFA. The VZV IFA test is a rapid and sensitive technique for detecting infection with VZV.

Antibodies, Monoclonal↗
PubMed1993

Secretory proteins in a ciliate cell: identification of epitopes common to numerous polypeptides.

Secretory vesicles of the ciliate Pseudomicrothorax dubius, called trichocysts, are separated into > 40 proteins by two-dimensional gel electrophoresis. The trichocyst, composed of a shaft and four arms, is in a condensed state when docked in the cell cortex, and it elongates into an extended state during exocytosis. Monoclonal antibodies (mAbs) were raised against trichocyst proteins. Their reactivities were analysed: 1) on Western blots of extended, isolated trichocysts by immunolabeling; 2) on entire cells and extended trichocysts by indirect immunofluorescent binding assay (IFA); 3) on semi-thin sectioned cells by IFA; and 4) on ultra-thin sections of cells by immunogold labeling. mAb IV 4E5 labels major trichocyst proteins at 15-19, 22 and 24 kDa, pI 4.6-6.6. The epitope recognized by mAb IV 4E5 is common to as many as 30 proteins and suggests a family of proteins with possible sequence homology. By IFA, the shafts of extended trichocysts are labeled. The shafts of condensed trichocysts are labeled on both semi-thin sections in Lowicryl and ultrathin sections. On semi-thin Epon sections, the part of the trichocyst which is labeled is arm-like. mAb VI 2D12 labels three major trichocyst proteins at 31 kDa, pI 5.0-5.4. The arms of extended trichocysts are labeled by IFA, but are only weakly labeled on ultrathin sections. The shaft of extended trichocysts is labeled by IFA, and the shaft of condensed trichocysts is labeled on ultrathin sections.

Animals↗
PubMed1983

Antibody responses of ponies to initial and challenge infections of Strongylus vulgaris.

An indirect fluorescent antibody assay (IFA) was developed using Strongylus vulgaris third stage larvae (L3) as antigens. Observations using the IFA indicate that a species-specific antibody response to S. vulgaris L3 develops in S. vulgaris-infected ponies and that some surface L3 antigens are shared by adult worms. Sequential antibody levels against S. vulgaris were measured in strongyle-naive and in immune ponies following initial and challenge infections using the IFA and an indirect hemagglutination assay (IHA). Antibody levels measured by IFA increased faster following initial infections than did levels measured by IHA. Antibody levels appear to increase following challenge infections of immune ponies when measured with the IFA, but not with the IHA. Significant differences in antibody titers were not seen between ponies which developed colic following challenge infections and those that did not develop colic. Antibodies were not detectable in ponies unexposed to larval migrations, but which received surgical implantation of S. vulgaris adults into the cecum.

Animals↗
PubMed1996

Evaluation of dot enzyme-linked immunosorbent assay (dot-ELISA) for the serodiagnosis of canine leishmaniosis as compared with indirect immunofluorescence assay.

A dot enzyme-linked immunosorbent assay (dot-ELISA) was developed and compared with a standard indirect immunofluorescence assay (IFA) for the rapid serodiagnosis of canine leishmaniosis. The two tests were used to examine sera from Leishmania infantum-infected and control dogs. Using the dot-ELISA, 137 of 149 sera (91.9%) from infected animals gave a clearly positive reaction, whereas in the standard IFA technique 147 (98.7%) were positive at a reciprocal titer of 40 or over (titer range 40-10,240). Control sera from 75 healthy dogs, not living in endemic areas, and 11 dogs with other diseases (babesiosis, cryptococcosis, ehrlichiosis, dermatitis, and chronic hepatitis) but Leishmania-negative were used to determine the specificity of the assays. All the sera were negative with IFA (100%), whereas using the dot-ELISA only 74 sera (86%) from controls gave a negative result. In the standard IFA no cross-reactivity was noted, in the dot-ELISA a weak cross-reaction was observed with a serum sample from a dog with babesiosis. The interpretation of dot-ELISA could be easily performed by visual inspection of the nitrocellulose disks. The most remarkable feature of dot-ELISA was the high sensitivity (91.9%) and positive predictive value (92.6%), even if the test showed a specificity lower than IFA. Because of its easy performance and high sensitivity, the dot-ELISA may be a useful test to be executed in the field for the diagnosis of canine leishmaniosis.

