Characteristic band pattern in western blots for specific detection of anti-Trichinella spiralis antibodies in different host species

Abstract Western blot (Wb) is considered to be the gold standard test for Trichinella infection serology, since this method allows specific Trichinella antigens to be distinguished from cross-reactive antigens. This is not the case with widely used antibody assay techniques - indirect immunofluorescence and ELISA - which are sensitive, but subject to crossreactions that make the interpretation of weakly positive results difficult. Application of Trichinella spiralis muscle larvae excretory-secretory (ES) antigens for the specific antibody detection in ELISA resulted in improved specificity compared to that of crude worm extract that was previously in use, but since production of ES has not yet been standardized, differences among laboratories occur. For this reason, the Wb profile of serum samples from different T. spiralis infected host species: human, horse, swine and dog, was investigated in the Serbian National Reference Laboratory for Trichinellosis (NRLT). The common feature of the obtained Wb profiles was the appearance of a triad of bands with molecular masses (Mw) of 45, 49, and 53 kDa. The very same triad was recognized by a monoclonal antibody (mAb) 7C2C5 specific for an immunodominant epitope unique to the muscle larvae stage of all species in the genus Trichinella. Inhibition studies confirmed that mAb and anti-Trichinella antibodies from sera competed for the same parasite epitope. Based on the obtained results, the NRLT introduced the recognition of the above mention triad as the basis for specific anti-Trichinella antibodies detection in the sera of infected hosts.

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Detection of circulating antigens in serum samples of mice experimentally infected with Trichinella spiralis by a sandwich ELISA based on IgY

Helminthologia ◽

10.2478/s11687-014-0227-6 ◽

2014 ◽

Vol 51 (3) ◽

pp. 181-189 ◽

Cited By ~ 1

Author(s):

F. Jing ◽

J. Cui ◽

R. Liu ◽

L. Liu ◽

P. Jiang ◽

...

Keyword(s):

Post Infection ◽

Trichinella Spiralis ◽

Sandwich Elisa ◽

Egg Yolk ◽

Mouse Serum ◽

Serum Samples ◽

Egg Yolk Immunoglobulin ◽

Muscle Larvae ◽

Sandwich Elisa Assay ◽

Circulating Antigens

AbstractIn the present study, a sandwich ELISA based on IgY (egg yolk immunoglobulin) was developed for detection of circulating antigens (CAg) in sere of mice experimentally infected with Trichinella spiralis. The IgY-sandwich ELISA assay involved the use of chicken antibody IgY against excretory-secretory (ES) antigens of Trichinella spiralis muscle larvae as a capture antibody and mouse polyclonal antibody IgG to ES antigens as a detecting antibody. This method was able to detect as little as 3 ng/ml of ES antigens added to normal mouse serum. A group of sixteen mice was orally inoculated with 500 T. spiralis muscle larvae per animal. The serum samples from the infected mice were taken during 1–35 days post-infection (dpi). The CAg was detectable as early as 8 dpi in the sera of infected mice. The level of CAg increased dramatically during 13–15 dpi and reached a peak at 22 dpi and remained a plateau for 3 days, then declined gradually. Another peak of CAg occurred at 31 dpi. The anti-Trichinella antibodies was first detected in 14.3 % of the infected mice at 2 weeks post-infection (wpi), and reached a peak positive rate of 100 % at 5 wpi. Moreover, the infected mice were treated with abendazole at 5 wpi and the serum CAg levels increased significantly during 2–6 days posttreatment (dpt) and then declined rapidly during 8–14 dpt. By 42 dpt, the CAg levels decreased to the undetected level, but the detection rate of antibodies was still 100 %. The IgY-sandwich ELISA appears to be a sensitive for detection of antigenemia of T. spiralis and valuable to judge the efficacy of chemotherapy in trichinellosis.

