Indirect Immunofluorescence Assay Results Research Articles

Comparison of the indirect immunofluorescence assay and a commercial ELISA to detect KSHV antibodies.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus involved in several diseases. The gold standard for KSHV sero diagnosis remains the indirect immunofluorescence assay (IFA), which is time-consuming and operator-dependent. We compared this method with an enzyme-linked immunosorbent assay (ELISA) targeting solubilized KSHV whole-genome extract among positive (n = 49, including 76% of HIV-infected patients) and negative (n = 14) control groups. We also included 14 sera with equivocal IFA results. ELISA showed better performance in detecting KSHV antibodies (McNemar's test, P = 0.0455). The sensitivity and specificity of both methods were 79% (64-89) and 100% (66-100) for the IFA, respectively, and 94% (83-99) and 100% (66-100) for ELISA, respectively. All IFA equivocal results were either negative or positive with ELISA. ELISA is more reliable and could be a good alternative for determining KSHV serological status, particularly in the context of immunocompromised patients and equivocal serology with the IFA.IMPORTANCEKaposi's sarcoma-associated herpesvirus (KSHV) sero status remains challenging because no perfect reference is available for the detection of KSHV antibodies. The current gold-standard method, the indirect immunofluorescence assay (IFA), has a very good specificity of close to 100%, but a lower sensitivity of around 80-85%, which decreases to 64-67% in immunocompromised patients. Additionally, this method is time-consuming and operator-dependent compared with new serological assays such as the enzyme-linked immunosorbent assay (ELISA). Thus, further research is still needed to improve KSHV sero diagnosis. Here, we compare the KSHV IgG ELISA kit assay (Advanced Biotechnologies Inc) with the gold-standard IFA, targeting the LANA-1 protein from latent BC-3 cell lines.

Preparation of a mouse monoclonal antibody against the NS1 protein of respiratory syncytial virus

The aim of this study was to prepare a mouse monoclonal antibody against the nonstructural protein 1 (NS1) of respiratory syncytial virus (RSV) to analyze its expression and distribution during transfection and infection. Additionally, we aimed to evaluate the antibody's application in immunoprecipitation assay. Firstly, the NS1 gene fragment was cloned into a prokaryotic plasmid and expressed in Escherichia coli. The resulting NS1 protein was then purified by affinity chromatography, and used to immunize the BALB/c mice. Subsequently, hybridoma cells capable of stably secreting the NS1 monoclonal antibody were selected using indirect enzyme linked immunosorbent assay (ELISA). This monoclonal antibody was employed in both indirect immunofluorescence assay (IFA) and Western blotting to analyze the expression and distribution of RSV NS1 in overexpressed and infected cells. Finally, the reliability of this monoclonal antibody was evaluated through the immunoprecipitation assay. The results showed that the RSV NS1 protein was successfully expressed and purified. Following immunization of mice with this protein, we obtained a highly specific RSV NS1 monoclonal antibody, which belonged to the IgG1 subtype with an antibody titer of 1:15 360 000. Using this monoclonal antibody, the RSV NS1 protein was identified in both transfected and infected cells. The IFA results revealed predominant distribution of NS1 in the cytoplasm and nucleus. Moreover, we confirmed that this monoclonal antibody could effectively bind specifically to NS1 protein in cell lysates, making it suitable as a capture antibody in immunoprecipitation assay. In conclusion, our study successfully achieved production of the RSV NS1 protein through a prokaryotic expression system and prepared a specific monoclonal antibody against NS1. This antibody demonstrates the ability to specifically identify the NS1 protein and can be used in the immunoprecipitation assay, thereby laying a foundation for the functional studies of the NS1 protein.

