Fluorescent bead-based serological detection of Toxoplasma gondii infection in chickens

Abstract Background: Especially free-ranging chickens are frequently exposed to Toxoplasma gondii. They are sensitive indicators for environmental contamination with T. gondii oocysts. The detection of infected birds relies primarily on serological assays. Methods: Here, we established a bead-based multiplex assay (BBMA) using the Luminex technology for the specific and sensitive detection of T. gondii infections in chickens. Recombinant biotinylated T. gondii surface antigen 1 (TgSAG1bio) bound to streptavidin-conjugated magnetic Luminex beads served as antigen. Specific serum antibodies were detected by a fluorophore-coupled secondary antibody. Beads of differing color codes were conjugated with anti-chicken IgY or chicken serum albumin and served for each individual sample as an internal positive or negative control, respectively. The assay was validated with sera from experimentally and naturally infected chickens. The results were compared to those from reference methods, including other serological tests and bioassay in mice.Results: In experimentally infected chickens, all chickens were positive by magnetic-capture PCR (100%, n=45/45) and the vast majority (98.5%, n=65/66) of inoculated birds tested seropositive in the BBMA. Most, but not all inoculated and TgSAG1bio-BBMA-positive chickens were also positive in two previously established TgSAG1-ELISAs, an immunofluorescence assay (IFAT) and in a modified agglutination test (MAT). All non-inoculated control animals (n=28) tested negative. In naturally exposed chickens, the TgSAG1bio-BBMA showed a high sensitivity (98.5%; 95% Confidence Interval: 90.7-99.9%) and specificity (100%; 85.0-100%) relative to a reference standard established using results obtained with ELISA, IFAT and MAT. Almost all naturally exposed chickens that were positive in the mouse bioassay or by PCR tested positive in the TgSAG1bio-BBMA (93.5%, 77.1-98.9%), while all mouse bioassay- or PCR-negative chickens remained negative (100%, 85.0-100%).Conclusions: The TgSAG1bio-BBMA represents a suitable method for the detection of T. gondii infections in chickens with high sensitivity and specificity, which is comparable or even superior to other tests. Since assays based on this methodology allow for the simultaneous analysis of a single biological sample with respect to multiple analytes, the described assay may represent one component in future multiplex assays for broad serological monitoring of poultry and other farm animals, including pigs or small ruminants, for various pathogens.

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Fluorescent bead-based serological detection of Toxoplasma gondii infection in chickens

10.21203/rs.3.rs-34121/v2 ◽

2020 ◽

Author(s):

Benedikt T. Fabian ◽

Fatima Hedar ◽

Martin Koethe ◽

Berit Bangoura ◽

Pavlo Maksimov ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Small Ruminants ◽

High Sensitivity ◽

Negative Control ◽

Immunofluorescence Assay ◽

Farm Animals ◽

Serological Tests ◽

Modified Agglutination Test ◽

Magnetic Capture ◽

Luminex Technology

Abstract Background: Free-ranging chickens are often infected with Toxoplasma gondii. Their infection indicates environmental contamination with T. gondii. The detection of infected birds relies primarily on serological assays. Methods: Here, we established a bead-based multiplex assay (BBMA) using the Luminex technology for the specific and sensitive detection of T. gondii infections in chickens. Recombinant biotinylated T. gondii surface antigen 1 (TgSAG1bio) bound to streptavidin-conjugated magnetic Luminex beads served as antigen. Serum antibodies were detected by a fluorophore-coupled secondary antibody. Beads of differing color codes were conjugated with anti-chicken IgY or chicken serum albumin and served for each sample as an internal positive or negative control, respectively. The assay was validated with sera from experimentally and naturally infected chickens. The results were compared to those from reference methods, including other serological tests and bioassay in mice.Results: In experimentally infected chickens, the vast majority (98.5%, n=65/66) of inoculated birds tested seropositive in the BBMA. This included all chickens positive by magnetic-capture PCR (100%, n=45/45). Most, but not all inoculated and TgSAG1bio-BBMA-positive chickens were also positive in two previously established TgSAG1-ELISAs (TgSAG1-ELISASL, n=61/65; or TgSAG1-ELISASH, n=60/65), or positive in an immunofluorescence assay (IFAT, n=64/65)) and in a modified agglutination test (MAT, n=61/65). All non-inoculated control animals (n=28/28, 100%) tested negative. In naturally exposed chickens, the TgSAG1bio-BBMA showed a high sensitivity (98.5%; 95% Confidence Interval: 90.7-99.9%) and specificity (100%; 85.0-100%) relative to a reference standard established using ELISA, IFAT and MAT. Almost all naturally exposed chickens that were positive in bioassay or by PCR tested positive in the TgSAG1bio-BBMA (93.5%; 77.1-98.9%), while all bioassay- or PCR-negative chickens remained negative (100%; 85.0-100%).Conclusions: The TgSAG1bio-BBMA represents a suitable method for the detection of T. gondii infections in chickens with high sensitivity and specificity, which is comparable or even superior to other tests. Since assays based on this methodology allow for the simultaneous analysis of a single biological sample with respect to multiple analytes, the described assay may represent one component in future multiplex assays for broad serological monitoring of poultry and other farm animals, including pigs or small ruminants, for various pathogens.