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A panel of antigens of muscle larvae of Trichinella spiralis and T. pseudospiralis as revealed by two-dimensional Western blot and immunoelectron microscopy

Parasitology ◽

10.1017/s0031182099004187 ◽

1999 ◽

Vol 118 (6) ◽

pp. 615-622 ◽

Cited By ~ 14

Author(s):

Z. WU ◽

I. NAGANO ◽

Y. TAKAHASHI

Keyword(s):

Western Blot ◽

Trichinella Spiralis ◽

Cross Reactivity ◽

Microscopical Study ◽

Detectable Level ◽

Antigenic Peptides ◽

Two Dimensional ◽

Western Blots ◽

Muscle Larvae ◽

Isoelectric Points

This study characterized antigens of Trichinella spiralis and T. pseudospiralis muscle larvae recognized by mice infected with the worms. Two-dimensional (2-D) Western blot analysis revealed some profile of antigenic peptides including: (1) molecular weight (MW); (2) isoelectric points (pI), (3) reactivity to well-defined monoclonal antibodies (mAb) and (4) cross-reactivity between the 2 species. Antigenic peptides of T. spiralis consisted of about 100 spots. The MW ranged from 22 to 80 kDa, and pI ranged from 4 to 7. The mAb against TSL-1 stained most of the T. spiralis excretory–secretory (E–S) peptides migrating at 40, 45 and 50 kDa, and the mAb against TSL-4 stained non-E–S peptides. Antigenic peptides of T. pseudospiralis consisted of about 20 to 30 peptide spots. The MW ranged from 25 to 80 kDa, and pI ranged from 4 to 7. The mAb against TSL-1 stained most of the T. pseudospiralis E–S peptides migrating at 35 and 45 kDa, and the mAb against TSL-4 stained non-E–S peptides. Two-dimensional Western blots showed that the E–S products of T. spiralis and T. pseudospiralis were highly cross-reactive with each other. The non-E–S peptides were, however, not recognized by T. pseudospiralis-infected sera but were recognized by T. spiralis-infected sera. An immunoelectron microscopical study showed the similar result that stichocyte granules and cuticle surface (known to contain E–S antigen) had cross-reactive antigens between the two species. T. pseudospiralis-infected sera stained very weakly the cuticle inner layers and haemolymph (known to contain non-E–S antigen). This evidence implies that mice infected with T. pseudospiralis do not evoke antibodies against non-E–S antigen at the detectable level.

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Use of Proficiency Samples To Assess Diagnostic Laboratories in France Performing a Trichinella Digestion Assay

Journal of Food Protection ◽

10.4315/0362-028x-70.7.1685 ◽

2007 ◽

Vol 70 (7) ◽

pp. 1685-1690 ◽

Cited By ~ 13

Author(s):

ISABELLE VALLÉE ◽

PAULINE MACÉ ◽

LORRY FORBES ◽

BRAD SCANDRETT ◽

BENOIT DURAND ◽

...

Keyword(s):

Trichinella Spiralis ◽

Reference Laboratory ◽

Test Sensitivity ◽

National Reference ◽

Large Samples ◽

Routine Diagnosis ◽

Muscle Larvae ◽

Artificial Digestion ◽

Meat Samples ◽

Subsequent Identification

Routine diagnosis of animal trichinellosis for food safety and trade relies on a method of artificial digestion to free Trichinella muscle larvae from meat for subsequent identification by microscopy. As part of a quality control system, the French National Reference Laboratory (NRL) initiated ring trials to determine the sensitivity of the test performed in the 72 routine diagnostic laboratories in France. A method was devised to obtain calibrated meat samples containing known numbers of capsules with Trichinella spiralis muscle larvae. This method was based on an incomplete artificial digestion of Trichinella-infected mice carcasses to allow the collection of intact Trichinella capsules. Capsules were placed into a meatball of 100 ± 2 g of pork and horsemeat to produce proficiency samples. Three categories of samples were prepared: small (3 to 5 capsules), medium (7 to 10), and large (12 to 15). The sensitivity was expressed as the percentage of muscle larvae recovered from each proficiency sample. Reproducibility was tested with ring trials organized between two NRLs (France and Canada), and a reference sensitivity of 84.9% was established. National ring trials were then organized in France, with the 72 routine diagnostic laboratories each receiving four proficiency samples per session. After five sessions, an improvement in the digest test sensitivity was observed. Results at the fifth session indicated sensitivities of 78.60% ± 23.70%, 81.19% ± 19.59%, and 80.52% ± 14.71% muscle larvae for small, medium, and large samples, respectively. This study supports the use of proficiency samples to accurately evaluate the performance of routine diagnostic laboratories that conduct digestion tests for animal trichinellosis diagnosis.