Detection of antinuclear antibodies: recommendations from EFLM, EASI and ICAPEFLM Paper

<br><b>Aim:</b> Antinuclear antibodies (ANA) are important for the diagnosis of various autoimmune diseases. ANA are usually detected by indirect immunofluorescence assay (IFA) using HEp-2 cells (HEp-2 IFA). There are many variables influencing HEp-2 IFA results, such as subjective visual reading, serum screening dilution, substrate manufacturing, microscope components and conjugate. Newer developments on ANA testing that offer novel features adopted by some clinical laboratories include automated computer-assisted diagnosis (CAD) systems and solid phase assays (SPA).</br> <br><b>Methods:</b> A group of experts reviewed current literature and established recommendations on methodological aspects of ANA testing. This process was supported by a two round Delphi exercise. International expert groups that participated in this initiative included (i) the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group "Autoimmunity Testing"; (ii) the European Autoimmune Standardization Initiative (EASI); and (iii) the International Consensus on ANA Patterns (ICAP).</br> <br><b>Results:</b> In total, 35 recommendations/statements related to (i) ANA testing and reporting by HEp-2 IFA; (ii) HEp-2 IFA methodological aspects including substrate/conjugate selection and the application of CAD systems; (iii) quality assurance; (iv) HEp-2 IFA validation/verification approaches and (v) SPA were formulated. Globally, 95% of all submitted scores in the final Delphi round were above 6 (moderately agree, agree or strongly agree) and 85% above 7 (agree and strongly agree), indicating strong international support for the proposed recommendations.</br> <br><b>Conclusions:</b> These recommendations are an important step to achieve high quality ANA testing.</br>

Detection of antinuclear antibodies: recommendations from EFLM, EASI and ICAP.

Antinuclear antibodies (ANA) are important for the diagnosis of various autoimmune diseases. ANA are usually detected by indirect immunofluorescence assay (IFA) using HEp-2 cells (HEp-2 IFA). There are many variables influencing HEp-2 IFA results, such as subjective visual reading, serum screening dilution, substrate manufacturing, microscope components and conjugate. Newer developments on ANA testing that offer novel features adopted by some clinical laboratories include automated computer-assisted diagnosis (CAD) systems and solid phase assays (SPA). A group of experts reviewed current literature and established recommendations on methodological aspects of ANA testing. This process was supported by a two round Delphi exercise. International expert groups that participated in this initiative included (i) the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group "Autoimmunity Testing"; (ii) the European Autoimmune Standardization Initiative (EASI); and (iii) the International Consensus on ANA Patterns (ICAP). In total, 35 recommendations/statements related to (i) ANA testing and reporting by HEp-2 IFA; (ii) HEp-2 IFA methodological aspects including substrate/conjugate selection and the application of CAD systems; (iii) quality assurance; (iv) HEp-2 IFA validation/verification approaches and (v) SPA were formulated. Globally, 95% of all submitted scores in the final Delphi round were above 6 (moderately agree, agree or strongly agree) and 85% above 7 (agree and strongly agree), indicating strong international support for the proposed recommendations. These recommendations are an important step to achieve high quality ANA testing.

The clinical characteristics of nuclear dense fine speckled pattern in 95 289 patients

To investigate the distribution and clinical significance of nuclear dense fine speckled (DFS) pattern in various diseases. A total of 95 289 patients who received DFS tests at Peking Union Medical College Hospital from January 2019 to December 2020 were included in this study. The results of indirect immunofluorescence assay (IIF) for detection of antinuclear antibody (ANA) were evaluated. The positive rates of ANA and DFS were 39.60% (37 733/95 289) and 1.19% (1 139/95 289) respectively. The positive rate of DFS in ANA-positive patients was 3.02% (1 139/37 733). DFS and ANA positivity were significantly different among different age groups rather than gender. The positivity rate of DFS reached the peak (55.57%, 633/1 139) in young patients between 21-40 years, while positive ANA with negative DFS was mainly observed in patients between 41-60 years (37.26%, 13 636/36 594). Additionally, single ANA-positivity were mainly detected in rheumatology department (59.23%, 18 402/31 066), whereas positive DFS was more common in obstetrics and gynecology department (3.08%, 49/1 593). There were 82.88% (944/1 139) patients with positive DFS diagnosed with non-autoimmune disease (non-AID), and 19.49%(222/1 139) with dermatosis. Positive DFS with higher titer (≥1∶320) was detected more frequently in autoimmune disease (AID) patients (5.13%, 10/195) than in non-AID patients (1.69%, 16/944) (P<0.05). The DFS pattern is rare in ANA positive patients, which is mainly observed in women between 21-49 years. High titer of DFS is prevalent in AID patients, but positive DFS is detected more in non-AID patients, especially those with dermatosis.