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Fluorescent bead-based serological detection of Toxoplasma gondii infection in chickens

10.21203/rs.3.rs-34121/v3 ◽

2020 ◽

Author(s):

Benedikt T. Fabian ◽

Fatima Hedar ◽

Martin Koethe ◽

Berit Bangoura ◽

Pavlo Maksimov ◽

...

Keyword(s):

Toxoplasma Gondii ◽

High Sensitivity ◽

Negative Control ◽

Immunofluorescence Assay ◽

Farm Animals ◽

Serological Tests ◽

Modified Agglutination Test ◽

Magnetic Capture ◽

Luminex Technology ◽

Toxoplasma Gondii Infection

Abstract Background: Free-ranging chickens are often infected with Toxoplasma gondii and seroconvert upon infection. This indicates environmental contamination with T. gondii. Methods: Here, we established a bead-based multiplex assay (BBMA) using the Luminex technology for the detection of T. gondii infections in chickens. Recombinant biotinylated T. gondii surface antigen 1 (TgSAG1bio) bound to streptavidin-conjugated magnetic Luminex beads served as antigen. Serum antibodies were detected by a fluorophore-coupled secondary antibody. Beads of differing color codes were conjugated with anti-chicken IgY or chicken serum albumin and served for each sample as an internal positive or negative control, respectively. The assay was validated with sera from experimentally and naturally infected chickens. The results were compared to those from reference methods, including other serological tests, PCRs and bioassay in mice.Results: In experimentally infected chickens, the vast majority (98.5%, n = 65/66) of birds tested seropositive in the BBMA. This included all chickens positive by magnetic-capture PCR (100%, n = 45/45). Most, but not all inoculated and TgSAG1bio-BBMA-positive chickens were also positive in two previously established TgSAG1-ELISAs (TgSAG1-ELISASL, n = 61/65; or TgSAG1-ELISASH, n = 60/65), or positive in an immunofluorescence assay (IFAT, n = 64/65)) and in a modified agglutination test (MAT, n = 61/65). All non-inoculated control animals (n = 28/28, 100%) tested negative. In naturally exposed chickens, the TgSAG1bio-BBMA showed a high sensitivity (98.5%; 95% confidence interval, CI: 90.7–99.9%) and specificity (100%; 95% CI: 85.0–100%) relative to a reference standard established using ELISA, IFAT and MAT. Almost all naturally exposed chickens that were positive in bioassay or by PCR tested positive in the TgSAG1bio-BBMA (93.5%; 95% CI: 77.1–98.9%), while all bioassay- or PCR-negative chickens remained negative (100%; 95% CI: 85.0–100%).Conclusions: The TgSAG1bio-BBMA represents a suitable method for the detection of T. gondii infections in chickens with high sensitivity and specificity, which is comparable or even superior to other tests. Since assays based on this methodology allow for the simultaneous analysis of a single biological sample with respect to multiple analytes, the described assay may represent a component in future multiplex assays for broad serological monitoring of poultry and other farm animals for various pathogens.