Animals↗
PubMed1988

Evaluation of a commercially developed indirect immunofluorescence system for the detection of antihuman immunodeficiency virus antibodies.

A commercially developed IFA system (Electro Nucleonics, Inc.) was compared to EIA (Abbott Laboratories) and WB (Bio-Rad, Inc.) for detection of HIV-1 antibodies. Of 91 EIA negative sera tested, 76 (83.5%) were negative, and 15 (16.5%) were equivocal by IFA on initial testing. Of 156 EIA positive sera tested, 18 were equivocal by either IFA or WB and could not be directly compared. Of 138 EIA positive sera directly comparable, IFA and WB agreed 137 times (99.3% correlation). Based on these findings, the commercially developed IFA system appears to be most useful as a supplementary test, similar to WB, rather than as a screening test.

Enzyme-Linked Immunosorbent Assay↗
PubMed2003

Estimation of sensitivity and specificity of two diagnostics tests for bovine immunodeficiency virus using Bayesian techniques.

The validation of assays for bovine immunodeficiency virus (BIV) in cattle is hampered by the absence of a gold standard. Two tests that often are used to detect BIV are the indirect fluorescent-antibody assay (IFA) and the nested-set polymerase chain-reaction assay (PCR). IFA detects an antibody response whereas PCR detects the provirus in white blood cells. Using Bayesian techniques performed simultaneously on animals from two different dairy herds, we estimated the performance of the IFA and PCR assays and infection prevalence. Bayesian techniques also were used to derive posterior distributions of sensitivities, specificities, and prevalences. The Bayesian estimates were IFA sensitivity=60%, IFA specificity=88%, PCR sensitivity=80%, PCR specificity=86%, Herd A prevalence=20%, and Herd B prevalence=71%. Although PCR was the more sensitive assay, substantial misclassification of infection would be expected in epidemiological studies of BIV regardless of which assay was used.

Animals↗
PubMed2004

Intermediate filaments are required for C. elegans epidermal elongation.

Cytoplasmic intermediate filaments (cIFs) are thought to provide mechanical strength to vertebrate cells; however, their function in invertebrates has been largely unexplored. The Caenorhabditis elegans genome encodes multiple cIFs. The C. elegans ifb-1 locus encodes two cIF isoforms, IFB-1A and IFB-1B, that differ in their head domains. We show that both IFB-1 isoforms are expressed in epidermal cells, within which they are localized to muscle-epidermal attachment structures. Reduction in IFB-1A function by mutation or RNA interference (RNAi) causes epidermal fragility, abnormal epidermal morphogenesis, and muscle detachment, consistent with IFB-1A providing mechanical strength to epidermal attachment structures. Reduction in IFB-1B function causes morphogenetic defects and defective outgrowth of the excretory cell. Reduction in function of both IFB-1 isoforms results in embryonic arrest due to muscle detachment and failure in epidermal cell elongation at the 2-fold stage. Two other cIFs, IFA-2 and IFA-3, are expressed in epidermal cells. We show that loss of function in IFA-3 results in defects in morphogenesis indistinguishable from those of embryos lacking ifb-1. In contrast, IFA-2 is not required for embryonic morphogenesis. Our data indicate that IFB-1 and IFA-3 are likely the major cIF isoforms in embryonic epidermal attachment structures.

Animals↗
PubMed2005

Protection against experimental autoimmune myocarditis is mediated by interleukin-10-producing T cells that are controlled by dendritic cells.

