Development of a Novel Cocktail Enzyme-Linked Immunosorbent Assay and a Field-Applicable Lateral-Flow Rapid Test for Diagnosis of Contagious Bovine Pleuropneumonia

Contagious bovine pleuropneumonia (CBPP) is a severe respiratory disease that is widespread in sub-Saharan Africa. It is caused byMycoplasma mycoidessubsp.mycoides, a bacterium belonging to theMycoplasma mycoidescluster. In the absence of an efficient CBPP vaccine, improved and easy-to-use diagnostic assays for recurrent testing combined with isolation and treatment of positive animals represent an option for CBPP control in Africa. Here we describe the comprehensive screening of 17 immunogenicMycoplasma mycoidessubsp.mycoidesproteins using well-characterized bovine sera for the development of a novel cocktail enzyme-linked immunosorbent assay (ELISA) for laboratory use. Two recombinantMycoplasmaimmunogens, MSC_0136 and MSC_0636, were used to set up a standardized cocktail ELISA protocol. According to the results from more than 100 serum samples tested, the sensitivity and specificity of the novel cocktail ELISA were 85.6% and 96.4%, respectively, with an overall diagnostic accuracy comparable to that of the Office International des Epizooties (OIE)-prescribed serological assays. In addition, we provide a proof of principle for a field-applicable, easy-to-use commercially produced prototype lateral-flow test for rapid (<30-min) diagnosis of CBPP.

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Detection of Francisella tularensis-Specific Antibodies in Patients with Tularemia by a Novel Competitive Enzyme-Linked Immunosorbent Assay

Clinical and Vaccine Immunology ◽

10.1128/cvi.00516-12 ◽

2012 ◽

Vol 20 (1) ◽

pp. 9-16 ◽

Cited By ~ 28

Author(s):

Neekun Sharma ◽

Akitoyo Hotta ◽

Yoshie Yamamoto ◽

Osamu Fujita ◽

Akihiko Uda ◽

...

Keyword(s):

Sensitivity And Specificity ◽

Francisella Tularensis ◽

Immunosorbent Assay ◽

Enzyme Linked Immunosorbent Assay ◽

The Novel ◽

Serum Samples ◽

Content Type ◽

Microagglutination Test ◽

Highly Sensitive ◽

High Degree

ABSTRACTA novel competitive enzyme-linked immunosorbent assay (cELISA) was developed and evaluated for detection of antibodies againstFrancisella tularensisin humans. The assay is based on the ability of serum antibodies to inhibit the binding of monoclonal antibodies (MAbs) directed againstF. tularensislipopolysaccharide antigens. The assay was evaluated using serum samples of tularemia patients, inactivatedF. tularensis-immunized rabbits, andF. tularensis-infected mice. Antibodies againstF. tularensiswere successfully detected in serum samples of tularemia patients as well as the immunized and infected animals. The cELISA method was compared to indirect ELISA (iELISA) and the commonly used microagglutination test (MA) using serum samples of 19 tularemia patients and 50 healthy individuals. The sensitivity and specificity of cELISA were 93.9 and 96.1%, respectively, in comparison to the iELISA. MA was less sensitive than cELISA with a sensitivity and specificity of only 81.8 and 98.0%, respectively. A high degree of correlation (R2= 0.8226) was observed between cELISA and iELISA results. The novel cELISA developed in this study appears to be highly sensitive and specific for serodiagnosis of human tularemia. The potential of the MAb-based cELISA to be used in both human and animal samples emphasizes its usefulness for serological survey of tularemia among multiple animal species.