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Serological Evaluation of Thin-Layer Immunoassay–Enzyme-Linked Immunosorbent Assay for Antibody Detection in Human Trichinellosis

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.7.5.810-812.2000 ◽

2000 ◽

Vol 7 (5) ◽

pp. 810-812 ◽

Cited By ~ 4

Author(s):

Alberto Gómez-Priego ◽

Lidia Crecencio-Rosales ◽

Jorge-Luis de-la-Rosa

Keyword(s):

Thin Layer ◽

Immunosorbent Assay ◽

Trichinella Spiralis ◽

Field Studies ◽

Antibody Detection ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Laboratory Equipment ◽

Specialized Equipment ◽

Human Trichinellosis

ABSTRACT A new immunoenzymatic test, named the thin-layer immunoassay–enzyme-linked immunosorbent assay (TIA-ELISA), was evaluated for antibody detection in human trichinellosis using excretion and secretion products prepared from Trichinella spiralis muscle larvae. Serum samples from people with positive muscle biopsies or symptoms compatible with the disease (n = 8 or 26, respectively), all reactive in enzyme-linked immunoelectrotransfer blot assay (EITB), as well as 67 serum samples from healthy, EITB-negative people, were tested in an ELISA and TIA-ELISA. TIA-ELISA was performed in polystyrene plastic petri dishes by adding dots of 10 μl each of antigen (7 μg/ml) followed by adding diluted serum and the conjugate. Finally, the substrate mixed with agar was added to develop the reaction. Enzymatic by-products were easily detected by the naked eye as defined dots. Sensitivity and specificity were 76 and 94% for ELISA, and both parameters were 91% for TIA-ELISA. The kappa correlation indices for both tests in relation to EITB were 0.73 and 0.80, respectively. The TIA-ELISA can be carried out with common laboratory equipment in 3 h and uses lower quantities of antigen than EITB and ELISA. Since TIA-ELISA is easy to perform, cheap, sensitive, and specific, the test could be an acceptable alternative to use in clinical laboratories lacking specialized equipment needed for ELISA and EITB and in field studies for antibody detection in human trichinellosis.

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Trichinella nativa and T. spiralis Induce Distinguishable Histopathologic and Humoral Responses in the Raccoon Dog (Nyctereutes procyonoides)

Veterinary Pathology ◽

10.1354/vp.39-2-257 ◽

2002 ◽

Vol 39 (2) ◽

pp. 257-265 ◽

Cited By ~ 14

Author(s):

A. Sukura ◽

A. Näreaho ◽

T. Mikkonen ◽

M. Niemi ◽

L. Oivanen

Keyword(s):

