Profiling Bovine Antibody Responses to Mycobacterium avium subsp. paratuberculosis Infection by Using Protein Arrays

ABSTRACT With the genome sequence of Mycobacterium avium subsp. paratuberculosis determined, technologies are now being developed for construction of protein arrays to detect the presence of antibodies against M. avium subsp. paratuberculosis in host serum. The power of this approach is that it enables a direct comparison of M. avium subsp. paratuberculosis proteins to each other in relation to their immunostimulatory capabilities. In this study, 93 recombinant proteins, produced in Escherichia coli, were arrayed and spotted onto nitrocellulose. These proteins include unknown hypothetical proteins and cell surface proteins as well as proteins encoded by large sequence polymorphisms present uniquely in M. avium subsp. paratuberculosis. Also included were previously reported or known M. avium subsp. paratuberculosis antigens to serve as a frame of reference. Sera from healthy control cattle (n = 3) and cattle infected with either M. avium subsp. avium and Mycobacterium bovis were exposed to the array to identify nonspecific or cross-reactive epitopes. These data demonstrated a degree of cross-reactivity with the M. avium subsp. avium proteins that was higher than the degree of cross-reactivity with the more distantly related M. bovis proteins. Finally, sera from naturally infected cattle (n = 3) as well as cattle experimentally infected with M. avium subsp. paratuberculosis (n = 3) were used to probe the array to identify antigens in the context of Johne's disease. Three membrane proteins were the most strongly detected in all serum samples, and they included an invasion protein, an ABC peptide transport permease, and a putative GTPase protein. This powerful combination of genomic information, molecular tools, and immunological assays has enabled the identification of previously unknown antigens of M. avium subsp. paratuberculosis.

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Development and Validation of a Novel ELISA for the Specific Detection of Antibodies against Mycobacterium avium Subspecies paratuberculosis Based on a Chimeric Polyprotein

Veterinary Medicine International ◽

10.1155/2021/7336848 ◽

2021 ◽

Vol 2021 ◽

pp. 1-10

Author(s):

Roberto Damián Moyano ◽

Magali Andrea Romero ◽

María Alejandra Colombatti Olivieri ◽

María Fiorella Alvarado Pinedo ◽

Gabriel Eduardo Traveria ◽

...

Keyword(s):

Mycobacterium Avium ◽

Roc Curve ◽

Bovine Serum ◽

Mycobacterium Avium Subspecies Paratuberculosis ◽

Cross Reactivity ◽

Specific Detection ◽

Serum Samples ◽

Specific Antigens ◽

Mycobacterium Avium Subsp Paratuberculosis ◽

Development And Validation

Bovine paratuberculosis (PTB) is caused by Mycobacterium avium subsp. paratuberculosis (MAP). The optimization of detection tests specific for MAP is crucial to improve PTB control. In this work, we aimed to develop and validate a diagnostic tool based on an ELISA to specifically detect anti-MAP antibodies from bovine serum samples. For that purpose, we designed a recombinant polyprotein containing four specific antigens from MAP and optimized the ELISA. The validation consisted of the assessment of 10 sera from PTB-infected and healthy bovines with different OD values. The diagnostic performance of the polyprotein-ELISA was evaluated by testing 130 bovine serum samples (47 healthy, 48 MAP-infected, and 35 M. bovis-infected bovines). The ELISA using the polyprotein yielded an area under the ROC curve (AUC) of 0.9912 (95% CI, 0.9758–1.007; P < 0.0001). Moreover, for this ELISA, the cut-off selected from the ROC curve based on the point with a sensitivity of 95.56% (95% CI, 0.8485–0.9946) and specificity of 97.92 (95% CI, 0.8893–0.9995) was 0.3328. Similar results were obtained with an ELISA using the commercial Paratuberculosis Protoplasmatic Antigen (PPA). However, the ELISA with the polyprotein antigen showed a better performance against sera from animals infected with Mycobacterium bovis compared to the ELISA with PPA: lower cross-reactivity (2.85% versus 25.71%). These results demonstrate a very low cross-reactivity of the polyprotein with antibodies present in serum samples from animals infected with M. bovis. The designed polyprotein and the validated ELISA could be very useful for the specific identification of MAP-infected animals in herds.