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Multiplex Screening of Surface Proteins from Mycoplasma mycoides subsp. mycoides Small Colony for an Antigen Cocktail Enzyme-Linked Immunosorbent Assay

Clinical and Vaccine Immunology ◽

10.1128/cvi.00223-09 ◽

2009 ◽

Vol 16 (11) ◽

pp. 1665-1674 ◽

Cited By ~ 12

Author(s):

Maja Neiman ◽

Carl Hamsten ◽

Jochen M. Schwenk ◽

Göran Bölske ◽

Anja Persson

Keyword(s):

Immunosorbent Assay ◽

False Negative ◽

Negative Control ◽

Surface Proteins ◽

Enzyme Linked Immunosorbent Assay ◽

Contagious Bovine Pleuropneumonia ◽

Mycoplasma Mycoides ◽

Small Colony ◽

Suspension Bead Array ◽

Selection Of

ABSTRACT A recombinant antigen cocktail enzyme-linked immunosorbent assay (ELISA) for diagnosis of contagious bovine pleuropneumonia (CBPP) was developed after careful selection of antigens among one-third of the surface proteome proteins of the infectious agent Mycoplasma mycoides subsp. mycoides small colony (M. mycoides SC). First, a miniaturized and parallelized assay system employing antigen suspension bead array technology was used to screen 97 bovine sera for humoral immune responses toward 61 recombinant surface proteins from M. mycoides SC. Statistical analysis of the data resulted in selection of eight proteins that showed strong serologic responses in CBPP-affected sera and minimal reactivity in negative control sera, with P values of <10−6. Only minor cross-reactivity to hyperimmune sera against other mycoplasmas was observed. When applied in an ELISA, the cocktail of eight recombinant antigens allowed a fivefold signal separation between 24 CBPP-affected and 23 CBPP-free sera from different geographical origins. No false-positive results and only two false-negative results were obtained. In conclusion, the selected recombinant mycoplasma antigens qualified as highly specific markers for CBPP and could be employed in both a suspension bead array platform and a cocktail ELISA setting. This set of proteins and technologies therefore offers a powerful combination to drive and further improve serological assays toward reliable, simple, and cost-effective diagnosis of CBPP.

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Development of an Enzyme-Linked Immunosorbent Assay for Serodiagnosis of Ringworm Infection in Cattle

Clinical and Vaccine Immunology ◽

10.1128/cvi.00243-13 ◽

2013 ◽

Vol 20 (8) ◽

pp. 1150-1154 ◽

Cited By ~ 6

Author(s):

Elena Tatiana Băguţ ◽

Ludivine Cambier ◽

Marie-Pierre Heinen ◽

Vasile Cozma ◽

Michel Monod ◽

...

Keyword(s):

Positive Predictive Value ◽

Negative Predictive Value ◽

Immunosorbent Assay ◽

Predictive Value ◽

Skin Lesions ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Predictive Values ◽

Content Type ◽

Ringworm Infection

ABSTRACTThe aim of this study was to develop an in-house enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of ringworm infection in cattle. We used available recombinant forms ofTrichophyton rubrumdipeptidyl peptidase V (TruDppV) andT. rubrumleucin aminopeptidase 2 (TruLap2), which are 98% identical toTrichophyton verrucosumorthologues. Field serum samples from 135 cattle with ringworm infection, as confirmed by direct microscopy, fluorescence microscopy, and PCR, and from 55 cattle without any apparent skin lesions or history of ringworm infection that served as negative controls were used. Sensitivities, specificities, and positive and negative predictive values were determined to evaluate the diagnostic value of our ELISA. Overall, the ELISAs based on recombinant TruDppV and TruLap2 discriminated well between infected animals and healthy controls. Highly significant differences (P< 0.0001, Mann-Whitney U test) were noted between optical density values obtained when sera from infected versus control cattle were tested. The ELISA developed for the detection of specific antibodies against DppV gave 89.6% sensitivity, 92.7% specificity, a 96.8% positive predictive value, and a 78.4% negative predictive value. The recombinant TruLap2-based ELISA displayed 88.1% sensitivity, 90.9% specificity, a 95.9% positive predictive value, and a 75.7% negative predictive value. To the best of our knowledge, this is the first ELISA based on recombinant antigens for assessing immune responses to ringworm infection in cattle; it is particularly suitable for epidemiological studies and also for the evaluation of vaccines and/or vaccination procedures.