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Genotyping of Toxoplasma gondii isolates from naturally infected Gallus domesticus in Santa Catarina state, Brazil

Arquivo Brasileiro de Medicina Veterinária e Zootecnia ◽

10.1590/1678-4162-8594 ◽

2017 ◽

Vol 69 (1) ◽

pp. 139-145 ◽

Cited By ~ 3

Author(s):

N. Trevisani ◽

L.D. Barros ◽

A. Vieira-Neto ◽

A.A. Sartor ◽

A.P. Souza ◽

...

Keyword(s):

Genetic Diversity ◽

Toxoplasma Gondii ◽

Immunofluorescence Assay ◽

Gallus Domesticus ◽

Mouse Bioassay ◽

Type I ◽

Santa Catarina ◽

High Genetic Diversity ◽

Clonal Population Structure ◽

Santa Catarina State

ABSTRACT Toxoplasmosis is a widespread zoonosis that can infect warm-blooded animals including birds and humans, and chickens are considered to be indicators of environmental contamination. In Brazil, Toxoplasma gondii has a non-clonal population structure composed of three lineages (I, II, and III), presenting high recombination, and resulting in wide genetic diversity. This study aimed to genetically characterize T. gondii isolates from naturally infected chickens (Gallus domesticus) in Santa Catarina state, southern Brazil region. Sera from 133 free-range chickens were analyzed by an immunofluorescence assay (IFA) to detect IgG antibodies against T. gondii. Brain and heart from 30 positive animals, based on IFA (≥ 1:64), were used to isolate the parasite using a mouse bioassay. Strain genotyping was performed by PCR-RFLP using 12 genetic markers (SAG1, 5´-3´SAG2, alt. SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico, and CS3). The results were classified according to the genotypes based on the ToxoDB (http://toxodb.org/toxo/). Of 133 chicken sera analyzed, 84 (63.16%) were positive, with antibody titers ranging from 16 to 1024. Eleven isolates were obtained from mouse bioassay (Ck3, Ck32, Ck35, Ck56, Ck63, Ck89, Ck102, Ck103, Ck125, Ck127, and Ck128). Genotyping revealed six genotypes; three were classified as #26, #53, and #120, and three (NEO1, NEO2, and NEO3) were had not been previously described. No clonal lineages of type I, II, or III were identified. The present study confirms the high genetic diversity of T. gondii in Brazil.

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Diagnostic-test evaluation of immunoassays for anti-Toxoplasma gondii IgG antibodies in a random sample of Mexican population

The Journal of Infection in Developing Countries ◽

10.3855/jidc.3858 ◽

2014 ◽

Vol 8 (05) ◽

pp. 642-647 ◽

Cited By ~ 7

Author(s):

Heriberto Caballero-Ortega ◽

Rocío Castillo-Cruz ◽

Sandra Murieta ◽

Luz Belinda Ortíz-Alegría ◽

Esther Calderón-Segura ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Western Blot ◽

Latin American ◽

High Sensitivity ◽

Reference Standards ◽

Igg Antibodies ◽

Serum Samples ◽

Serological Tests ◽

Western Blots ◽

Open Population

Introduction: There are few articles on evaluation of Toxoplasma gondii serological tests. Besides, commercially available tests are not always useful and are expensive for studies in open population. The aim of this study was to evaluate in-house ELISA and western blot for IgG antibodies in a representative sample of people living in Mexico. Methodology: Three hundred and five serum samples were randomly selected from two national seroepidemiological survey banks; they were taken from men and women of all ages and from all areas of the country. ELISA cut-off was established using the mean plus three standard deviations of negative samples. Western blots were analysed by two experienced technicians and positivity was established according to the presence of at least three diagnostic bands. A commercial ELISA kit was used as a third test. Two reference standards were built up: one using concordant results of two assays leaving the evaluated test out and the other in which the evaluated test was included (IN) with at least two concordant results to define diagnosis. Results: the lowest values of diagnostic parameters were obtained with the OUT reference standards: in-house ELISA had 96.9% sensitivity, 62.1% specificity, 49.6% PPV, 98.1% NPV and 71.8% accuracy, while western blot presented 81.8%, 89.7%, 84.0%, 88.2% and 86.6% values and the best kappa coefficient (0.72-0.82). Conclusions: The in-house ELISA is useful for screening people of Mexico, due to its high sensitivity, while western blot may be used to confirm diagnosis. These techniques might prove useful in other Latin American countries.

