Hepcidin-to-ferritin ratio: A potential novel index to predict iron overload-liver fibrosis in ß-thalassemia major.

OBJECTIVES: We aimed to determine a threshold cutoff for hepcidin, ferritin, and the hepcidin-to-ferritin ratio in the diagnosis of liver fibrosis caused by iron overload in chronic hepatitis C virus (HCV)-free ß-thalassemia major patients . METHODS: This 1:1-matched case-control study included 102 individuals (3-30 yr.); 51 ß-thalassemia major patients with iron overload , and 51 apparently healthy individuals. RESULTS: The highest areas under the receiver operating characteristic curves (AUC-ROCs) for the diagnosis of patients vs. controls had overlapping 95% confidence intervals (CIs): serum hepcidin (0.758; 0.64-0.87; P Ë‚ 0.001), serum ferritin (1.000; 1.00-1.00; P˂0.001), and the hepcidin/ferritin ratio (1.000; 1.00-1.00; P˂0.001). For differentiation of patients with liver fibrosis stages of F0-F1 vs. F2-F4 and F0-F1 vs. F3-F4, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) with P-values˂0.001 were the only statistically significant parameters, while the AUC-ROCs of the hepcidin/ferritin ratio (0.631, P=0.188 and 0.684, P=0.098) exhibited 90% and 89.5% sensitivity, respectively, in staging liver fibrosis. CONCLUSION: Our results showed that the hepcidin/ferritin ratio is as effective as the APRI and maybe a better predictor for the diagnosis of liver fibrosis and discriminating its stages, with excellent sensitivity and specificity compared to its components.

Elevated levels of the endogenous retrovirus ERV3 in human sebaceous glands.

ERV3 (HERV-R) is a complete human endogenous retrovirus located on the long arm of chromosome 7. Long terminal repeat-envelope (env) gene spliced mRNAs of 9 and 3.5 kb are widely expressed in human tissues and cells, but gag-pol mRNAs have not been found. Furthermore, the env gp70 gene contains an open reading frame throughout its length. The highest expression of ERV3 mRNA detected so far is in placenta and the lowest in choriocarcinoma cell lines. We have previously shown that the human monoblastic cell line U-937 and some normal and neoplastic tissues also express high levels of ERV3 env message by Northern blot analysis; however, this method does not distinguish between mRNA expression in different cell types in tissues. In this report, we have studied the ERV3 mRNA expression in specific cell types of human skin by in situ hybridization. We found high levels expression of ERV3 env mRNA in human sebaceous glands in normal skin and dermoid cysts of the ovary. In all glands, the expression is maximal in the periphery of the lobule and ceases towards the center in the region of characteristic holocrine secretion. Since it is known that the regulation of sebaceous glands is primarily via steroid hormones, particularly androgens, it is possible that expression of ERV3 is hormone dependent.

Serologic evidence of Puumala virus infection in wild moose in northern Sweden.

Puumala (PUU) virus is the causative agent of nephropathia epidemica, the Scandinavian form of hemorrhagic fever with renal syndrome. The infection is acquired by airborne transmission of PUU virus from its rodent reservoir, the bank vole. Besides serologic data indicating that the virus may spread also to heterologous rodents, there is little information on the susceptibility of wild living animals to PUU virus. We studied the occurrence of antibodies to PUU virus in serum samples from 427 wild-living moose, of which 260 originated from the PUU virus-endemic northern and central parts of Sweden and 167 originated from the southern, nonendemic part of Sweden. Samples from 5 animals showed reactivity in an ELISA for recombinant PUU virus nucleocapsid protein, an immunofluorescent assay, and a neutralization test. These 5 animals all originated from the PUU virus-endemic northern part of Sweden. In conclusion, 5 of 260 moose from the endemic region showed convincing serologic evidence of past PUU virus infection. The seroprevalence was low, suggesting that the moose is subjected to endstage infection rather than being part of an enzootic transmission cycle.

Immunoaffinity purification of two major proteins of bovine leukemia virus (gp51 and p24) and their use for discrimination between vaccinated and infected animals.