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Recent Developments in Vaccines for Bovine Mycoplasmoses Caused by Mycoplasma bovis and Mycoplasma mycoides subsp. mycoides

Vaccines ◽

10.3390/vaccines9060549 ◽

2021 ◽

Vol 9 (6) ◽

pp. 549

Author(s):

Katarzyna Dudek ◽

Ewelina Szacawa ◽

Robin A. J. Nicholas

Keyword(s):

Vaccine Coverage ◽

Sub Saharan Africa ◽

Economic Losses ◽

Mycoplasma Bovis ◽

Contagious Bovine Pleuropneumonia ◽

Cattle Industry ◽

Mycoplasma Mycoides ◽

Recent Developments ◽

Sub Saharan ◽

Cattle Rearing

Two of the most important diseases of cattle are caused by mycoplasmas. Mycoplasma bovis is a world-wide bovine pathogen that can cause pneumonia, mastitis and arthritis. It has now spread to most, if not all, cattle-rearing countries. Due to its increasing resistance to antimicrobial therapy, vaccination is the principal focus of the control of infection, but effective vaccines are currently lacking. Despite being eradicated from most parts of the world, Mycoplasma mycoides subsp. mycoides, the cause of contagious bovine pleuropneumonia (CBPP), continues to plague sub-Saharan Africa, affecting at least 25 countries. Numerous new experimental vaccines have been developed over the last 20 years to improve on protection afforded by the T1/44, a live vaccine in continuous use in Africa for over 60 years, but none so far have succeeded; indeed, many have exacerbated the disease. Tools for diagnosis and control are adequate for eradication but what is necessary are resources to improve vaccine coverage to levels last seen in the 1970s, when CBPP was restricted to a few countries in Africa. This paper summarizes the results of the main studies in the field of experimental mycoplasma vaccines, reviews data on commercially available bacterin vaccines and addresses issues relating to the search for new candidates for effective vaccines to reduce economic losses in the cattle industry caused by these two mycoplasmas.

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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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Prevalence of peste des petits ruminants in the arid zone in the Republic of Niger

Onderstepoort Journal of Veterinary Research ◽

10.4102/ojvr.v80i1.544 ◽

2013 ◽

Vol 80 (1) ◽

Cited By ~ 16

Author(s):

Souaibou Farougou ◽

Mariama Gagara ◽

Guy A. Mensah

Keyword(s):

Arid Zone ◽

Small Ruminants ◽

Sub Saharan Africa ◽

Effective Control ◽

Enzyme Linked Immunosorbent Assay ◽

Peste Des Petits Ruminants ◽

Serum Samples ◽

Significant Difference ◽

Sub Saharan ◽

The Republic

The study aimed to determine the prevalence of peste des petits ruminants in the arid zone (Niamey, Tillabéry and Tahoua) of the Republic of Niger. A serological survey was conducted and 519 serum samples were collected from 253 unvaccinated sheep and 266 unvaccinated goats. The sample included 340 female animals (168 sheep and 172 goats) and 160 kids and lambs (78 lambs and 82 kids). A competitive enzyme-linked immunosorbent assay yielded an overall seroprevalence of 45.0%. The prevalence in sheep was 42.0% compared with 47.9% in goats. The seroprevalence observed amongst small ruminants in Tahoua (49.8%) and Tillabéry (46.6%) was significantly higher (p = 0.001) than that observed in animals from Niamey (25.1%). It was also higher (p = 0.04) in sheep younger than two years (51.8%) than in adults (37.6%). Conversely, the seroprevalence showed no significant difference between male animals (35.8% in sheep; 50.1% in goats) and female animals (45.1% in sheep; 46.4% in goats). The prevalence of the disease observed amongst the sheep and goat populations confirms the continued danger of this disease in the areas studied. It is therefore necessary to develop strategies such as improving livestock services, providing effective vaccines and implementing a vaccination programme for an effective control of the disease in sub-Saharan Africa.

