Enzyme-Linked Immunosorbent Assays Based on Neospora caninum Dense Granule Protein 7 and Profilin for Estimating the Stage of Neosporosis

ABSTRACTNeospora caninumis an intracellular protozoan parasite that causes bovine and canine neosporosis, characterized by fetal abortion and neonatal mortality and by neuromuscular paralysis, respectively. Although many diagnostic methods to detect parasite-specific antibodies or parasite DNA have been reported, to date no effective serodiagnostic techniques for estimating pathological status have been described. Our study aimed to elucidate the relationship between the parasite-specific antibody response, parasite activation, and neurological symptoms caused byN. caninuminfection by using a recombinant antigen-based enzyme-linked immunosorbent assay. Among experimentally infected mice, anti-N. caninumprofilin (NcPF) antibody was only detected in neurologically symptomatic animals. Parasite numbers within the brains of the symptomatic mice were significantly higher than those in asymptomatic animals. In addition, anti-NcPF and anti-NcGRA7 antibodies were mainly detected at the acute stage in experimentally infected dogs, while anti-NcSAG1 antibody was produced during both acute and chronic stages. Furthermore, among anti-NcSAG1 antibody-positive clinical dogs, the positive rates of anti-NcGRA7 and anti-NcPF antibodies in the neurologically symptomatic dogs were significantly higher than those in the non-neurologically symptomatic animals. Our results suggested that the levels of anti-NcGRA7 and anti-NcPF antibodies reflect parasite activation and neurological symptoms in dogs. In conclusion, antibodies against NcGRA7 and NcPF may have potential as suitable indicators for estimating the pathological status of neosporosis.

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Development of an Immunochromatographic Assay Based on Dense Granule Protein 7 for Serological Detection of Toxoplasma gondii Infection

Clinical and Vaccine Immunology ◽

10.1128/cvi.00747-12 ◽

2013 ◽

Vol 20 (4) ◽

pp. 596-601 ◽

Cited By ~ 32

Author(s):

Mohamad Alaa Terkawi ◽

Kyohko Kameyama ◽

Nazim Hamza Rasul ◽

Xuean Xuan ◽

Yoshifumi Nishikawa

Keyword(s):

Toxoplasma Gondii ◽

Neospora Caninum ◽

Latex Agglutination ◽

Dense Granule ◽

Diagnostic Tools ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Content Type ◽

Specificity And Sensitivity ◽

Field Samples

ABSTRACTDense granule antigen proteins derived fromToxoplasma gondii(TgGRAs) are potential antigens for the development of diagnostic tools. TgGRA7 and TgGRA14 were detected in the peritoneal fluid ofT. gondii-infected mice, suggesting that TgGRAs may be highly antigenic proteins. Here, TgGRA7 and TgGRA14 were evaluated as candidates for the development of a marker for a rapid diagnostic test. The specificity and sensitivity of purified recombinant proteins of TgGRA7 and TgGRA14 were compared in an indirect enzyme-linked immunosorbent assay (iELISA) using a series of serum samples fromT. gondii-experimentally infected mice and using recombinantT. gondiimajor surface antigen 2 (TgSAG2) as a reference control. The iELISA with TgGRA7 showed the greatest diagnostic accuracy and could detect anti-TgGRA7 antibody in acute and chronic infections. A total of 59 field samples from pigs were also examined by the iELISAs, and the results compared with those of the latex agglutination test (LAT). Among the three recombinant antigens, TgGRA7 had the highest rates of positivity, with significant concordance (88.14) and kappa value (0.76) in comparison with the results using LAT. Furthermore, an immunochromatographic test (ICT) based on recombinant TgGRA7 was developed for rapid detection of antibodies to the infection. The ICT differentiated clearly between sera fromT. gondii-infected mice and uninfected orNeospora caninum-infected mice. Pig sera were examined with the ICT, and the results compared favorably with those of LAT and iELISA for TgGRA7, with kappa values of 0.66 and 0.70 to 0.79, respectively. These data suggest that the ICT based on TgGRA7 is a promising diagnostic tool for routine testing in the clinic and mass screening of samples in the field.

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Matrix metalloproteinase—9 concentration after spontaneous intracerebral hemorrhage

Journal of Neurosurgery ◽

10.3171/jns.2003.99.1.0065 ◽

2003 ◽

Vol 99 (1) ◽

pp. 65-70 ◽

Cited By ~ 119

Author(s):

Sònia Abilleira ◽

Joan Montaner ◽

Carlos A. Molina ◽

Jasone Monasterio ◽

José Castillo ◽

...