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Detection of Mycobacterium avium subsp. paratuberculosis (MAP) from Subclinical Caprine Paratuberculosis Cases of Odisha

Indian Journal of Animal Research ◽

10.18805/ijar.b-3691 ◽

2020 ◽

Author(s):

Sangram Biswal ◽

Adya Prakash Rath ◽

Shoor Vir Singh ◽

Niranjana Sahoo ◽

Saurabh Gupta ◽

...

Keyword(s):

Mycobacterium Avium ◽

Animal Health ◽

Restriction Endonuclease Analysis ◽

Mycobacterium Avium Subsp ◽

Serum Samples ◽

Blood Samples ◽

Blind Review ◽

Faecal Samples ◽

Goat Population ◽

Mycobacterium Avium Subsp Paratuberculosis

Paratuberculosis is caused by Mycobacterium avium subsp. Paratuberculosis (MAP) and is a chronic, intestinal tract infection in the ruminant sector globally. A total of 122 EDTA mixed blood samples, 121 serum and 16 pooled faecal samples were collected from farms of 4 different districts i.e. Nayagarh, Cuttack, Khordha and Angul and a blind review was conducted at the Animal Health Division, CIRG, Mathura. Microscopic examination of 16 pooled faecal samples revealed +2 reactivity to Acid-Fast Bacilli. All the serum samples were subjected to indirect ELISA. Out of them, 23 (19.01%), 85 (70.25%), shows strongly positive, positive, antibody titre respectively. EDTA blood samples of 23 ELISA-strongly positive were subjected to 413 bp IS900 PCR and 11 (9%) of them were found positive for Mycobacterium avium subsp. Paratuberculosis (MAP). MAP isolates were further subjected to genotyping using 608 bpIS1311 PCR and restriction endonuclease analysis (IS1311 PCR-REA) and 2 (1.64%) of them matched with “Indian Bison Type”. Genotyping of the isolates using IS1311 PCR-REA revealed that goat population of Odisha are primarily infected with “Indian Bison Type” strains.

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Multiplexed High-Throughput Serological Assay for Human Enteroviruses

Microorganisms ◽

10.3390/microorganisms8060963 ◽

2020 ◽

Vol 8 (6) ◽

pp. 963

Author(s):

Niila V. V. Saarinen ◽

Jussi Lehtonen ◽

Riitta Veijola ◽

Johanna Lempainen ◽

Mikael Knip ◽

...

Keyword(s):

High Throughput ◽

Simultaneous Detection ◽

Cross Reactivity ◽

Antibody Responses ◽

Serum Samples ◽

Single Well ◽

Specific Variation ◽

Enterovirus Type ◽

Immunological Assays ◽

High Throughput Assay

Immunological assays detecting antibodies against enteroviruses typically use a single enterovirus serotype as antigen. This limits the ability of such assays to detect antibodies against different enterovirus types and to detect possible type-specific variation in antibody responses. We set out to develop a multiplexed assay for simultaneous detection of antibodies against multiple enterovirus and rhinovirus types encompassing all human infecting species. Seven recombinant VP1 proteins from enteroviruses EV-A to EV-D and rhinoviruses RV-A to RV-C species were produced. Using Meso Scale Diagnostics U-PLEX platform we were able to study antibody reactions against these proteins as well as non-structural enterovirus proteins in a single well with 140 human serum samples. Adults had on average 33-fold stronger antibody responses to these antigens (p < 10−11) compared to children, but children had less cross-reactivity between different enterovirus types. The results suggest that this new high-throughput assay offers clear benefits in the evaluation of humoral enterovirus immunity in children, giving more exact information than assays that are based on a single enterovirus type as antigen.