Trichinella Spiralis ◽

Total Surface Area ◽

Enzyme Linked Immunosorbent Assay ◽

Control Group ◽

Nyctereutes Procyonoides ◽

Raccoon Dog ◽

Triceps Brachii ◽

Serum Samples ◽

Western Blots ◽

Homologous Antigen

Three experimental groups of six male raccoon dogs ( Nyctereutes procyonoides) each were formed by placing one of three littermates from six litters into each group. One group was inoculated with pigorigin Trichinella spiralis, the second was inoculated with raccoon dog-origin T. nativa, and the third served as a control group. The infective dose was 1,000 larvae/kg of body weight. Every third week, biopsies from M. triceps brachii were taken, and serum samples were collected for up to 12 weeks postinfection. In the early phase of the infection, cysts of both parasites were elongated cylinders that later became more spherical. However, at the end of the experiment, the cysts of T. nativa were more rounded than those of T. spiralis (mean length/width = 2.5 versus 1.5 in T. spiralis versus T. nativa, respectively). Both species accumulated a collagen-rich capsule around the nurse cell, but the capsule was thicker in T. nativa. In both parasites, the total surface area of the sagittal section of the cyst was equal. Inflammation was more intense around T. nativa cysts. Specific antibodies were recognizable 2 weeks after infection by both enzyme-linked immunosorbent assay (ELISA) and western blot. In western blots, serum from both T. nativa- and T. spiralis-infected animals recognized the same components, but reaction with the homologous antigen was stronger. The same pattern was also seen in the ELISA. Immunoreactive epitopes were localized only in internal organs and cuticula of larvae in muscle.

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Molecular cloning and expression of the full-length tropomyosin gene from Trichinella spiralis

Journal of Helminthology ◽

10.1079/joh2002153 ◽

2003 ◽

Vol 77 (1) ◽

pp. 57-63 ◽

Cited By ~ 7

Author(s):

T. Nakada ◽

I. Nagano ◽

Z. Wu ◽

Y. Takahashi

Keyword(s):

Trichinella Spiralis ◽

Expression System ◽

Muscle Layer ◽

Full Length ◽

Cloning And Expression ◽

Western Blots ◽

Reading Frame ◽

Muscle Larvae ◽

Taxonomical Position ◽

Good Agreement

AbstractA clone, designated as TsTM, was selected from the cDNA library of newborn larvae (NBL) of Trichinella spiralis through immunoscreening against infected sera. The clone contained a cDNA transcript of 855 bp in length with a single open reading frame, which encoded 285-amino acids (33 kDa in the estimated molecular weight). A sequence analysis revealed that the clone TsTM encoded the full-length of tropomyosin gene. The phylogenetic analysis of the tropomyosin gene was in good agreement with the classical taxonomical position of T. spiralis. The fusion proteins encoded by the clone TsTM were produced in an Escherichia coli expression system and affinity purified, and the antibody was raised against the protein for the following studies. The antibody against the fusion protein positively bound to the hypodermal muscle layer in immunolocalization analysis, and the 35 kDa band in crude extracts of muscle larvae but not in excretory and secretory (ES) products on Western blots. The antigenicity of the clone TsTM was recognized by host mice but exhibited little species specificity.

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Immunoproteomic analysis of Trichinella spiralis and Trichinella britovi excretory-secretory muscle larvae proteins recognized by sera from humans infected with Trichinella

PLoS ONE ◽

10.1371/journal.pone.0241918 ◽

2020 ◽

Vol 15 (11) ◽

pp. e0241918

Author(s):

Sylwia Grzelak ◽

Anna Stachyra ◽

Jerzy Stefaniak ◽

Karolina Mrówka ◽

Bożena Moskwa ◽

...

Keyword(s):