Keyword(s):

Intracerebral Hemorrhage ◽

Neurological Diseases ◽

Neurological Status ◽

Acute Stage ◽

Ct Scans ◽

Enzyme Linked Immunosorbent Assay ◽

Content Type ◽

Blood Brain Barrier Disruption ◽

Brain Barrier Disruption ◽

Fine Print

Object. Matrix metalloproteinases (MMPs) are overexpressed in the presence of some neurological diseases in which blood—brain barrier disruption exists. The authors investigated the MMP-9 concentration in patients after acute intracerebral hemorrhage (ICH) and its relation to perihematomal edema (PHE). Methods. Concentrations of MMP-9 and related proteins were determined in plasma by performing an enzyme-linked immunosorbent assay of samples drawn after hospital admission (< 24 hours after stroke) from 57 patients with ICH. The diagnosis of ICH was made on the basis of findings on computerized tomography (CT) scans. The volumes of ICH and PHE were measured on baseline and follow-up CT scans at the same time that the patient's neurological status was assessed using the Canadian Stroke Scale and the Glasgow Coma Scale. Increased expression of MMP-9 was found among patients with ICH. In cases of deep ICH, MMP-9 was significantly associated with PHE volume (r = 0.53; p = 0.01) and neurological worsening (237.4 compared with 111.3 ng/ml MMP-9; p = 0.04). A logistic regression model focusing on the study of absolute PHE volume showed ICH volume as an independent predictor (odds ratio [OR] 3.37; 95% confidence interval [CI] 1.1–10.3; p = 0.03). A second analysis of relative PHE volume (absolute PHE volume/ICH volume) in patients with deep ICH demonstrated that the only factor related to it was MMP-9 concentration (OR 11.6; 95% CI 1.5–89.1; p = 0.018). Conclusions. Expression of MMP-9 is raised after acute spontaneous ICH. Among patients with deep ICH this increase is associated with PHE and the development of neurological worsening within the acute stage.

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Evidence of Neospora caninum exposure among native Korean goats (Capra hircus coreanae)

Veterinární Medicína ◽

10.17221/7824-vetmed ◽

2014 ◽

Vol 59 (No. 12) ◽

pp. 637-640 ◽

Cited By ~ 5

Author(s):

BY Jung ◽

SH Lee ◽

D. Kwak

Keyword(s):

Neospora Caninum ◽

Control Strategies ◽

Integrated Control ◽

Protozoan Parasite ◽

Capra Hircus ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Caninum Infection ◽

One Year ◽

And Control

Neospora caninum is a protozoan parasite that causes abortion in ruminants, including goats. The objective of the present study was to determine the seroprevalence of N. caninum in native Korean goats (Capra hircus coreanae). A commercial enzyme-linked immunosorbent assay kit was used to analyse 464 serum samples for the presence of N. caninum antibodies. Four samples (0.9%, 95% confidence intervals – CI: 0.0–1.7) were found to be positive for N. caninum antibodies. The seroprevalence was analysed according to age (less than to one year, young; more than or equal one year, adult; and unknown), sampling season (April to September, warm; October to March, cold), and region (northern, central, and southern). However, there were no statistically significant differences in seroprevalence according to age, season, and region (P > 0.05). This is the first report on the seroprevalence of N. caninum in native Korean goats. The results of this study indicate a nationwide distribution of N. caninum among goats, with a relatively low prevalence. Therefore, the implementation of integrated control strategies as well as measures for prevention and control of N. caninum infection among goats is recommended.

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Diagnosis of inflammatory peri-implant diseases using an immunochromatographic assay for calprotectin in peri-implant crevicular fluid

International Journal of Implant Dentistry ◽

10.1186/s40729-021-00386-z ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Rie Kido ◽

Jun-ichi Kido ◽

Yasufumi Nishikawa ◽

Eijiro Sakamoto ◽

Yoritoki Tomotake ◽

...