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Mycobacterium avium subsp. paratuberculosis Antibody Response, Fecal Shedding, and Antibody Cross-Reactivity to Mycobacterium bovis in M. avium subsp. paratuberculosis-Infected Cattle Herds Vaccinated against Johne's Disease

Clinical and Vaccine Immunology ◽

10.1128/cvi.00032-14 ◽

2014 ◽

Vol 21 (5) ◽

pp. 698-703 ◽

Cited By ~ 15

Author(s):

Deepanker Tewari ◽

Ernest Hovingh ◽

Rick Linscott ◽

Edmond Martel ◽

John Lawrence ◽

...

Keyword(s):

Mycobacterium Bovis ◽

Mycobacterium Avium ◽

Cross Reactivity ◽

Johne's Disease ◽

Antibody Responses ◽

Johne’S Disease ◽

Serum Samples ◽

Content Type ◽

Fecal Shedding ◽

Cattle Herds

ABSTRACTVaccination for Johne's disease with killed inactivated vaccine in cattle herds has shown variable success. The vaccine delays the onset of disease but does not afford complete protection. Johne's disease vaccination has also been reported to interfere with measurements of cell-mediated immune responses for the detection of bovine tuberculosis. Temporal antibody responses and fecal shedding ofMycobacterium aviumsubsp.paratuberculosis, the causative agent of Johne's disease, were measured in 2 dairy cattle herds using Johne's disease vaccine (Mycopar) over a period of 7 years. Vaccination against Johne's disease resulted in positive serumM. aviumsubsp.paratuberculosisantibody responses in both herds, and the responses persisted in vaccinated cattle up to 7 years of age. Some vaccinated animals (29.4% in herd A and 36.2% in herd B) showed no serological reactivity toM. aviumsubsp.paratuberculosis.M. aviumsubsp.paratuberculosis-specific antibody responses were also detected in milk from Johne's disease-vaccinated animals, but fewer animals (39.3% in herd A and 49.4% in herd B) had positive results with milk than with serum samples. With vaccination againstM. aviumsubsp.paratuberculosis, fecal shedding in both dairy herds was reduced significantly (P< 0.001). In addition, when selected Johne's disease-vaccinated and -infected animals were investigated for serological cross-reactivity toMycobacterium bovis, no cross-reactivity was observed.

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Serological and Molecular Characterization of Mycobacterium avium Subsp. paratuberculosis (MAP) from Sheep, Goats, Cattle and Camels in the Eastern Province, Saudi Arabia

Animals ◽

10.3390/ani11020323 ◽

2021 ◽

Vol 11 (2) ◽

pp. 323

Author(s):

Ibrahim Elsohaby ◽

Mahmoud Fayez ◽

Mohamed Alkafafy ◽

Mohamed Refaat ◽

Theeb Al-Marri ◽

...

Keyword(s):

Saudi Arabia ◽

Molecular Characterization ◽

Mycobacterium Avium ◽

Mycobacterium Avium Subsp ◽

Type I ◽

Serum Samples ◽

Eastern Province ◽

Fecal Samples ◽

Mycobacterium Avium Subsp Paratuberculosis

The objectives of the present study were to characterize Mycobacterium avium subsp. paratuberculosis (MAP) infection using serological and molecular tools and investigate the distribution and molecular characterization of MAP strains (cattle (C) and sheep (S) types) in sheep, goat, cattle, and camel herds in Eastern Province, Saudi Arabia. Serum and fecal samples were collected from all animals aged >2 years old in 31 herds (sheep = 8, goats = 6, cattle = 8 and camels = 9) from January to December 2019. Serum samples were tested by ELISA for the detection of MAP antibodies. Fecal samples were tested by PCR for the detection of MAP IS900 gene and the identification of MAP strains. MAP antibodies were detected in 19 (61.3%) herds. At the animal level, antibodies against MAP were detected in 43 (19.5%) sheep, 21 (17.1%) goats, 13 (19.7%) cattle and 22 (9.1%) camels. The IS900 gene of MAP was detected in 23 (74.2%) herds and was directly amplified from fecal samples of 59 (26.8%) sheep, 34 (27.6%) goats, 20 (30.3%) cattle and 36 (15.0%) camels. The S-type was the most prevalent MAP type identified in 15 herds, and all were identified as type-I, while the C-type was identified in only 8 herds. The IS900 sequences revealed genetic differences among the MAP isolates recovered from sheep, goats, cattle and camels. Results from the present study show that MAP was prevalent and confirm the distribution of different MAP strains in sheep, goat, cattle and camel herds in Eastern Province, Saudi Arabia.