Experimental autoimmune myocarditis (EAM) can be induced in the Lewis rat by cardiac myosin or its cryptic S2-16 peptide epitope (amino acids 1052 to 1076). To investigate cellular mechanisms and the role of antigen-presenting cells in regulation of myocarditis, we induced protection against EAM in Lewis rats by administration of S2-16 peptide in incomplete Freund's adjuvant (IFA). Protection to EAM was associated with activation of S2-16-reactive splenocytes secreting high levels of interleukin (IL)-10 and reduced levels of interferon-gamma and IL-2. Adoptive transfer of S2-16:IFA-induced splenocytes producing IL-10 suppressed myocarditis induction in syngeneic recipients, suggesting their regulatory cell nature. However, exposure of S2-16:IFA-induced cells to inflammatory cytokine IL-12 converted them to Th1 effectors that transferred EAM. Differentiated function of S2-16-reactive T cells in protected rats resulted from increased IL-10 production by dendritic cells (DCs). Purified DCs from S2-16:IFA-treated rats promoted S2-16-reactive CD4+ T cells to produce increased IL-10 and reduced interferon-gamma. In addition, adoptive transfer of IL-10-producing DCs from S2-16:IFA-treated rats also induced protection to EAM in recipient rats. These studies demonstrated DCs and key cytokines, such as IL-10 and IL-12, regulated the fate of T cells in myocarditis development in the Lewis rat.

Adoptive Transfer↗
PubMed2003

Associations between dogs that were serologically positive for Rickettsia conorii relative to the residences of two human cases of Mediterranean spotted fever in Piemonte (Italy).

A geographic information system and K-function analysis were used to evaluate the spatial association of canine serological results for Rickettsia conorii, the causative agent of Mediterranean spotted fever (MSF), and clinical cases of MSF in humans in Piemonte, northwest Italy. The residences of dog owners were clustered in two rural villages in the province of Cuneo, where two human cases of MSF occurred in 1997 and 1998. Eighteen out of 116 dogs examined were positive by indirect immunofluorescent assay (IFA+, titre > or =1:160) for MSF. K-functions were compared for IFA+ dogs and for all dogs sampled. Monte Carlo and bootstrap simulations demonstrated that clustering of IFA+ dogs was significantly greater than clustering of all dogs, at distances of less than 0.6 km from human cases of MSF. Logistic regression analysis indicated that the risk of being IFA+ was highest for dogs residing within the first quartile of distance (0.7 km) from human cases of the disease, and for dogs that were not confined. However, year and season of blood collection were not associated with IFA status. It was concluded that a relatively high dog population density along with a rural or semi-rural environment favours the occurrence of emergent foci of MSF in the province of Cuneo.

Age Factors↗
PubMed2003

Effect of gilt seropositivity to Lawsonia intracellularis (LI) on their offspring's seropositivity to LI and on diarrhoea after a pure-culture challenge.

This study was done in a large Croatian pig-production unit. Blood samples from late-pregnancy gilts were tested by an indirect immunofluorescence-antibody (IFA) serum assay for Lawsonia intracellularis (LI). Ten IFA-positive and 10 IFA-negative gilts were selected. The 2-week-old piglets (97 from positive gilts, and 98 from negative gilts) were inoculated intragastrically with pure culture of LI. All pigs were tested until slaughter for seroprevalence of LI. Blood samples were collected at 5, 8, 11, 14, 17, 20, 23 and 26 weeks of age. Seropositivity in offspring of IFA-negative gilts was highest at age 5 weeks (84%) and declined gradually from week 11 to only 10% at week 26. At the same time, the offspring of IFA-positive gilts showed lower and faster-decaying seroprevalence: only 32% at week 5 and 0% starting at week 17. There was no difference in the pen-days of diarrhoea between the offspring of LI-seropositive and -seronegative gilts.

Animals↗
PubMed2003

Comparison of immunofluorescence assay and immunomagnetic electrochemiluminescence in detection of Cryptosporidium parvum oocysts in karst water samples.