The outer envelope glycoprotein gp51 and the core protein p24 of bovine leukemia virus (BLV), were purified from culture media of FLK-BLV cells by a single-step procedure, using immunoaffinity chromatography based on monoclonal antibodies to the respective proteins. About 90% of the envelope glycoprotein in the culture medium was recovered as a highly purified product. Both purified protein (gp51 and p24) preparations, were found to be highly specific antigens by ELISA, and did not cross-react with sera raised against the other antigen. The conformational epitopes on the purified gp51 were preserved as judged by their reactions with the corresponding monoclonal antibodies. The p24 ELISA reacted only with sera from naturally infected animals and not with sera from animals immunized with an experimental gp51-iscom vaccine. The p24 antigen is therefore useful for discriminating between BLV-infected animals and those immunized with a gp51 subunit vaccine.

Evaluation of electrophoretic immunoblotting for the detection of antibodies against the bovine leukosis virus in cattle.

Six antigen preparations of bovine leukemia virus, including affinity-purified glycoprotein gp51, gradient-purified fetal lamb kidney-bovine leukemia virus antigen, and four crude antigens, were used in combination with several groups of cattle sera, for the evaluation of electrophoretic immunoblotting as a serological test method. Sera (89) from cattle naturally-infected with bovine leukosis virus, a panel of reference sera from infected and uninfected cattle (18), and serial bleedings from experimentally-infected cows (4) were used. Major differences between the six antigen preparations were observed in their reactivity with the various sera. The immunological variabilities of these antigens were confirmed further by their reactions with a gp51-specific monoclonal antibody. The known immunodominant gp51 failed as a reliable indicator for the serological status of the sera in blots when compared to the results on the same sera, two gp51-specific ELISAs and the agar gel immunodiffusion test were used as reference tests. There was a lack of staining of gp51 antigen by many sera, probably due to the labile nature of the gp51 molecule. On the other hand, non-specific staining in the gp51 region appeared with high frequency in some antigens. Antibody staining of the internal viral protein p24 correlated well with the results of the three reference tests. Other bands stained infrequently and were of no diagnostic value.

Equine herpesvirus type 2 (EHV-2) as a predisposing factor for Rhodococcus equi pneumonia in foals: prevention of the bifactorial disease with EHV-2 immunostimulating complexes.

Equine herpesvirus type 2 (EHV-2), a member of the Gammaherpesvirinae subfamily, was studied in a two-phase respiratory disease complex of young foals as a predisposing factor for the secondary bacterial invasion of lungs with Rhodococcus equi (R. equi). Foals were immunized against EHV-2 on a farm where R. equi pneumonia regularly occurred during the last years. The immunizations were performed by using a subunit vaccine which selectively presents envelope glycoproteins of EHV-2 in a multimeric form of immunostimulating complexes (iscoms). The etiological role of EHV-2 was estimated by observing the occurrence of the respiratory disease complex in groups of foals immunized against the virus, in comparison to non-immunized controls. The immunization trials of young animals revealed that the iscom subunit vaccine formulation is able to overcome the interference of maternal antibodies. Active immunization of foals with a single dose of the iscoms provided a certain degree of protection, while two injections of iscoms yielded complete protection against the disease complex in the majority of the treated animals, by preventing the manifestation of R. equi pneumonia. The present findings strongly support the hypothesis that EHV-2 is a predisposing factor for the R. equi invasion of the respiratory tract. The EHV-2 iscom formulation provides highly specific and effective means to prevent the disease complex.

Envelope glycoprotein gp51 of bovine leukemia virus is differently glycosylated in cells of various species and organ origin.

The carbohydrate moiety of the envelope glycoprotein gp51 of bovine leukemia virus, American strain, was studied. The virus was grown in ovine, bovine, porcine, bat and rat cells of various organ specificities. The gp51 was purified by immunoaffinity chromatography from virions of ten different virus-producing cells derived from various body organs of different species. Highly purified glycoproteins (single band in PAGE) were compared for their electrophoretic mobility, for the presence of epitopes by a battery of monoclonal antibodies, and for the glycosylation pattern by lectin blot analysis. Electrophoretic analysis of all tested glycoproteins deglycosylated by glycopeptidase F detected the same polypeptide backbone according to PAGE. The glycoproteins produced in rat cells migrated faster in PAGE, as detected in cells or in virions, than those produced in ovine cells. The pattern of their glycosylation was found to be dependent on the type of cells used for virus production. The differences in glycosylation were most pronounced when comparing the glycoprotein produced in ovine cells versus bat or rat cells. Changes in epitope expression were also detected. The differences in the patterns of glycosylation and in the accessibility of epitopes owing to the virus production in various kind of cells are discussed from virus infectivity and vaccine points of view.