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Sensitive and Specific Enzyme-Linked Immunosorbent Assay for Detecting Serum Antibodies against Mycobacterium avium subsp. paratuberculosis in Fallow Deer

Clinical and Vaccine Immunology ◽

10.1128/cvi.00159-14 ◽

2014 ◽

Vol 21 (8) ◽

pp. 1077-1085 ◽

Cited By ~ 8

Author(s):

José M. Prieto ◽

Ana Balseiro ◽

Rosa Casais ◽

Naiara Abendaño ◽

Liam E. Fitzgerald ◽

...

Keyword(s):

Immunosorbent Assay ◽

Mycobacterium Avium ◽

Cost Effective ◽

Fallow Deer ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Igg Antibody ◽

Tissue Samples ◽

Content Type ◽

Mycobacterium Avium Subsp Paratuberculosis

ABSTRACTThe enzyme-linked immunosorbent assay (ELISA) is the diagnostic test most commonly used in efforts to control paratuberculosis in domestic ruminants. However, commercial ELISAs have not been validated for detecting antibodies againstMycobacterium aviumsubsp.paratuberculosisin wild animals. In this study, we compared the sensitivities and specificities of five ELISAs using individual serum samples collected from 41 fallow deer with or without histopathological lesions consistent with paratuberculosis. Two target antigenic preparations were selected, an ethanol-treated protoplasmic preparation obtained from a fallow deerM. aviumsubsp.paratuberculosisisolate (ELISAs A and B) and a paratuberculosis protoplasmic antigen (PPA3) (ELISAs C and D). Fallow deer antibodies bound to the immobilized antigens were detected by using a horseradish peroxidase (HRP)-conjugated anti-fallow deer IgG antibody (ELISAs A and C) or HRP-conjugated protein G (ELISAs B and D). A commercially available assay, ELISA-E, which was designed to detectM. aviumsubsp.paratuberculosisantibodies in cattle, sheep, and goats, was also tested. Although ELISAs A, C, and E had the same sensitivity (72%), ELISAs A and C were more specific (100%) for detecting fallow deer with lesions consistent with paratuberculosis at necropsy than was the ELISA-E (87.5%). In addition, the ELISA-A was particularly sensitive for detecting fallow deer in the latent stages of infection (62.5%). The antibody responses detected with the ELISA-A correlated with both the severity of enteric lesions and the presence of acid-fast bacteria in gut tissue samples. In summary, our study shows that the ELISA-A can be a cost-effective diagnostic tool for preventing the spread of paratuberculosis among fallow deer populations.

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Detection of Mycobacterium avium subsp. paratuberculosis in Iranian patients with type 1 diabetes mellitus by PCR and ELISA

The Journal of Infection in Developing Countries ◽

10.3855/jidc.7473 ◽

2016 ◽

Vol 10 (08) ◽

pp. 857-862 ◽

Cited By ~ 4

Author(s):

Sonia Hesam Shariati ◽

Anoosha Alaei ◽

Rouhollah Keshavarz ◽

Nader Mosavari ◽

Ali Rabbani ◽

...

Keyword(s):