Clinical Performance of Two Automated Immunoassays, EliA CTD Screen and QUANTA Flash CTD Screen Plus, for Antinuclear Antibody Screening.

BackgroundRecently, two fully automated immunoassays for antinuclear antibody (ANA) screening were introduced EliA CTD Screen (Thermo Fisher Scientific, Freiburg, Germany) and QUANTA Flash CTD Screen Plus (Inova Diagnostics, San Diego, USA). We evaluated their clinical performance in comparison with the indirect immunofluorescence assay (IIFA) and analyzed samples with discrepant results.MethodsIn total, 406 serum samples (206 from patients undergoing routine checkups and 200 from rheumatology clinic patients) were assayed using EliA, QUANTA Flash, and IIFA. We evaluated assay concordance and agreement and confirmed the presence of anti-extractable nuclear antigen (ENA) antibodies in samples with discrepant automated immunoassay and IIFA results. Additionally, we compared the clinical performance of each assay in diagnosing ANA-associated rheumatic disease (AARD) and adjusted the cut-off values.ResultsIn rheumatology clinic samples, the concordance and agreement were 91.5% and strong between EliA and QUANTA Flash, 79.0% and weak between EliA and IIFA, and 80.5% and moderate between QUANTA Flash and IIFA, respectively. In automated immunoassay-positive, IIFA-negative samples (N=15), all anti-ENA antibodies detected (6/15) were anti-Sjögren’s syndrome antigen A/Ro (Ro60) antibodies. The automated immunoassays and IIFA showed high accuracy for diagnosing AARD, and adjusted cut-off values improved their sensitivities (EliA with 0.56 ratio, 82.9% sensitivity; QUANTA Flash with 9.7 chemiluminescent units, 87.8% sensitivity).ConclusionsThe two automated immunoassays showed reliable performance compared with IIFA and can be efficiently used with the IIFA in clinical immunology laboratories. Clinical cut-off values can be adjusted according to the workflow in each laboratory.

Preparation and Characterization of a New Monoclonal Antibody Specific Against Lawsonia intracellularis and Its Application in Indirect Immunofluorescence and Immunocytochemistry Assay.

Proliferative enteropathy (PE) is an infectious enteric disease caused by Lawsonia intracellularis (L. intracellularis) and is endemic in pig herds worldwide. However, a L. intracellularis-specific monoclonal antibody plays an important role in the evaluation of L. intracellularis infection in vitro. Therefore, the objective of this study was to produce and identify the characteristics of a new monoclonal antibody against the outer membrane protein (Omp2) of L. intracellularis and apply it in an indirect immunofluorescence assay (IFA) and immunocytochemistry (IHC). The results indicated that three highly specific monoclonal antibodies against the Omp2 protein (4D9, 3G2, and 7G5) of L. intracellularis were obtained by using purified Omp2 as an immunogen, the titers of ascitic fluids of 4D9, 3G2, and 7G5 cells were 1:2,048,000, 1:512,000, and 1:256,000, respectively. IFA analysis showed that the 4D9, 3G2, and 7G5 have no cross-reactivity with other enteric bacteria commonly found in the ilea of pigs or closely related to L. intracellularis, such as Desulfovibrio, Bilophila wadsworthia (B. wadsworthia), Salmonella choleraesuis (S. choleraesuis), Salmonella typhimurium (S. typhimurium), Escherichia coli (E. coli), and Brachyspira hyodysenteriae (B. hyodysenteriae). IFA and IHC results indicated that the monoclonal antibodies can be successfully used as primary antibodies to detect L. intracellularis in infected cells and in the crypt of the ileum from infected tissues of PE. Our findings suggested that the new monoclonal antibody specific against L. intracellularis will be useful for the evaluation of L. intracellularis infection in vivo and in vitro.