Testing of bovine sera by ELISA for IgG, IgM and IgA rheumatoid factors.

Sera from 19 colostrum-deprived calves less than 1 week old, 24 colostrum-supplemented calves less than 1 week old, 36 3-5-month-old calves and 200 females greater than 9 months of age were tested by ELISA for the presence of IgM, IgG and IgA rheumatoid factors (RF). An increasing level of IgM- and IgG-RF with age was found. IgG-RF levels in the colostrum-supplemented calves were significantly higher than in the non-supplemented calves (p < 0.001). Individual IgG-RF values correlated with serum IgG levels, as determined by zinc sulphate turbidity testing (r=0.59, p < 0.01). No IgA-RF was detected. The cross-reactivity of IgM-RF with heterologous IgG was found to be greatest with rabbit IgG, followed by mouse and chicken IgG. The significance of rheumatoid factors in relation to diagnostic testing is discussed.

Detection of IgM responses to bovine respiratory syncytial virus by indirect ELISA following experimental infection and reinfection of calves: abolition of false positive and false negative results by pre-treatment of sera with protein-G agarose.

The IgM responses in three panels of sera generated by infection and reinfection of calves with bovine respiratory syncytial virus (BRSV) were measured by indirect ELISA (I-ELISA). The effect of depleting serum IgG by pre-treatment with protein G agarose (PGA) was evaluated. Following primary infection a weak IgM response was detected in the untreated sera of 3 out of 4 calves with maternally derived antibody (MDA). Both the magnitude and duration of the specific IgM responses in these calves were increased by pre-treatment with PGA. In addition, the fourth infected calf tested gave a single positive IgM result following PGA treatment. Transient or persistent IgM responses which were abolished by pre-treatment of sera with PGA were detected in 4/8 calves following reinfection. These were considered to be false positive results, consistent with the influence of IgM rheumatoid factor (IgM-RF). One of these calves and two additional calves showed transient increases in IgM which were resistant to PGA treatment. These were considered to represent specific IgM responses to reinfection. The results indicate the ability of PGA treatment to eliminate both false positive and false negative results and emphasise the necessity for controlling the influence of IgM-RF in IgM-specific indirect ELISAs.

The use of a neutralizing monoclonal antibody to detect infections of equine herpesvirus type 2 (EHV-2).

A blocking enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies to equine herpesvirus 2 in serum samples of horses. By measuring the binding to a single epitope, this blocking ELISA gives a good picture of the antibody status in the animal. The test is based on a monoclonal antibody with neutralizing activity and had a sensitivity of 94% and a specificity of 100%. Antibodies due to newly acquired infection in foals were successfully detected with this blocking ELISA.

Evaluation of an IgM-specific indirect enzyme-linked immunosorbent assay for serodiagnosis of bovine respiratory syncytial virus infection: influence of IgM rheumatoid factor on test results with field sera.

A commercially available indirect enzyme-linked immunosorbent assay for measuring bovine respiratory syncytial virus (BRSV)-specific IgG was adapted to measure virus-specific IgM. Using this assay, the development of rapid IgM responses in experimentally infected calves was observed 7-9 days postinfection, with peak absorbance values ranging from 1.698 to 2.873. When absorbance values were expressed as a percentage of a positive reference serum, a positive/negative threshold of 22% was determined by testing serum samples from 59 healthy 3-5-month-old calves. Acute and convalescent serum samples collected from 151 calves during 38 outbreaks of respiratory disease were tested, and 130 sera were positive. To determine the number of false-positive results due to the presence of IgM rheumatoid factor, a method for depleting serum IgG by pretreatment of sera with a suspension of protein-G-agarose was developed. All sera that initially tested IgM positive were retested following depletion of serum IgG. False-positive IgM reactions were detected in 23 sera (17.7%). Specific IgM responses were confirmed in 107 sera from 84 calves. Evidence of BRSV infection was detected in 34 of 38 outbreaks. In contrast, seroconversion was detected in 69 calves from 24 outbreaks, confirming the diagnostic potential of the IgM assay. Overall correlation between IgM and seroconversion results was 74.2%. Intra- and interassay reproducibility were 12.50% and 17.48%, respectively (mean coefficients of variation).