Diabetes Mellitus ◽

Type 1 Diabetes ◽

Type 1 Diabetes Mellitus ◽

Mycobacterium Avium ◽

Mycobacterium Avium Subsp ◽

Blood Samples ◽

Control Subjects ◽

Healthy Control ◽

Mycobacterium Avium Subsp Paratuberculosis

Introduction: Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis or Johne’s disease in ruminants. Its role in triggering autoimmunity, including type 1 diabetes mellitus (T1DM), has been reported in recent years. Due to the high contamination rate of MAP in Iran’s livestock and the increasing outbreak of T1DM, we investigated this association in a small group of patients with T1DM in Iran. Methodology: Blood samples of 29 T1DM patients and 29 healthy control subjects were tested through enzyme-linked immunosorbent assay (ELISA) to detect antibodies against MAP3865c and ZnT8 homologous epitopes and the presence of MAP DNA. Blood samples were also cultured in mycobacterial growth indicator tubes and Herrold's egg yolk medium containing mycobactin J. Results: The results of ELISA showed a significant difference between T1DM patients and healthy group. IS900 was also detected in 51.72% of T1DM patients but in none of the control group. None of the samples grew in culture media. Conclusions: Due to the presence of MAP DNA and antibodies against MAP peptides in a significant number of T1DM patients compared with healthy control subjects, we may consider MAP as a possible trigger of T1DM in Iran. This indicates that exposure to MAP occurred in the positive subjects. Identifying the sources of contamination and routes of MAP transmission to humans seems necessary to prevent and reduce the burden of MAP infection in Iran.

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A Comparative Study on the Efficiency of Two Mycobacterium avium subsp. paratuberculosis (MAP)-Derived Lipopeptides of L3P and L5P as Capture Antigens in an In-House Milk ELISA Test

Vaccines ◽

10.3390/vaccines9090997 ◽

2021 ◽

Vol 9 (9) ◽

pp. 997

Author(s):

Sepideh Hosseiniporgham ◽

Franck Biet ◽

Christelle Ganneau ◽

John P. Bannantine ◽

Sylvie Bay ◽

...

Keyword(s):

Mycobacterium Avium ◽

Elisa Test ◽

Cross Reactivity ◽

Mycobacterium Avium Subsp ◽

Enzyme Analysis ◽

Restriction Enzyme Analysis ◽

Individual Level ◽

Milk Samples ◽

Milk Elisa ◽

Mycobacterium Avium Subsp Paratuberculosis

Mycobacterium avium subsp. paratuberculosis (MAP) surface-exposed lipopeptides could be specific capture-antigen molecules targeting antibodies against MAP, in milk, through ELISA. Previous studies have revealed that MAP strains, isolated from sheep (S) or cow (C), could produce specific lipopeptides, L3P or L5P, respectively. In this study, we used L3P and L5P as capture antigens in an in-house milk ELISA (H-MELISA) to assess how these antigens perform, in comparison with other ELISA tests, on well-defined milk samples from MAP-infected sheep. The overall positivity rates of H-MELISA via L3P and L5P varied by the source of milk samples, in which, at bulk-tank-milk (BTM) level, the majority of positive cases (63.83%) reacted more against L5P, whereas a predominant number (69.14%) of milk samples were more responsive against L3P at the individual level. To clarify whether the positivity status of milk samples in H-MELISA L3P/L5P were predictive of MAP strain-types (S/C), strain-typing was carried out using PCR IS1311-restriction enzyme analysis. Although the presence of three MAP strains (S/C/bison types) was detected among the milk samples, the C-type (46.67%) and S-type (75%) MAP strains were detected with higher incidence among BTMs and individual milk samples, respectively. However, further examination on the H-MELISA L3P/L5P-positivity pattern of each C/S-type-MAP sample revealed that some samples had a reverse reactivity against both L3P and L5P. These results could be the consequence of either cross-reactivity between L3P and L5P (due to the similarity in the structures of the two epitopes) or simply a within-herd mixed infection with MAP strains of C and S types. These findings suggest that lipopeptide antigens could contribute a diagnostic test with optimal performance, considering the diversity of MAP strains.

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A 38-Kilobase Pathogenicity Island Specific for Mycobacterium avium subsp. paratuberculosis Encodes Cell Surface Proteins Expressed in the Host

Infection and Immunity ◽

10.1128/iai.72.3.1265-1274.2004 ◽

2004 ◽

Vol 72 (3) ◽

pp. 1265-1274 ◽

Cited By ~ 28

Author(s):

Janin Stratmann ◽

Birgit Strommenger ◽

Ralph Goethe ◽

Karen Dohmann ◽

Gerald-F. Gerlach ◽

...