A serological and hematological study on Rift valley fever and associated risk factors in aborted sheep at Kurdistan province in west of Iran

Rift Valley fever (RVF) is a disease caused by RVF virus (RVFV) which can cause infections in a range of wild and domestic ruminants as well as in humans and characterized by an increased incidence of abortion in ruminants. This study aims to survey the seroprevalence and risk factors of this zoonose among aborted sheep in Kurdistan province, the west of Iran. 182 blood samples were collected from aborted sheep during the past one month under age groups <1, ≥1−3, >3−5 year in four seasons in two groups of border and non-border cities of Kurdistan province. The presence of RVFV-specific Antibodies was investigated by using competitive ELISA. Indirect immunofluorescence assay (IIFA) was used to confirm positive samples, after separation of serum, as well as blood samples were analyzed for description of hematological parameters. Of a total sheep sampled 1.65 % (n = 3) were positive for RVFV antibodies in both test. The results of IIFA were correlated with the ELISA results. All of the positive samples showed leucopenia and had significant relation with seroprevalence of RVF (P < 0.05). The seroprevalence of RVF in the border cities were significantly higher than other group (P < 0.05) Age of sheep and season had no significant effect on prevalence of RVF (P > 0.05). Results obtained in this study indicated the presence of low-level RVFV circulation among the sheep of Kurdistan Province in Iran, so it is necessary to carry out further studies in other areas of Iran. Doing an epidemiologically study aimed at isolating RVFV in the ruminants of Kurdistan province is recommended. The risk factor of bordering with Iran's western neighbor (Iraq) requires seriously control of the exchange of animals and the relevant products between the two countries.

C-C Chemokine receptor-like 2 (CCRL2) acts as coreceptor for human immunodeficiency virus-2.

Most of the typical chemokine receptors (CKRs) have been identified as coreceptors for a variety of human and simian immunodeficiency viruses (HIVs and SIVs). This study evaluated CCRL2 to examine if it was an HIV/SIV coreceptor. The Human glioma cell line, NP-2, is normally resistant to infection by HIV and SIV. The cell was transduced with amplified cluster of differentiation 4 (CD4) as a receptor and CCR5, CXCR4 and CCRL2 as coreceptor candidates to produce NP-2/CD4/coreceptor cells (). The cells were infected with multiplicity of infection (MOI) 1.0. Infected cells were detected by indirect immunofluorescence assay (IFA). Multinucleated giant cells (MGC) in syncytia were quantified by Giemsa staining. Proviral DNA was detected by polymerase chain reaction (PCR), and reverse transcriptase (RT) activity was measured. IFA detected viral antigens of the primary isolates, HIV-1HAN2 and HIV-2MIR in infected NP-2/CD4/CCRL2 cells, indicated CCRL2 as a functional coreceptor. IFA results were confirmed by the detection of proviral DNA and measurement of RT-activity in the spent cell supernatants. Additionally, MGC was detected in HIV-2MIR-infected NP-2/CD4/CCCRL2 cells. HIV-2MIR were found more potent users of CCRL2 than HIV-1HAN2. Moreover, GWAS studies, gene ontology and cell signaling pathways of the HIV-associated genes show interaction of CCRL2 with HIV/SIV envelope protein. In vitro experiments showed CCRL2 to function as a newly identified coreceptor for primary HIV-2 isolates conveniently. The findings contribute additional insights into HIV/SIV transmission and pathogenesis. However, its in vivo relevance still needs to be evaluated. Confirming in vivo relevance, ligands of CCRL2 can be investigated as potential targets for HIV entry-inhibitor drugs.

Antinuclear antibodies in follicular fluid may reduce efficacy of in vitro fertilization and embryo transfer by invading endometrium and granular cells.