Keyword(s):

Operating Characteristic ◽

Inflammatory Marker ◽

Diagnostic System ◽

Diagnostic Methods ◽

Enzyme Linked Immunosorbent Assay ◽

Roc Curve Analysis ◽

Implant Placement ◽

Probing Depth ◽

Crevicular Fluid ◽

The Relationship

Abstract Background The incidence rate of peri-implant diseases is increasing with implant placement. Early detection of peri-implant diseases is important to prevent and treat these diseases, and a simple and objective diagnostic method is expected. Immunochromatographic (IC) assays are used for rapid diagnostic methods for some diseases. The aim of this clinical study was to determine the amount of calprotectin, an inflammatory marker, in peri-implant crevicular fluid (PICF) using an IC chip, and estimate the possibility of this diagnostic system. Methods Forty-six individuals with dental implants participated in a pilot study. PICF samples were collected from the peri-implant sites with or without inflammation after clinical examinations including probing depth (PD), bleeding on probing (BOP) and gingival index (GI). Calprotectin in PICF was determined by an IC chip and enzyme-linked immunosorbent assay (ELISA) for calprotectin. The density of calprotectin line on the IC chip was measured using an IC reader (IC reader value). The relationship between IC reader value and ELISA value or clinical parameters was investigated. A receiver operating characteristic (ROC) curve analysis of IC reader value of calprotectin was performed to predict inflammation in peri-implant diseases. Results IC reader value of calprotectin was significantly correlated with its ELISA value and PD. IC reader values of calprotectin in PICF samples from periodontal sites with GI-1 and GI-2, and with BOP-positive sites were significantly higher than those of PICF samples from GI-0 sites, and BOP-negative sites, respectively. The IC reader value for calprotectin in PICF samples from inflammatory diseased sites was significantly higher than that of non-diseased sites. ROC analysis suggested that the IC reader value of PICF calprotectin was useful for predicting inflammatory peri-implant diseases. Conclusion IC assay for PICF calprotectin may be a possible system for diagnosing the inflammatory peri-implant diseases.

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Comparative study between ECL and ELISA to determine the reliable range of Estradiol in the treatment of infertility

Bionatura ◽

10.21931/rb/2020.05.04.12 ◽

2020 ◽

Vol 5 (4) ◽

pp. 1352-1355

Author(s):

Ali Sadeghitabar ◽

Narges Maleki ◽

Maryam Armand ◽

Nasiri Reza

Keyword(s):

Human Subjects ◽

Pearson Correlation ◽

Diagnostic Methods ◽

Enzyme Linked Immunosorbent Assay ◽

P Value ◽

Vitro Fertilization ◽

The Relationship ◽

Different Levels ◽

Estradiol Concentration

Estradiol is one factor that can alter the outcome of the treatment of infertile couples following the application of in vitro fertilization techniques. Currently, the estradiol level is measured by two diagnostic methods Enzyme-Linked Immunosorbent Assay (ELISA) and Electrochemiluminescence (ECL). Accordingly, this study determines ELISA and ECL's sensitivity and consistency to measure different levels of estradiol and determine its reliable range and provide this information to laboratories and gynecologists. This study is performed on 250 patients of the Avicenna Fertility Center. The data of the study are analyzed in SPSS18 and MiniTab. Consent was obtained for experimentation with human subjects. Pearson correlation coefficient was used to investigate the relationship between these two methods. The results indicated a strong correlation between the two variables ECL and ELISA ( r= 0.735, P-value<0.001). High numbers indicate that the decrease and increase of one variable are proportional to the other variable's fluctuation. This study shows that the results of estradiol obtained from both ECL and ELISA are similar. In the ELISA method, due to the linear values' limitation, samples with estradiol concentration above the highest standard level should be diluted and the dilution coefficient should then be applied.

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A study of Neospora caninum antibody seroprevalence ın dairy cows in Turkey

Journal of the Hellenic Veterinary Medical Society ◽

10.12681/jhvms.22950 ◽

2020 ◽

Vol 71 (1) ◽

pp. 2018

Author(s):

S. KASAP ◽

S. ERTUNC ◽

E. M. TEMIZEL ◽

S. SENTURK

Keyword(s):

Blood Serum ◽

Dairy Cows ◽

Neospora Caninum ◽

Protozoan Parasite ◽

Total Sample ◽

Enzyme Linked Immunosorbent Assay ◽

Marmara Region ◽

Serum Samples ◽

Caninum Antibody ◽

Intracellular Protozoan Parasite

Neospora caninum is a intracellular protozoan parasite and is one of the major causes of repeated abortions, foetal malformations, pre-term deliveries, stillbirth and possible loss of milk yield in livestock. The presence of specific antibodies against N. caninum in the blood serum of dairy cows is investigated in the present study. A total of 184 blood serum samples of dairy cows were examined in Bursa province in the Marmara Region. N. caninum antibodies were measured using an indirect enzyme-linked immunosorbent assay (ELISA) (The Svanovir Neospora-Ab ELISA). From the total sample, antibodies to N. caninum were detected in 62 of the 184 examined cows (33.3%) and neurological findings were seen in a calf.