Serine Protease ◽

Trichinella Spiralis ◽

Clinical Manifestations ◽

Immunoblot Analysis ◽

Hypothetical Protein ◽

Secretory Proteins ◽

Serum Samples ◽

Ms Analysis ◽

Muscle Larvae ◽

Human Sera

The present study compares the immunogenic patterns of muscle larvae excretory-secretory proteins (ML E-S) from T. spiralis and T. britovi recognized by Trichinella-infected human sera. Samples were analyzed using two-dimensional electrophoresis (2-DE) coupled with 2D-immunoblot and liquid chromatography-tandem mass spectrometry LC-MS/MS analysis, two ELISA procedures and a confirmatory 1D-immunoblot test. Sera were obtained from nine patients with a history of ingestion of raw or undercooked meat who presented typical clinical manifestations of trichinellosis and from eleven healthy people. Specific anti-Trichinella IgG antibodies were detected in all samples tested with the Home-ELISA kits, but in only four samples for the commercially-available kit. The 1D-immunoblot results indicated that all nine serum samples were positive for T. spiralis ML E-S antigens, expressed as the presence of specific bands. In contrast, eight of the serum samples with T. britovi E-S ML antigens were positive, with one serum sample taken from a patient at 33dpi (days post infection) being negative. To identify immunoreactive proteins that are specifically recognized by host antibodies, both species of ML E-S proteins were subjected to 2D-immunoblotting with human serum taken at 49 dpi. The sera recognized 22 protein spots for T. spiralis and 18 for T. britovi in 2D-immunoblot analysis. Their molecular weights (MW) ranged from 50 to 60 kDa. LC-MS/MS analysis identified both common and specifically-recognized immunoreactive proteins: transmembrane serine protease 9, serine protease, antigen targeted by protective antibodies and Actin-1 partial were shared for both Trichinella species; hypothetical protein T01_7775 and P49 antigen, partial those specific to T. spiralis; deoxyribonuclease-2-alpha and hypothetical protein T03_17187/T12_7360 were specific to T. britovi. Our results demonstrate the value of 2-DE and 2D-immunblot as versatile tools for pinpointing factors contributing to the parasite-host relationship by comparing the secretomes of different Trichinella species.

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Development of a rapid and sensitive immunochromatographic strip based on EuNPs-ES fluorescent probe for the detection of early Trichinella spiralis-specific IgG antibody in pigs

Veterinary Research ◽

10.1186/s13567-021-00951-9 ◽

2021 ◽

Vol 52 (1) ◽

Author(s):

Xinyu Wang ◽

Bin Tang ◽

Ying Zhao ◽

Jing Ding ◽

Nan Wang ◽

...

Keyword(s):

Test Line ◽

Trichinella Spiralis ◽

Parasite Infection ◽

Igg Antibody ◽

Immunochromatographic Strip ◽

Muscle Larvae ◽

Zoonotic Parasite ◽

Circulating Antibodies ◽

Novel Method ◽

Specific Igg

AbstractTrichinellosis, which is caused by nematodes of the genus Trichinella, is one of the most important zoonotic parasite diseases in the world. A rapid and sensitive immunochromatographic strip (ICS) based on Eu (III) nanoparticles (EuNPs) was developed for the detection of Trichinella spiralis (T. spiralis) infection in pigs. T. spiralis muscle larvae excretory secretory or preadult worm excretory secretory (ML-ES or PAW-ES) antigens were conjugated with EuNPs probes to capture T. spiralis-specific antibodies in pig sera, after which the complex bound to mouse anti-pig IgG deposited on the test line (T-line), producing a fluorescent signal. In the pigs infected with 100, 1000 and 10 000 ML, seroconversion was first detectable for the EuNPs-ML-ES ICS at 30, 25 and 21 days post-infection (dpi) and for the EuNPs-PAW-ES ICS at 25, 21 and 17 dpi. These results show that EuNPs-PAW-ES ICS detects anti-Trichinella IgG in pigs 4–5 days earlier that test using ML-ES antigens. Our ICS have no cross reaction with other parasite infection sera. Furthermore, the detection process could be completed in 10 min. This study indicated that our ICS can be used for the detection of the circulating antibodies in early T. spiralis infection and provide a novel method for on-site detection of T. spiralis infection in pigs.

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Prevalence and clinical correlation of hepatitis E virus antibody in the patients’ serum samples from a tertiary care hospital in Thailand during 2015–2018

Virology Journal ◽

10.1186/s12985-021-01616-x ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Atiporn Boonyai ◽

Anchalee Thongput ◽

Thidarat Sisaeng ◽

Parisut Phumchan ◽

Navin Horthongkham ◽

...