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Monoclonal Antibodies Specific for Immunorecessive Epitopes of Glucuronoxylomannan, the Major Capsular Polysaccharide of Cryptococcus neoformans, Reduce Serotype Bias in an Immunoassay for Cryptococcal Antigen

Clinical and Vaccine Immunology ◽

10.1128/cvi.05052-11 ◽

2011 ◽

Vol 18 (8) ◽

pp. 1292-1296 ◽

Cited By ~ 28

Author(s):

Ann Percival ◽

Peter Thorkildson ◽

Thomas R. Kozel

Keyword(s):

Monoclonal Antibodies ◽

Cryptococcus Neoformans ◽

Capsular Polysaccharide ◽

Polyclonal Antibodies ◽

Sub Saharan Africa ◽

Enzyme Linked Immunosorbent Assay ◽

Content Type ◽

Screening Strategies ◽

Sub Saharan ◽

High Degree

ABSTRACTImmunoassay for detection of glucuronoxylomannan (GXM), the major capsular polysaccharide ofCryptococcus neoformans, is an important tool for diagnosis of cryptococcosis. However, immunoassays that are based solely or in part on detection with polyclonal antibodies may show serotype bias in detection of GXM, particularly limited sensitivity for serotype C. In this study, we describe detection of GXM in an antigen capture sandwich enzyme-linked immunosorbent assay (ELISA) that used a cocktail of two monoclonal antibodies (MAbs). MAb F12D2 was previously produced by immunization with GXM that had been treated to removeO-acetyl groups, a major source of serotype specificity. MAb F12D2 has a high degree of reactivity with GXM of serotypes A, B, C, and D, but the reactivity with serotype D was less than was found with other MAbs. MAb 339 is highly reactive with GXM of serotypes A and D. Use of a combination of the two MAbs produced an immunoassay that had the best properties of both MAbs, including good reactivity with serotype C, which is an emerging threat in sub-Saharan Africa. These results suggest that next-generation immunoassays for diagnosis of cryptococcosis may be formulated by (i) use of immunization and hybridoma screening strategies that are designed to prospectively meet the needs of immunoassay performance and (ii) careful selection of MAbs that span the expected polysaccharide serotypes in the subject patient population.

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Evaluation of the Dynamiker Cryptococcal Antigen Lateral Flow Assay for the Diagnosis of HIV-Associated Cryptococcosis

Journal of Clinical Microbiology ◽

10.1128/jcm.02421-20 ◽

2020 ◽

Author(s):

Richard Kwizera ◽

Denis Omali ◽

Kiiza Tadeo ◽

John Kasibante ◽

Morris K. Rutakingirwa ◽

...

Keyword(s):

Predictive Value ◽

Diagnostic Performance ◽

Point Of Care ◽

Lateral Flow ◽

Lateral Flow Assay ◽

Sub Saharan Africa ◽

Serum Samples ◽

Cryptococcal Antigen ◽

Asymptomatic Patients ◽

Sub Saharan

Background: Cryptococcal meningitis is a leading cause of meningitis in sub-Saharan Africa. Given the need for rapid point of care testing, we evaluated the diagnostic performance of the Dynamiker cryptococcal antigen (CrAg) lateral flow assay (LFA). Methods: We assessed the diagnostic performance of the Dynamiker CrAg-LFA compared to the IMMY CrAg-LFA as the reference standard. We tested 150 serum, 115 plasma, 100 cerebrospinal fluid (CSF) samples from HIV patients with symptomatic meningitis and 113 serum samples from patients with suspected asymptomatic cryptococcal antigenemia. Results: Compared to the IMMY CrAg-LFA, sensitivity of Dynamiker CrAg-LFA was 98% in serum, 100% in plasma, 100% in CSF from symptomatic patients and 96% in serum from asymptomatic patients. Specificity was 66% in serum, 61% in plasma, 91% in CSF from symptomatic patients, and 86% in serum from asymptomatic patients. The positive predictive value was 85% in serum, 82% in plasma, 96% in CSF from symptomatic patients, and 69% in serum from asymptomatic patients. The negative predictive value was 94% in serum, 100% in plasma, 100% in CSF from symptomatic patients, and 99% in serum from asymptomatic patients. The inter-assay reproducibility was 100% across the four sample types with no observed discordant results when Dynamiker CrAg-LFA was tested in duplicate. However, a high number of false positives were observed on serum of symptomatic patients (11%), serum of asymptomatic patients (11%) and plasma of symptomatic patients (14%). Conclusion: The Dynamiker CrAg-LFA had excellent sensitivity but poor specificity, particularly when tested on serum and plasma.