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Toxoplasma gondii Recombinant antigen AMA1: Diagnostic Utility of Protein Fragments for the Detection of IgG and IgM Antibodies

Pathogens ◽

10.3390/pathogens9010043 ◽

2020 ◽

Vol 9 (1) ◽

pp. 43 ◽

Cited By ~ 2

Author(s):

Bartłomiej Ferra ◽

Lucyna Holec-Gąsior ◽

Justyna Gatkowska ◽

Bożena Dziadek ◽

Katarzyna Dzitko

Keyword(s):

Toxoplasma Gondii ◽

Expression System ◽

High Sensitivity ◽

Recombinant Antigen ◽

Full Length ◽

Diagnostic Utility ◽

Intermediate Hosts ◽

Serological Tests ◽

Apical Membrane Antigen 1 ◽

Diagnostic Potential

Toxoplasma gondii is an important zoonotic protozoan that infects a wide variety of vertebrates as intermediate hosts. For this reason, the diagnosis of this disease is very important and requires continuous improvement. One possibility is to use recombinant antigens in serological tests. Apical membrane antigen 1 (AMA1), a protein located in specific secretory organelles (micronemes) of T. gondii, is very interesting in regard to its potential diagnostic utility. In the present study, we attempted to identify a fragment of the AMA1 protein with a high sensitivity and specificity for the serological diagnosis of human toxoplasmosis. The full-length AMA1 and two different fragments (AMA1N and AMA1C) were produced using an Escherichia coli expression system. After purification by metal affinity chromatography, recombinant proteins were tested for their utility as antigens in enzyme-linked immunosorbent assays (ELISAs) for the detection of IgG and IgM anti-T. gondii antibodies in human and mouse immune sera. Our data demonstrate that the full-length AMA1 recombinant antigen (corresponding to amino acid residues 67–569 of the native protein) has a better diagnostic potential than its N- or C-terminal fragments. This recombinant protein strongly interacts with specific anti-T. gondii IgG (99.4%) and IgM (80.0%) antibodies, and may be used for developing new tools for diagnostics of toxoplasmosis.

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EVALUATION OF IGM ELISA, WEIL FELIX TEST AND INDIRECT IMMUNOFLUORESCENCE ASSAY IN THE DIAGNOSIS OF SCRUB TYPHUS.

10.36106/ijar/5405204 ◽

2020 ◽

pp. 1-4

Author(s):

Pooja Raghunath

Keyword(s):

Diagnostic Test ◽

Scrub Typhus ◽

Febrile Illness ◽

High Sensitivity ◽

Immunofluorescence Assay ◽

Research Centre ◽

Medical Sciences ◽

Serological Tests ◽

Igm Elisa ◽

Highly Sensitive

Scrub typhus is an acute febrile illness caused by Orientia tsutsugamushi. There are reports of resurgence of scrub typhus in different parts of India including Kerala. Many cases go undiagnosed due to the lack of knowledge regarding the diagnostic test to be used. This study was undertaken at Pushpagiri Institute of Medical Sciences and Research Centre, Tiruvalla in the Department of Microbiology from January 2014 to September 2015, to assess the validity of various tests in diagnosing scrub typhus. A total of 94 samples were subjected to IgM ELISA, Weil Felix (WF) test and Indirect Immunouorescence assay(IFA). The agreement between ELISA, IFA and Weil Felix test was assessed by κ (kappa) coefcient of Cohen using SPSS version 17. The sensitivity and specicity of ELISA with respect to IFA was 100% and 94.67% respectively, and that of IFA with respect to ELISA was 82.61% and 100% respectively. The sensitivity and specicity of WF test with respect to ELISA was 43.48 and 100% respectively and that of WF test with respect to IFA was 52.63% and 100% respectively. Although IgM ELISA is highly sensitive, it cannot be relied upon as the sole diagnostic test due to high false positivity. WF test is not a sensitive test, but when positive it is rather specic. IFA has high sensitivity and specicity, but is unsuitable for moderately equipped laboratories. Hence, we suggest that scrub typhus could be diagnosed using a combination of IgM ELISA and WF test and these tests should be included in the panel of serological tests ordered for patients presenting with undifferentiated febrile illnesses.