Keyword(s):

Mycobacterium Avium ◽

Pathogenicity Island ◽

Surface Proteins ◽

Open Reading Frames ◽

Representational Difference Analysis ◽

Specific Phage ◽

Expression Studies ◽

Specific Locus ◽

Mycobacterium Avium Subsp Paratuberculosis ◽

Phage Peptide Library

ABSTRACT We have used representational difference analysis to identify a novel Mycobacterium avium subsp. paratuberculosis-specific ABC transporter operon (mpt), which comprises six open reading frames designated mptA to -F and is immediately preceded by two putative Fur boxes. Functional genomics revealed that the mpt operon is flanked on one end by a fep cluster encoding proteins involved in the uptake of Fe3+ and on the other end by a sid cluster encoding non-ribosome-dependent heterocyclic siderophore synthases. Together these genes form a 38-kb M. avium subsp. paratuberculosis-specific locus flanked by an insertion sequence similar to IS1110. Expression studies using Western blot analyses showed that MptC is present in the envelope fraction of M. avium subsp. paratuberculosis. The MptD protein was shown to be surface exposed, using a specific phage (fMptD) isolated from a phage-peptide library, by differential screening of Mycobacterium smegmatis transformants. The phage fMptD-derived peptide could be used in a peptide-mediated capture PCR with milk from infected dairy herds, thereby showing surface-exposed expression of the MptD protein in the host. Together, these data suggest that the 38-kb locus constitutes an M. avium subsp. paratuberculosis pathogenicity island.

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A Novel Enzyme-Linked Immunosorbent Assay for Diagnosis of Mycobacterium avium subsp. paratuberculosis Infections (Johne's Disease) in Cattle

Clinical and Vaccine Immunology ◽

10.1128/cvi.13.5.535-540.2006 ◽

2006 ◽

Vol 13 (5) ◽

pp. 535-540 ◽

Cited By ~ 25

Author(s):

C. A. Speer ◽

M. Cathy Scott ◽

John P. Bannantine ◽

W. Ray Waters ◽

Yasuyuki Mori ◽

...

Keyword(s):

Sensitivity And Specificity ◽

Mycobacterium Avium ◽

Johne's Disease ◽

Johne’S Disease ◽

Diagnostic Sensitivity ◽

Enzyme Linked Immunosorbent Assay ◽

Formaldehyde Concentration ◽

Serum Samples ◽

Mycobacterium Avium Subsp Paratuberculosis ◽

Diagnostic Sensitivity And Specificity

ABSTRACT Enzyme-linked immunosorbent assays (ELISAs) for the diagnosis of Johne's disease (JD), caused by Mycobacterium avium subsp. paratuberculosis, were developed using whole bacilli treated with formaldehyde (called WELISA) or surface antigens obtained by treatment of M. avium subsp. paratuberculosis bacilli with formaldehyde and then brief sonication (called SELISA). ELISA plates were coated with either whole bacilli or sonicated antigens and tested for reactivity against serum obtained from JD-positive and JD-negative cattle or from calves experimentally inoculated with M. avium subsp. paratuberculosis, Mycobacterium avium subsp. avium, or Mycobacterium bovis. Because the initial results obtained from the WELISA and SELISA were similar, most of the subsequent experiments reported herein were performed using the SELISA method. To optimize the SELISA test, various concentrations (3.7 to 37%) of formaldehyde and intervals of sonication (2 to 300 s) were tested. With an increase in formaldehyde concentration and a decreased interval of sonication, there was a concomitant decrease in nonspecific binding by the SELISA. SELISAs prepared by treating M. avium subsp. paratuberculosis with 37% formaldehyde and then a 2-s burst of sonication produced the greatest difference (7×) between M. avium subsp. paratuberculosis-negative and M. avium subsp. paratuberculosis-positive serum samples. The diagnostic sensitivity and specificity for JD by the SELISA were greater than 95%. The SELISA showed subspecies-specific detection of M. avium subsp. paratuberculosis infections in calves experimentally inoculated with M. avium subsp. paratuberculosis or other mycobacteria. Based on diagnostic sensitivity and specificity, the SELISA appears superior to the commercial ELISAs routinely used for the diagnosis of JD.

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