Standardization of enzyme-linked immunosorbent assays (ELISAs) for quantitative estimation of antibodies specific for infectious bovine rhinotracheitis virus, respiratory syncytial virus, parainfluenza-3 virus, and bovine viral diarrhea virus.

Commercial enzyme-linked immunosorbent assays (ELISAs) for detection of serum antibodies to bovine viral diarrhea virus (BVDV), parainfluenza-3 virus (PI3V), respiratory syncytial virus (RSV), and infectious bovine rhinotracheitis virus (IBRV) were standardized to give a quantitative result when testing was performed at a single optimum dilution. For each test, serum samples were titrated and their end point titers calculated by an algebraic method directly from a plot of each titration series and also from a regression line fitted to this plot. The corrected optical density (COD) of each sample when tested at dilutions of 1/25, 1/50, and 1/100 was expressed as a percentage of the COD of a positive reference serum included on each plate, this value was the sample/positive (S/P) ratio. For each test, the linear relationship between the S/P ratio obtained at a dilution of 1/25, 1/50, and 1/100 and the end point titer calculated by each method was determined. In each case, the best linear relationship existed when samples were tested at a dilution of 1/100 (r = 0.973 for BVDV, 0.962 for PI3V, 0.961 for RSV, 0.947 for IBRV). From the equation of these lines, an increase in the S/P ratio between acute and convalescent serum samples of 31%, 23%, 21%, and 35% would correspond to a 4-fold rise in ELISA titer to BVDV, PI3V, RSV, and IBRV, respectively. ELISA titers calculated from S/P ratios at 1/100 were significantly related to virus neutralization titers to BVDV, RSV, and IBRV and to hemagglutination inhibition titers to PI3V (P < < 0.001 in all cases). Samples with low S/P ratios had the greatest intraassay and interassay variation. Intraassay reproducibility ranged from 3.5% to 22.3% (coefficient of variation), with a median value of 9.5%. Interassay reproducibility was lower, ranging from 6.0% to 50.6%, with a median of 17.4%.

Evaluation of a single dilution ELISA system for detection of seroconversion to bovine viral diarrhea virus, bovine respiratory syncytial virus, parainfluenza-3 virus, and infectious bovine rhinotracheitis virus: comparison with testing by virus neutralization and hemagglutination inhibition.

A single-dilution quantitative enzyme-linked immunosorbent assay (ELISA) system, based on commercial ELISA kits, for the simultaneous detection of seroconversion to bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), parainfluenza-3 virus (PI3V), and infectious bovine rhinotracheitis virus (IBRV) was evaluated by testing acute and convalescent serum pairs from 564 cattle in 145 outbreaks of respiratory disease. Seroconversion to BVDV, BRSV, PI3V and IBRV was detected in 8.0%, 19.0%, 13.7%, and 7.4%, respectively, of serum pairs tested. Seroconversion was detected in 60.7% of herds and 34.6% of animals tested. Infection with 2 or more viruses was found in 46.6% of these herds and in 27.2% of these animals. The majority of BVDV infections (62%) were associated with other virus infections, suggesting that BVDV may potentiate infection with other agents rather than being a primary pathogen of the respiratory tract. The results were compared with those obtained by virus neutralization and hemagglutination inhibition testing, and the sensitivity, specificity, and overall correlation were calculated. Sensitivities of 92%, 95%, 100%, and 100% were obtained for BVDV, BRSV, PI3V, and IBRV, respectively. The corresponding specificity values were 89%, 92%, 86%, and 91%. The overall correlation for each virus was 90%, 93%, 90%, and 93%, respectively. These results demonstrate that this ELISA system may be used successfully to detect seroconversion in serum pairs, highlight the frequency of multiple viral infections in outbreaks of respiratory disease, and provide further evidence of an immunosuppressive role for BVDV infections.