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Seroprevalence of Neospora caninum infection in dairy cows in Northern provinces, Thailand

Acta Parasitologica ◽

10.2478/s11686-014-0245-z ◽

2014 ◽

Vol 59 (2) ◽

Cited By ~ 8

Author(s):

Tawin Inpankaew ◽

Sathaporn Jittapalapong ◽

Thomas Mitchell ◽

Chainirun Sununta ◽

Ikuo Igarashi ◽

...

Keyword(s):

Dairy Cows ◽

Neospora Caninum ◽

Fluorescent Antibody ◽

Protozoan Parasite ◽

Antibody Test ◽

Current Status ◽

Enzyme Linked Immunosorbent Assay ◽

Caninum Infection ◽

Slow Growth Rate ◽

The Impact

AbstractNeospora caninum, an obligate intracellular protozoan parasite, is the causative agent of neosporosis, recognized as a major cause of bovine abortion around the world. Thailand is a developing agricultural country located in Southeast Asia. Livestock developments particularly in dairy cows of this country have been hampered by low productivity including milk and slow growth rate due to the impact of many pathogens including N. caninum. Currently, there is no effective method for control of neosporosis since there is less information regarding current status of infections. The objective of this study was to investigate the seroprevalence of neosporosis in dairy cows of the northern part of Thailand. During 2006–2007, the sera of 642 cows from 42 small farm holders with the top three highest consensus of dairy farms in the northern provinces, such as Chiang Rai, Chiang Mai and Lumpang were collected and performed tests. Antibodies to N. caninum were assayed by enzyme-linked immunosorbent assay (ELISA) with recombinant N. caninum surface antigen 1 (NcSAG1) and indirect fluorescent antibody test (IFAT). The overall prevalence of N. caninum infection in this study was 46.9% (301/642) by ELISA and 34.3% (220/642) by IFAT.

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Toll-Like Receptor 3–TRIF Pathway Activation by Neospora caninum RNA Enhances Infection Control in Mice

Infection and Immunity ◽

10.1128/iai.00739-18 ◽

2019 ◽

Vol 87 (4) ◽

Cited By ~ 5

Author(s):

Vanessa dos Santos Miranda ◽

Flávia Batista Ferreira França ◽

Mylla Spirandelli da Costa ◽

Vanessa Resende Souza Silva ◽

Caroline Martins Mota ◽

...

Keyword(s):

Neospora Caninum ◽

Parasitophorous Vacuole ◽

Protozoan Parasite ◽

Interferon Response ◽

Economic Losses ◽

Toll Like Receptor ◽

Content Type ◽

Pathway Activation ◽

Th1 Immune Responses

ABSTRACT Neospora caninum is a protozoan parasite closely related to Toxoplasma gondii and has been studied for causing neuromuscular disease in dogs and abortions in cattle. It is recognized as one of the main transmissible causes of reproductive failure in cattle and consequent economic losses to the sector. In that sense, this study aimed to evaluate the role of Toll-like receptor 3 (TLR3)–TRIF-dependent resistance against N. caninum infection in mice. We observed that TLR3−/− and TRIF−/− mice presented higher parasite burdens, increased inflammatory lesions, and reduced production of interleukin 12p40 (IL-12p40), tumor necrosis factor (TNF), gamma interferon (IFN-γ), and nitric oxide (NO). Unlike those of T. gondii, N. caninum tachyzoites and RNA recruited TLR3 to the parasitophorous vacuole (PV) and translocated interferon response factor 3 (IRF3) to the nucleus. We also observed that N. caninum upregulated the expression of TRIF in murine macrophages, which in turn upregulated IFN-α and IFN-β in the presence of the parasite. Furthermore, TRIF−/− infected macrophages produced lower levels of IL-12p40, while exogenous IFN-α replacement was able to completely restore the production of this key cytokine. Our results show that the TLR3-TRIF signaling pathway enhances resistance against N. caninum infection in mice, since it improves Th1 immune responses that result in controlled parasitism and reduced tissue inflammation, which are hallmarks of the disease.