Immunofluorescence assay (IFA) and immunomagnetic electrochemiluminescence (IM-ECL) were used for comparison of the percent recovery of Cryptosporidium parvum in environmental water samples obtained from a spring draining a karst basin. The monoclonal antibodies to C. parvum, isotype IgG3 were used for optimization of the IM-ECL protocol. The combination of biotinylated and TAG-labeled anti-C. parvum antibodies with the streptavidin beads gave a linear regression slope for log ECL vs. log fresh oocysts of 0.79 (from 5 to 5,000 oocysts), which indicates a constant ECL signal per oocyst. Standard curves gave a dynamic range of 5 to 5,000 oocysts/ml (fresh) and 10 to 100,000 cells/ml (4-month-old oocysts) with the maximum limit of linear detection higher than 100,000. The linear slope of 4-month-old oocysts decreased to 0.62, which indicates that ECL signal is a function of oocyst age. The experiment associated with bead storage time shows that even after 4 months of storage of the biotinylated antibodies, the complex retains the ability for binding the oocysts and generating the ECL signal. Based on the IFA results in the experiment evaluating different protocols for oocysts recovery from karst water samples, the most efficient protocol involved dispersion, followed by flotation and immunomagnetic separation (IMS) (24% recovery). The ECL results obtained in that experiment were very similar to the results obtained in the IFA method, which indicates that the IM-ECL method is accurate. Results of the IFA in the study of the prevalence of C. parvum in the groundwater showed that oocysts were present in 78% of 1 L water samples with average number of oocysts of 6.4+/-5.5 and ranged from 0 (13 samples) to 23.3 (2 samples). The ECL signal generated from these water samples ranged from 3,771 to 622 (average 1,620+/-465). However, the background value estimated in groundwater samples with low number of oocysts detected by IFA was highly variable and elevated (from 3,702 to 272, average 1,503+/-475). The background value as a result of nonspecific binding to beads by unidentified organic components in the water can inhibit or even completely mask the signal generated by oocysts. Our investigations showed that the IM-ECL method appears to be promising for the qualitative and quantitative detection of C. parvum from the environmental water; however, the method requires further development to improve sensitivity and account for background signals.

Animals↗
PubMed1987

Production and characterization of monoclonal antibodies specific for pathogenic serogroups O:3, O:8, and O:9 of Yersinia enterocolitica.

A panel of 7 murine hybridoma derived monoclonal antibodies (MAbs) to Yersinia enterocolitica were produced and characterized by indirect fluorescence assay (IFA) and agglutination reactions. One MAb designated 2D8 (IgG 3) showed specific reactivity both in the IFA and agglutination assays with 70 of the 70 strains belonging to serogroup O:3 of Y. enterocolitica. Three MAbs, 8E9 (IgG 3b), 10G11 (IgG3) and 11G2 (IgG 3), gave unequivocal positive reactions in the IFA and agglutination tests with all the strains representing serogroup O:9 (56/56), but not with serogroup O:3 or O:8 strains. Another MAb coded 1G2 (IgG 1) reacted specially in the IFA test (but not in the agglutination test) with all the strains representing serogroup O:3 (70/70) and O:9 (56/56) indicating that this antibody recognizes an immunodeterminant shared by serogroups O:3 and O:9. Finally, 2 MAbs 4C2 (IgM) and 6G5 (IgM), showed reactivity both in IFA and agglutination assays with "esculin negative" biogroups 1 (pathogenic American strains) strains of serogroup O:8 (12/12), whereas the strains assigned to "esculin positive" biogroup 1 (nonpathogenic) failed to react (6/6). However, 4C2 and 6G5 exhibited narrow cross reactivity with type strains assigned to serogroups O:18, O:20, and O:22. The results of absorption tests and the clear out cell wall immunofluorescence imply that the antigenic molecule(s) recognized by these MAbs are exposed on the bacterial cell surfaces. Some of the MAbs described in this report are useful reagents for precise serotyping of clinical isolates of Y. enterocolitica by simple and rapid slide agglutination assay. They may also allow the development of specific and sensitive tests for probing the presence of pathogenic Y. enterocolitica organism in clinical and food materials.

Agglutination Tests↗