Evaluation of an immunofluorescent antibody test to detect bovine herpesvirus 1-specific IgM.

An indirect immunofluorescent antibody test (IIFAT) was developed to detect bovine herpesvirus 1 (BHV-1)-specific IgM. All sera were treated with protein-G agarose prior to testing to eliminate the possibility of false-positive results due to IgM-isotype rheumatoid factor (IgM-RF). Specific IgM was first detected 8 days after experimental infection of 3 calves free of maternally derived antibody, with peak responses occurring 2-7 days later. Seroconversion was detected in all 3 calves using a single-dilution enzyme-linked immunosorbent assay. Following reinfection at 30 days postinfection, a low-level IgM response was detected in only 1 calf. Seroconversion was detected in 2 calves. There was no evidence of activation of IgM-RF by infection or reinfection with BHV-1. When 87 acute and convalescent serum pairs collected from 21 outbreaks of respiratory disease were tested, specific IgM was detected in 58 animals (66.6%) from 19 (90.5%) outbreaks. Seroconversion was detected in 44 of these animals (50.6%) from 17 outbreaks (81.0%). The correlations between these 2 assays on a calf and outbreak basis were 79.3% and 90.5%, respectively. Specific IgM was detected in 17/20 sera (85.0%) collected from an additional outbreak. No virus was detected by virus isolation or immunofluorescent staining in nasal mucus samples collected at the same time. Detection of specific IgM by IIFAT is a useful technique for the serodiagnosis of BHV-1 infection.

Isotype- and subclass-specific responses to infection and reinfection with parainfluenza-3 virus: comparison of the diagnostic potential of ELISAs detecting seroconversion and specific IgM and IgA.

Isotype- and subclass-specific indirect enzyme-linked immunosorbent assays were developed to detect parainfluenza-3 virus-specific IgG1, IgG2, IgM, and IgA responses. Sera were treated with protein G-agarose prior to testing for specific IgM and IgA to eliminate the possibility of false-positive results due to IgM-rheumatoid factor and to remove interisotypic competition due to specific IgG. IgM and IgA absorbance values were expressed as a percentage of the absorbance values of positive reference sera included on each plate (S/P%), and respective positive/negative threshold values of 15.0% and 28.0% were determined. The mean interval between experimental infection of 3 calves and initial detection of specific IgG1 and IgG2 responses was 8.0 and 9.3 days respectively, rising rapidly to an initial plateau 13.7 and 11.0 days postinfection (dpi). Reinfection of these calves at 30 dpi resulted in further rapid increases, with higher plateau values reached 13.0 (IgG1) and 13.7 (IgG2) days later. The mean interval between infection and the first positive IgM and IgA responses was 6.7 and 12.3 days, respectively. IgM S/P% values peaked at 13.0 dpi, with all 3 calves showing a secondary anamnestic response to reinfection, peaking 4.7 days later. The IgA response to initial infection was weak, with only 2 calves showing an obvious peak response at 15.0 dpi. A strong anamnestic IgA response to reinfection occurred in 2 calves, with a peak response 9.5 days later. Apparent biphasic and triphasic IgM and IgA responses were evident in some calves. Acute and convalescent serum samples from 80 calves involved in 17 outbreaks of respiratory disease were tested for specific IgM and IgA. Positive IgM results were detected in 15 outbreaks, with 71 sera from 44 calves testing positive. Although IgA-positive results were detected in the same 15 outbreaks, only 42 sera from 31 calves were positive. In a previous study, seroconversion was detected in 21 of these calves from 10 outbreaks. Thus the diagnostic potential of the assays was in the order IgM > IgA > seroconversion. The correlations between IgM and IgA, IgM and seroconversion, and IgA and seroconversion results for each calf were 73.8%, 58.8% and 62.5%, respectively.

A comparative study on the use of virus and antibody detection techniques for the diagnosis of La Piedad Michoacan paramyxovirus (LPMV) infection in pigs.