The mechanism(s) by which antinuclear antibodies (ANA) induce implantation failure are notclear, and little information regarding the function of autoantibodies in reproductive tissues is available. A total of 380 patients who underwent in vitro fertilization and embryo transfer (IVF-ET) were divided into control, serum positive, and follicular fluid (FF) positive groups based on the results of indirect immunofluorescence assay for ANA in the serum and FF. Immunofluorescence assay was performed to evaluate the existence of ANA in granular cells and endometrial tissues. Presence in FF of soluble apoptotic markers, including Bcl-2, Caspase-3, cleaved PARP, Cytochrome C, GAPDH, and p53, was assessed using magnetic bead based assays. The patients in the FF positive group had the lowest numbers of retrieved oocytes, fertilizations, and high-quality embryos. The fertilization rate, and the proportion of two pronuclear (2PN) embryos in patients in the FF positive group were significantly lower than those in the other two groups. The FF positive group also had the lowest clinical pregnancy rate, and the highest early miscarriage rate. Granulosa cells and endometrial tissues in patients in the FF positive group were ANA positive. High levels of BCL-2, Caspase-3, Cytochrome C, GAPDH, and p53 were found in the FF of patients in the FF positive group. Antinuclear antibodies in FF and endometrial tissues may cause imbalanced apoptosis, resulting in poor IVF-ET treatment outcomes. Local autoimmunity and cell apoptosis in reproductive tissues could be considered new therapeutic targets for improving IVF-ET treatment efficacy.

Cloning and expression of duck C4BPα and verification of its interaction with Riemerella anatipestifer

To study the interaction between C4b-binding protein (C4BP) and Riemerella anatipestifer (RA), we cloned duck C4BPα, conducted prokaryotic expression and prepared the polyclonal antibody by immunizing mice. Then indirect immunofluorescence assay and dot blotting hybridization assay were used to verify the interaction between C4BP and RA. The full length of duck C4BPα nucleotide sequence was 1 230 bp, with the highest similarity to chicken C4BPα (82.1%). Phylogenetic tree analysis showed that duck C4BPα and chicken C4BPα were on the same phylogenetic tree branch and the genetic evolution relationship between them was the closest. C4BPα was efficiently expressed in Escherichia coli BL21 (DE3). The recombinant proteins existed in intracellular soluble form. The titer of polyclonal antibody was more than 1:10 000 and polyclonal antibodies could specifically recognize the recombinant proteins. The results of indirect immunofluorescence assay and dot blot hybridization assay showed that RA could interact with duck C4BP. The results provide a basis to further reveal the pathogenesis of RA.

Assessment of antinuclear antibodies by indirect immunofluorescence assay: report from a survey by the American Association of Medical Laboratory Immunologists.

The indirect immunofluorescence assay (IFA) using HEp-2 cell substrates is the preferred method by some for detecting antinuclear antibodies (ANA) as it demonstrates a number of characteristic staining patterns that reflect the cellular components bound as well as semi-quantitative results. Lack of harmonized nomenclature for HEp-2 IFA patterns, subjectivity in interpretation and variability in the number of patterns reported by different laboratories pose significant harmonization challenges. The main objectives of this study were to assess current practice in laboratory assessment of HEp-2 IFA, identify gaps and define strategies to improve reading, interpretation and reporting. We developed and administered a 24-item survey based on four domains: educational and professional background of participants, current practice of HEp-2 IFA testing and training, gap assessment and the perceived value of International Consensus on Antinuclear Antibody Patterns (ICAP) and other factors in HEp-2 IFA assessment. The Association of Medical Laboratory Immunologists (AMLI) and American Society for Clinical Pathology administered the survey from April 1 to June 30, 2018, to members involved in ANA testing. This report summarizes the survey results and discussion from a dry workshop held during the 2019 AMLI annual meeting. One hundred and seventy-nine (n = 179) responses were obtained where a significant number were clinical laboratory scientists (46%), laboratory directors (24%), supervisors (13%) or others (17%). A majority of respondents agreed on the need to standardize nomenclature and reporting of HEp-2 IFA results. About 55% were aware of the ICAP initiative; however, among those aware, a significant majority thought its guidance on HEp-2 IFA nomenclature and reporting is of value to clinical laboratories. To improve ICAP awareness and further enhance HEp-2 IFA assessment, increased collaboration between ICAP and the clinical laboratory community was suggested with emphasis on education and availability of reference materials. Based on these suggestions, future efforts to optimize HEp-2 IFA reading, interpretation and reporting would benefit from more hands-on training of laboratory personnel as well as continuous collaboration between professional organizations, in vitro diagnostic manufacturers and clinical laboratories.