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Recombinant Antigen-Based Enzyme-Linked Immunosorbent Assay for Diagnosis of Baylisascaris procyonis Larva Migrans

Clinical and Vaccine Immunology ◽

10.1128/cvi.00083-11 ◽

2011 ◽

Vol 18 (10) ◽

pp. 1650-1655 ◽

Cited By ~ 14

Author(s):

Sriveny Dangoudoubiyam ◽

Ramesh Vemulapalli ◽

Momar Ndao ◽

Kevin R. Kazacos

Keyword(s):

Immunosorbent Assay ◽

Cross Reactivity ◽

Larva Migrans ◽

Enzyme Linked Immunosorbent Assay ◽

Parasitic Infections ◽

Serum Samples ◽

Western Blot Assay ◽

Human Patient ◽

Content Type ◽

Baylisascaris Procyonis

ABSTRACTBaylisascarislarva migrans is an important zoonotic disease caused byBaylisascaris procyonis, the raccoon roundworm, and is being increasingly considered in the differential diagnosis of eosinophilic meningoencephalitis in children and young adults. Although aB. procyonisexcretory-secretory (BPES) antigen-based enzyme-linked immunosorbent assay (ELISA) and a Western blot assay are useful in the immunodiagnosis of this infection, cross-reactivity remains a major problem. Recently, a recombinantB. procyonisantigen, BpRAG1, was reported for use in the development of improved serological assays for the diagnosis ofBaylisascarislarva migrans. In this study, we tested a total of 384 human patient serum samples in a BpRAG1 ELISA, including samples from 20 patients with clinicalBaylisascarislarva migrans, 137 patients with other parasitic infections (8 helminth and 4 protozoan), and 227 individuals with unknown/suspected parasitic infections. A sensitivity of 85% and a specificity of 86.9% were observed with the BpRAG1 ELISA, compared to only 39.4% specificity with the BPES ELISA. In addition, the BpRAG1 ELISA had a low degree of cross-reactivity with antibodies toToxocarainfection (25%), while the BPES antigen showed 90.6% cross-reactivity. Based on these results, the BpRAG1 antigen has a high degree of sensitivity and specificity and should be very useful and reliable in the diagnosis and seroepidemiology ofBaylisascarislarva migrans by ELISA.

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Contagious bovine pleuropneumonia (lungsickness) in Africa : historical overview : Onderstepoort and veterinary research in Africa

Onderstepoort Journal of Veterinary Research ◽

10.4102/ojvr.v76i1.55 ◽

2009 ◽

Vol 76 (1) ◽

Cited By ~ 22

Author(s):

W. Amanfu

Keyword(s):

Central Africa ◽

Sub Saharan Africa ◽

Contagious Bovine Pleuropneumonia ◽

Beef Industry ◽

Resource Base ◽

Disease Epidemiology ◽

Prime Concern ◽

Mycoplasma Mycoides ◽

Sub Saharan ◽

Proactive Measures

Contagious bovine pleuropneumonia (CBPP) or lung sickness, is an insidious pneumonic disease of cattle caused by Mycoplasma mycoides subspecies mycoides small colony variant (MmmSC) and it is one of the major diseases affecting cattle in Africa. With the imminent eradication of rinderpest from Africa (Somali ecosystem) CBPP has become the disease of prime concern in terms of epizootics that affect cattle on the continent. The control and/or eradication of the disease have suffered from unsustained control actions due to lack of operational funds to support such actions and deterioration in the quality of veterinary services in many countries affected by the disease. Stamping out procedures which were adopted by Botswana to control the disease (1995-1997) cannot be carried out by many countries currently affected by CBPP due to the high financial cost, the widespread nature of disease, animal welfare considerations and the potential loss of a valuable genetic resource base. The current scenario of CBPP disease epidemiology in sub-Saharan Africa requires that proactive measures are taken to safeguard countries in southern Africa which are currently free from CBPP from being contaminated by the disease thus affecting the beef industry and people's livelihoods ; and to progressively control the disease in endemic zones of Western and Central Africa.

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