La Piedad Michoacan paramyxovirus (LPMV) is newly recognized paramyxovirus that has been associated with neurologic and reproductive disorders in pigs in Mexico. To date, no comparative study of methods for the diagnosis of infection with this virus has been published. In this study, we identified tissues containing maximum virus load to optimize virus isolation procedures, and we compared this method to a rapid diagnostic test employing immunostaining of impression smears for LPMV antigens. In addition, several of the available tests for detecting LPMV antibodies were compared for their sensitivity in detecting seroconversion. Pigs used for the study of virus load in tissues and serologic studies were inoculated at 17 days of age with 10(7.00) TCID50 of LPMV. Serial blood samples were collected from selected pigs, and selected pigs were necropsied over a 14-day period. Pigs used in the investigation comparing standard virus isolation techniques to immunostaining of impression smears were inoculated at 3 days of age as described above and necropsied over an 8-day period. The results demonstrate that in the 17-day-old pigs maximum virus titers were detected in olfactory bulb at 5 days postinoculation (PI) and in midbrain at 9 days PI. In addition, the most consistent recovery of high titer virus was from tonsil (3-9 days PI) and olfactory bulb (4-9 days PI). Immunostaining of impression smears was as sensitive as virus isolation when selected tissues (lung, midbrain, olfactory bulb) were compared, with virus detected by both methods in 11/13 samples and in 1 sample each by immunostaining and virus isolation, respectively. All of the serology tests investigated detected seroconversion in pigs by 8 days PI. The identification of target organs where highest virus titers are found combined with immunofluorescent methods for the detection of LPMV antigens and a comparative study of the available serologic tests should facilitate the selection of techniques suitable for any laboratory to diagnose LPMV infection in pigs.

Development of a blocking ELISA for screening antibodies to porcine rubulavirus, La Piedad Michoacan Virus.

A blocking enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies to porcine rubulavirus (La Piedad Michoacan Virus [LPMV]) in serum samples from pigs. The test, based on a monoclonal antibody against the LPMV hemagglutinin-neuraminidase glycoprotein, had a sensitivity of 99% and a specificity of 97%. The results of this test were in agreement with those obtained by an indirect ELISA and hemagglutination inhibition, indirect immunofluorescence, and virus neutralization tests. The blocking ELISA is considered the most suitable test for routine screening for antibodies against LPMV.

Seroprevalence to bovine virus diarrhoea virus and other viruses of the bovine respiratory complex in Venezuela (Apure State).

Six hundred and fifteen serum samples obtained from cows in five districts of Apure State, Venezuela, were tested by ELISA for antibodies to bovine virus diarrhoea virus (BVDV). The same samples were also ELISA-tested for antibodies to bovine herpesvirus type 1 (BHV-1) and bovine respiratory syncytial virus (BRSV). Additionally, the haemagglutination-inhibition (HI) test was used for detecting antibodies to parainfluenza virus type 3 (PIV-3). Overall, seroprevalence to BVDV was 36+/-7% (SE); seroprevalence varied by district (19-42%). BHV-1 seroprevalence was 67+/-4%; variation by district was similar to that of BVDV. However, the first 80 serum samples tested by BHV-1 ELISA all had a strong background reaction with the control antigen. Therefore, these sera were adsorbed to a homogenate of non-infected bovine kidney cell line (MDBK) and retested by ELISA. The non-specific reactivity was significantly reduced (p<0.001 by Wilcoxon's signed-rank test). Compared to the virus-neutralisation (VN) test, the adsorbed BHV-1 ELISA showed 94% agreement and gave a kappa value of 0.84, indicating that the adsorption did not interfere with test accuracy. Seroprevalence against BRSV was 85+/-3%, and showed differences across districts. Most of the cows (94+/-2%) were seropositive to PIV-3, and there were no significant differences among districts.

Temporary suppression of cell-mediated immunity in standardbred horses with decreased athletic capacity.

Eighty Standardbred horses, originating from 5 training campuses, with decreased athletic performance in association with symptoms such as intermittent fever and mild pharyngitis were examined. As control animals, 10 horses from a stable with normally performing horses were used. Virus isolation and clinico-chemical and serological tests were performed. Lymphocyte proliferation tests were carried out to evaluate the capacity of the cell-mediated immunity. In addition, a bioassay for equine type I interferon, as a marker for early viral infections, was established. No specific microbe could be linked to these symptoms, but there was a temporary suppression of the cell-mediated immunity, which might be explained by the serological evidence of an EHV-2 and/or rhinovirus infection.