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Chimeric Protein Designed by Genome-Scale Immunoinformatics Enhances Serodiagnosis of Bovine Neosporosis

Journal of Clinical Microbiology ◽

10.1128/jcm.01343-19 ◽

2020 ◽

Vol 58 (7) ◽

Author(s):

Higor Sette Pereira ◽

Ludmila Tavares e Almeida ◽

Vitória Fernandes ◽

Renato Lima Senra ◽

Patrícia Pereira Fontes ◽

...

Keyword(s):

Neospora Caninum ◽

Leukemia Virus ◽

Clinical Signs ◽

Chimeric Protein ◽

Cross Reactivity ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Noninvasive Test ◽

Content Type ◽

Bovine Neosporosis

ABSTRACT Neosporosis has become a concern since it is associated with abortion in cattle. Currently, in situ diagnosis is determined through anamnesis, evaluation of the history, and perception of the clinical signs of the herd. There is no practical and noninvasive test adapted to a large number of samples, which represents a gap for the use of new approaches that provide information about infections and the risks of herds. Here, we performed a search in the Neospora caninum genome by linear B-cell epitopes using immunoinformatic tools aiming to develop a chimeric protein with high potential to bind specifically to antibodies from infected cattle samples. An enzyme-linked immunosorbent assay with the new chimeric antigen was developed and tested with sera from natural field N. caninum-infected bovines. The cross-reactivity of the new antigen was also evaluated using sera from bovines infected by other abortive pathogens, including Trypanosoma vivax, Leptospira sp., Mycobacterium bovis, and Brucella abortus, and enzootic bovine leucosis caused by bovine leukemia virus, as well as with samples of animals infected with Toxoplasma gondii. The assay using the chimeric protein showed 96.6% ± 3.4% of sensitivity in comparison to healthy animal sera. Meanwhile, in relation to false-positive results provided by cross-reactivity with others pathogens, the specificity value was 97.0% ± 2.9%. In conclusion, immunoinformatic tools provide an efficient platform to build an accurate protein to diagnose bovine neosporosis based on serum samples.

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B-Cell-Specific Peptides of Leptospira interrogans LigA for Diagnosis of Patients with Acute Leptospirosis

Clinical and Vaccine Immunology ◽

10.1128/cvi.00456-13 ◽

2014 ◽

Vol 21 (3) ◽

pp. 354-359 ◽

Cited By ~ 11

Author(s):

Murugesan Kanagavel ◽

Santhanam Shanmughapriya ◽

Kumarasamy Anbarasu ◽

Kalimuthusamy Natarajaseenivasan

Keyword(s):

Early Diagnosis ◽

B Cell ◽

Predictive Value ◽

Acute Stage ◽

Antibody Detection ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Serological Tests ◽

Content Type

ABSTRACTLeptospirosis is a reemerging infectious disease that is underdiagnosed and under-recognized due to low-sensitivity and cumbersome serological tests. Rapid reliable alternative tests are needed for early diagnosis of the disease. Considering the importance of the pathogenesis-associated leptospiral LigA protein expressedin vivo, we have evaluated its application in the diagnosis of the acute form of leptospirosis. The C-terminal coding sequence ofligA(ligA-C) was cloned into pET15b and expressed inEscherichia coli. Furthermore, the B-cell-specific epitopes were predicted and were synthesized as peptides for evaluation along with recombinant LigA-C. Epitope 1 (VVIENTPGK), with a VaxiJen score of 1.3782, and epitope 2 (TALSVGSSK), with a score of 1.2767, were utilized. A total of 140 serum samples collected from leptospirosis cases during the acute stage of the disease and 138 serum samples collected from normal healthy controls were utilized for evaluation. The sensitivity, specificity, positive predictive value, and negative predictive value were calculated for the recombinant LigA-C-specific IgM enzyme-linked immunosorbent assay (ELISA) and were found to be 92.1%, 97.7%, 92.8%, and 97.5%, respectively. Epitopes 1 and 2 used in the study showed 5.1 to 5.8% increased sensitivity over recombinant LigA-C in single and combination assays for IgM antibody detection. These findings suggest that these peptides may be potential candidates for the early diagnosis of leptospirosis.

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