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Prevalence of Toxoplasma gondii infection in the dairy goat population from an organic farm

Medycyna Weterynaryjna ◽

10.21521/mw.5797 ◽

2017 ◽

Vol 73 (11) ◽

pp. 736-738

Author(s):

Dawid Jańczak ◽

Marcin Świątek ◽

Żaneta Szymańska ◽

Roman Niżnikowski ◽

Elżbieta Gołąb

Keyword(s):

Toxoplasma Gondii ◽

Public Health Problem ◽

Farm Animals ◽

Dairy Goat ◽

Economic Losses ◽

Dairy Goats ◽

Serological Tests ◽

Organic Farm ◽

Goat Population ◽

Toxoplasma Gondii Infection

Protozoal infection of T. gondii is a public health problem and also causes serious economic losses in livestock production in many countries. Farm animals from organic farms are more likely to be infected. The aim of the study was to determine the prevalence of Toxoplasma gondii among 50 dairy goats from an organic farm in the northwestern Poland region and to assess the prevalence of parasite DNA in the milk of infected animals. Serological tests performed by direct agglutination of IgG antibodies against T. gondii were positive in 10% of the tested animals. No parasite DNA was detected in the milk from the seropositive goats. However, the number of tested animals was too small to draw significant epidemiological conclusions.

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Occurrence of anti-Neospora caninum and anti-Toxoplasma gondii IgG antibodies in goats and sheep in western Maranhão, Brazil

Revista Brasileira de Parasitologia Veterinária ◽

10.1590/s1984-29612011000400010 ◽

2011 ◽

Vol 20 (4) ◽

pp. 312-317 ◽

Cited By ~ 17

Author(s):

Larissa Martins de Brito Moraes ◽

Juliana Macedo Raimundo ◽

Andresa Guimarães ◽

Huarrisson Azevedo Santos ◽

Gilberto de Lima Macedo Junior ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Neospora Caninum ◽

Small Ruminants ◽

Immunofluorescence Assay ◽

Northeastern Brazil ◽

Food Supplementation ◽

Igg Antibodies ◽

Etiologic Agents ◽

Western Area ◽

Significant Difference

Neosporosis and toxoplasmosis are parasitic diseases which can cause reproductive problems in goats and sheep. The current study aimed to determine the occurrence of anti-Neospora caninum and anti-Toxoplasma gondii IgG antibodies in goats and sheep from the districts of Amarante do Maranhão and Buritirana, Imperatriz microregion, western area of Maranhão State, northeastern Brazil, and to assess factors associated to infection by these etiologic agents. Blood samples from 110 animals (46 goats and 64 sheep) from five herds were collected, and indirect immunofluorescence assay was used for serological testing. Of 46 goat samples, 17.39% (n = 8) showed anti-N. caninum antibodies and 4.35% (n = 2) anti-T. gondii, while of 64 sheep samples 4.69% (n = 3) and 18.75% (n = 12) showed anti-N. caninum and anti-T. gondii antibodies, respectively. No significant difference regarding the presence of domestic cats and/or dogs on the property and veterinary care was seen for both etiologic agents studied. However, food supplementation and animal reproductive failure were significantly (p < 0.05) for N. caninum among sheep and goats, respectively. The current study showed that goats and sheep in western Maranhão are exposed to N. caninum and T. gondii. It is the first evidence of these agents in small ruminants in this region.