Monoclonal Antibody Against HA Protein of the European Avian-Like H1N1 Swine Influenza Virus.

The purified whole-virus proteins derived from A/swine/Shanghai/1/2014 (H1N1) (SH1) were chosen to immunize BALB/c mice to prepare the monoclonal antibody (MAb) against hemagglutinin (HA) protein of an European avian-like (EA) H1N1 swine influenza virus (SIV). After cloning three times by limiting dilution, one strain of hybridoma cells named 3C7 secreting anti-HA protein MAb was obtained by hybridoma technique. The results of indirect immunofluorescence assay and western blot analyses showed that the MAb 3C7 specifically reacted with the HA protein of EA H1N1 SIV. This work indicated that the MAb 3C7 would be a valuable tool as a specific diagnostic reagent for SIV epidemiological surveys and identification of HA protein epitopes of the EA H1N1 SIVs in the future.

Analysis of antigen-antibody cross-reactivity among lineages and sublineages of Babesia microti parasites using human babesiosis specimens.

Human babesiosis is caused mainly by Babesia microti and has recently become a public health concern due to an increase in transfusion-transmitted infection. Thus, the development of an antibody detection method with high specificity and sensitivity is a priority. Seroreactivity against B. microti has been reported to be highly specific not only to B. microti lineages but also to sublineages. This study aimed to elucidate the human antibody reactivity against various lineages, including US, Kobe, and Hobetsu, and sublineages (North America and East Asia) in the US lineage. Twenty samples obtained from individuals infected with B. microti in the United States were tested for the presence of anti-B. microti antibodies using indirect immunofluorescence assay (IFA) and Western blotting (WB) to indicate antigens of each (sub-)lineage. By IFA, 20 samples showed reactivity to the North America sublineage (titer range, 64-4096), 16 to the East Asia sublineage (64-512), 10 to the Kobe (64-128), and five to the Hobetsu (64). Antibody titers to the East Asia sublineage, Kobe, and Hobetsu were significantly lower than those to the North America sublineage (p < 0.01). By WB, in parallel with the IFA results, 18 samples showed strong reactions to the North America sublineage, weak reactions to the East Asia sublineage, and near-zero reactions to the Kobe and Hobetsu. Human antibodies induced by B. microti infection are highly specific against B. microti lineages and sublineages with low cross-reactivity. Developing a precise antibody detection method may require specific antigens based on B. microti lineages and sublineages.

The first positive serological study on rift valley fever in ruminants of Iran.

Rift Valley fever (RVF) is a zoonotic vector-borne disease that primarily affects domestic animals but can also infect humans. The purpose of the present study was to investigate the presence of antibodies against RVF virus (RVFV) in ruminants, viz. cattle, sheep, and goats in Kurdistan Province of western Iran. Blood samples were collected from 288 ruminants (118 cattle, 142 sheep and 28 goats) of both sexes, under age groups ≤1, 1-3, 3-5 and ≥5 yr, from January 2016 to December 2016. Clinical symptoms and history of abortion were recorded. The presence of RVFV-specific antibodies was investigated by using ELISA (competitive) and indirect immunofluorescence assay (IIFA) after separation of serum. The results of two tests were positive for five (1.74%) of total 288 animals which included two cattle of 118 (1.7%), and three sheep of 142 (2.11%). The results of IIFA were correlated with the ELISA results. All animals were clinically normal. No significant relationship between the RVFV infection rate and the variable considered, i.e. season, animal's age or sex, and the species of the animal (p ≥ 0.05), although there were four seropositive animals in the age group 1-3 and five seropositive animals in the spring season. The results of the study revealed the presence of low-level RVFV circulation among the ruminants of Kurdistan Province in Iran indicating that they are at risk of exposure to the virus during their lifetime. Since the present study was the first serological study on RVF in Iran with positive results, further studies are suggested including other areas of Iran.