Keyword(s):

Pregnant Women ◽

Tertiary Care ◽

Laboratory Data ◽

Organ Transplant ◽

Hospital Setting ◽

Antibody Detection ◽

Enzyme Linked Immunosorbent Assay ◽

Care Hospital ◽

Serum Samples ◽

Igm Antibodies

Abstract Background Prevalence and incidence of hepatitis caused by HEV infection are usually higher in developing countries. This study demonstrated the HEV seroprevalence and incidence of HEV infection in patients with clinical hepatitis in a tertiary hospital in Thailand. Methods A laboratory-based cross-sectional study was conducted using 1106 serum samples from patients suspected of HEV infection sent to the Serology laboratory, Siriraj Hospital, for detecting HEV antibodies during 2015–2018. Prevalence of anti-HEV IgG and IgM antibodies in general patients, including organ transplant recipients and pregnant women in a hospital setting, were determined using indirect enzyme-linked immunosorbent assay (ELISA) kits. Comparison of laboratory data between groups with different HEV serological statuses was performed. Results HEV IgG antibodies were detected in 40.82% of 904 serum samples, while HEV IgM antibodies were detected in 11.75% of 1081 serum samples. Similar IgG and IgM antibody detection rates were found in pregnant women. Interestingly, anti-HEV IgM antibodies were detected in 38.5% of patients who underwent organ transplantation. Patients who tested positive for anti-HEV IgM antibodies had higher alanine aminotransferase levels than those who had not. In contrast, patients who tested positive for anti-HEV IgG had more elevated levels of total bilirubin than those who tested negative. Conclusions HEV seroprevalence and incidence in patients with clinical hepatitis were relatively high in the Thai population, including the pregnancy and organ transplant subgroups. The results potentially benefit the clinicians in decision-making to investigate HEV antibodies and facilitating proper management for patients.

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Diagnosis of Dengue Virus Infection by Detection of Specific Immunoglobulin M (IgM) and IgA Antibodies in Serum and Saliva

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.10.2.317-322.2003 ◽

2003 ◽

Vol 10 (2) ◽

pp. 317-322 ◽

Cited By ~ 83

Author(s):

Angel Balmaseda ◽

María G. Guzmán ◽

Samantha Hammond ◽

Guillermo Robleto ◽

Carolina Flores ◽

...

Keyword(s):

Dengue Virus ◽

Immunoglobulin M ◽

Antibody Detection ◽

Enzyme Linked Immunosorbent Assay ◽

Control Group ◽

Serum Samples ◽

Diagnostic Modality ◽

Iga Antibodies ◽

Specific Immunoglobulin ◽

Feasible Alternative

ABSTRACT To evaluate alternative approaches to the serological diagnosis of dengue virus (DEN) infection, the detection of DEN-specific immunoglobulin M (IgM) and IgA antibodies in serum and saliva specimens was assessed in 147 patients with symptoms of DEN infection seen at the Ministry of Health in Nicaragua. Seventy-two serum samples were determined to be positive for anti-DEN antibodies by IgM capture enzyme-linked immunosorbent assay, the routine diagnostic procedure. Serum and saliva specimens were obtained from 50 healthy adults as additional controls. IgM was detected in the saliva of 65 of the 72 serum IgM-positive cases, 6 of the 75 serum IgM-negative cases, and none of the control group, resulting in a sensitivity of 90.3% and a specificity of 92.0% and demonstrating that salivary IgM is a useful diagnostic marker for DEN infection. Detection of IgA in serum may be another feasible alternative for the diagnosis of DEN infection, with serum IgA found in 68 (94.4%) of the IgM-positive cases. In contrast, detection of IgA in saliva was not found to be a useful tool for DEN diagnosis in the present study. Further studies of the kinetics of antibody detection in another set of 151 paired acute- and convalescent-phase serum samples showed that DEN-specific IgA antibodies were detected in more acute-phase samples than were IgM antibodies. Thus, we conclude that DEN-specific IgA in serum is a potential diagnostic target. Furthermore, given that saliva is a readily obtainable, noninvasive specimen, detection of DEN-specific salivary IgM should be considered a useful, cheaper diagnostic modality with similar sensitivity and specificity to IgM detection in serum.

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