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Development of a Multiplex PCR and Magnetic DNA Capture Assay for Detecting Six Species Pathogens of the Genera Anaplasma and Ehrlichia in Canine, Bovine, Caprine and Ovine Blood Samples from Grenada, West Indies

Pathogens ◽

10.3390/pathogens10020192 ◽

2021 ◽

Vol 10 (2) ◽

pp. 192

Author(s):

Bhumika Sharma ◽

Roman R. Ganta ◽

Diana Stone ◽

Andy Alhassan ◽

Marta Lanza-Perea ◽

...

Keyword(s):

Sensitivity And Specificity ◽

Clinical Signs ◽

Small Ruminants ◽

High Sensitivity ◽

Molecular Evidence ◽

Blood Samples ◽

Persistent Problem ◽

Magnetic Capture ◽

Low Sensitivity ◽

Species Specific

Infections with tick-borne pathogens belonging to Anaplasma/Ehrlichia in various vertebrate hosts are a persistent problem resulting in nonspecific clinical signs during early infection. Diagnosis of single and multi-infections with these pathogens, causing diseases in companion/agricultural animals and people, remains a challenge. Traditional methods of diagnosis, such as microscopy and serology, have low sensitivity and specificity. Polymerase chain reaction (PCR) assays are widely used to detect early-phase infections, since these have high sensitivity and specificity. We report the development and validation of an assay involving PCR followed by magnetic capture method using species-specific oligonucleotides to detect six Anaplasma/Ehrlichia species pathogens in canine, bovine, caprine, and ovine blood samples. Overall, the assay application to 455 samples detected 30.1% (137/455) positives for one or more out of six screened pathogens. Single-pathogen infections were observed in 94.9% (130/137) of the positive samples, while co-infections were detected in 5.1% (7/137). Anaplasma marginale infection in cattle had the highest detection rate (34.4%), followed by canines positive for Anaplasma platys (16.4%) and Ehrlichia canis (13.9%). The assay aided in documenting the first molecular evidence for A. marginale in cattle and small ruminants and Ehrlichia chaffeensis and Ehrlichia ewingii in dogs in the Caribbean island of Grenada.

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CLINICAL, HEMATOLOGICAL, AND SEMINAL ALTERATIONS AND PARASITEMIA OF MALE GOATS EXPERIMENTALLY INFECTED WITH Toxoplasma gondii

Ciência Animal Brasileira ◽

10.1590/1089-6891v16i326476 ◽

2015 ◽

Vol 16 (3) ◽

pp. 399-409 ◽

Cited By ~ 2

Author(s):

Luís Fernando Santana ◽

Roberta Cordeiro Gaspar ◽

Gabriel Augusto Marques Rossi ◽

Gabriel Antônio Nogueira Nascentes ◽

Eliana Aparecida Rodrigues ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Small Ruminants ◽

Control Group ◽

Serological Tests ◽

Seminal Parameters ◽

Handling Methods ◽

Toxoplasmic Infection ◽

Clinical Changes ◽

Animal Handling ◽

And Control

<title>Abstract:</title>Toxoplasmosis is a parasitic disease that affects reproductive performance in small ruminants. Although the <italic>Toxoplasma gondii</italic> life cycle is well understood since 1960s, several aspects related to its infection remains unclear. This study aimed to determine the effects of <italic>T. gondii</italic>experimental infection, and the influence on clinical, hematological, parasitemia and seminal parameters in male goats. Nine animals were selected and distributed in three groups: GI (n=3) – control group (placebo) orally inoculated with saline solution; GII (n=3) – subcutaneously inoculated with 1 x 106 tachyzoites of <italic>T. gondii</italic>; and GIII (n=3) – orally inoculated with 2 x 105 oocysts of <italic>T. gondii</italic>. After that, clinical exams, serological tests, hemograms, parasitemia determination and semen evaluation were performed. Reciprocal serological titers had highest values of 4096 in both groups of goats infected with <italic>T. gondii,</italic>confirming the experimental infections. However, we could not observe clinical changes (except for mild hyperthermia on the 5th DAI in one of the animals - GIII) or in hematimetric parameters. Although there were some statistically significant changes (P <0.05) on the percentages of pathology and sperm concentrations in some of the dates between the infected and control animals, these changes were not associated with toxoplasmic infection. Infection was associated with animal handling methods and environmental factors.

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