Effect of nicotinic acetylcholine receptor α7 subunit gene on liver inflammatory reaction in mice with nonalcoholic steatohepatitis and related mechanisms

Objective: To investigate the effect of nicotinic acetylcholine receptor α7 (α7nAChR) subunit gene on liver inflammation in mice with nonalcoholic steatohepatitis (NASH) and related mechanisms. Methods: C57BL/6J mice and α7nAChR gene knockout mice were fed for 24 weeks to establish the NASH model, and the mice were sacrificed to isolate and culture the primary liver macrophages. After the treatment with nicotine and endotoxin, ELISA was used to measure the levels of the inflammatory factors interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in supernatant; indirect immunofluorescence assay and Western blot were used to observe the effect on the NF-κB signaling pathway, and quantitative PCR was used to measure the mRNA expression of Toll-like receptor-4 (TLR-4) in macrophages. An analysis of variance was used for comparison of means between multiple groups. Results: The results of ELISA showed that compared with the endotoxin+nicotine group of C57 NASH mice, the endotoxin+nicotine group of gene knockout NASH mice had significantly higher levels of IL-6 and TNF-α in supernatant (IL-6: 1 599±65 pg/ml vs 1 465±45 pg/ml, P < 0.05; TNF-α: 1 567±66 pg/ml vs 1 433±50 pg/ml, P < 0.05). The results of Western blot showed that compared with the endotoxin+nicotine group of C57 NASH mice, the endotoxin+nicotine group of gene knockout NASH mice had significantly higher relative protein expression of phosphorylated NF-κB and TLR-4 (NF-κB: 69 425±600 vs 51 133±200, P < 0.05; TLR-4: 93 387±684 vs 64 198±630, P < 0.05). The results of indirect immunofluorescence assay showed that the endotoxin+nicotine group of gene knockout NASH mice had a significantly higher fluorescence intensity of NF-κB than the endotoxin+nicotine group of C57 NASH mice. The results of PCR showed that the endotoxin+nicotine group of gene knockout NASH mice had significantly higher relative mRNA expression of TLR-4 than the endotoxin+nicotine group of C57 NASH mice (4.13±0.13 vs 2.93±0.14, P < 0.05). Conclusion: The α7nAChR gene knockout can aggravate the degree of inflammatory reaction in NASH, and its mechanism may be related to the fact that the NF-κB signaling pathway cannot be inhibited, which aggravates inflammatory reaction.

Antibodies for Rickettsia spp. in patients with negative serology for dengue virus, leptospirosis, and meningococcal disease in municipalities of São Paulo State, Brazil.

Brazilian spotted fever is an infectious disease with a high mortality rate if not treated early. Differential diagnosis is difficult, as the first clinical signs are non-specific and can be confused with other diseases. The aim of the study was to investigate evidence of infection with Rickettsia rickettsii and Rickettsia parkeri in negative sera samples, collected in 2014, from patients with suspected leptospirosis, dengue fever, and meningococcal disease in Atibaia and Bragança Paulista municipalities of the State of São Paulo. The samples stored at the Institute Adolfo Lutz in Campinas were tested using an indirect immunofluorescence assay (IFA) with IgG and IgM against R. rickettsii and R. parkeri. Real-time polymerase chain reaction (PCR) testing was performed for the sera samples of patients who died (n = 3), those with initial suspicion of meningococcal disease (n = 6), and those with positive IFA results. Of 258 samples from Bragança Paulista, 4 (1.6%) were positive, with IgG titers of 1:64 and 1:128 against R. rickettsii and R. parkeri, respectively. Of 155 samples from Atibaia, 2 (1.3%) were positive, with IgG titers of 1:64 and 1:128 against R. rickettsii and R. parkeri, respectively. No sample showed positive PCR results. This serological investigation suggests there is evidence of exposure to Rickettsia spp. in residents of areas that have environmental conditions favorable to the spread of bacteria, in which Brazilian spotted fever incidence was not previously confirmed.

Horses seropositive for Toxoplasma gondii, Sarcocystis spp. and Neospora spp.: Possible risk factors for infection in Brazil

Many parasitic diseases are considered asymptomatic, even though some studies have shown that they may cause pathological changes in the host. Therefore, the aim of this study was to evaluate the occurrence of antibodies against Toxoplasma gondii, Neospora spp. and Sarcocystis spp. in horses, and to identify the risk factors for disease. For this, 174 horses were studied, 90 males and 84 females aged between two and 20 years old. Blood samples were collected and stored in tubes without anticoagulant to obtain serum, which was subjected to serological tests for T. gondii, Sarcocystis spp., and Neospora spp. using indirect immunofluorescence assay (IFA). IFA results were as follows: Sarcocystis spp. 41.37% (72/174) (CI95%-34.05–49.09); T. gondii 32.18% (56/174) (CI95%-25.42–39.74) and Neospora spp. 48.27% (84/174) (CI95%-40.68.50–55.93). Out of 174 horses, 81 had simple infection, 61 had mixed infections with two or three of these pathogens, and therefore, only 32 horses showed no antibodies to any of these pathogens. No risk factors for Sarcocystis spp. and T. gondii infection were identified. However, there was a significant (1.22-CI95%-1.02–1.52) relationship between animal age and Neospora spp. infection, since older animals showed higher prevalence. Therefore, it was possible to conclude that T. gondii and Neospora spp. affect horses in Southern Brazil, however all the animals studied were asymptomatic without reproductive, neurological or locomotor problems.

The assembly of virus-like particles of porcine transmissible gastroenteritisvirus in vitro by baculovirus expression system

Virus-like particles (VLPs) produced by recombinant expression of the major viral structural proteins could be an attractive method for transmissible gastroenteritis virus (TGEV) assembly and release. In this study, the M gene, sM gene, and N gene of TGEV were inserted into donor plasmid pFastBac Dual. Those recombinant donor plasmids were transformed into E.coli DH10Bac respectively. Recombinant baculoviruses were obtained by transfection of the Sf9 cells with Bacmid-M, Bacmid-sM, and Bacmid-N. The results of indirect immunofluorescence assay showed that recombinant M protein, sM protein, and N protein were expressed successfully. In vitro experiments of VLP assembly of TGEV were performed by infection of insect cells with various combinations of recombinant baculoviruses identified. It is shown that expression of M protein alone, coexpression of M plus sM protein, and coexpression of M plus N protein in Sf9 insect cells infected by recombinant baculovirus could result in the occurrence of a virion morphologically similar to TGEV. However, the sM and N protein could not generate a structure similar to the virion of TGEV when expressed alone. This study provides a theoretical foundation for further study on the role of TGEV structural proteins in the assembly process of virions.

Protective efficacies and immune responses induced by recombinant HCD, atpD and gdhA against bacterial cold-water disease in ayu (Plecoglossus altivelis)

Protective efficacies of three antigenic proteins (3-hydroxyacyl-CoA dehydrogenase (HCD), ATP synthase beta subunit (atpD), and glutamate dehydrogenase (gdhA)) against Flavobacterium psychrophilum were investigated in ayu (Plecoglossus altivelis). Recombinant proteins of HCD, atpD, and gdhA were expressed in Escherichia coli BL21 cells. Ayu were then vaccinated with inactivated cells via the intraperitoneal route. Compared with the empty BL21- and PBS-injected groups, the vaccinated group had a significantly longer survival time after challenge with F. psychrophilum. The antibody titers against each recombinant protein were significantly higher in serum from vaccinated fish, compared with serum from control fish. Results of indirect immunofluorescence assays using serum indicated that the HCD, atpD, and gdhA proteins are located on the surface of F. psychrophilum. These results suggest that these three surface proteins are protective antigens and are good candidates for development of vaccines against bacterial cold-water disease in ayu.