Improved Enzyme-Linked Immunosorbent Assay To Reveal Mycoplasma agassizii Exposure: a Valuable Tool in the Management of Environmentally Sensitive Tortoise Populations

ABSTRACT The precarious status of desert (Gopherus agassizii) and gopher (Gopherus polyphemus) tortoises has resulted in research and conservation efforts that include health assessments as a substantial component of management decision-making. Therefore, it is critical that available diagnostic tests for diseases impacting these species undergo rigorous standardization and validation. Since 1992, analysis of exposure of tortoises to Mycoplasma agassizii, an etiological agent of upper respiratory tract disease, has relied on the detection of specific M. agassizii antibody by enzyme-linked immunosorbent assay (ELISA). We report here substantive refinements in the diagnostic assay and discuss the implications of its use in wildlife conservation and management. The ELISA has been refined to include more stringent quality control measures and has been converted to a clinically more meaningful titer reporting system, consistent with other diagnostic serologic tests. The ELISA results for 5,954 desert and gopher tortoises were plotted, and a subset of these serum samples (n = 90) was used to determine end-point titers, to establish an optimum serum dilution for analyzing samples, and to construct a standard curve. The relationship between titer and A 405 was validated using 77 serum samples from known positive (n = 48) and negative (n = 29) control tortoises from prior transmission studies. The Youden index, J, and the optimal cut point, c, were estimated using ELISA results from the 77 control sera. Based on this evaluation, the refinement has substantially improved the performance of the assay (sensitivity of 0.98, specificity of 0.99, and J of 0.98), thus providing a clinically more reliable diagnostic test for this important infection of tortoises.

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Mycoplasma agassizii Strain Variation and Distinct Host Antibody Responses Explain Differences between Enzyme-Linked Immunosorbent Assays and Western Blot Assays

Clinical and Vaccine Immunology ◽

10.1128/cvi.00215-10 ◽

2010 ◽

Vol 17 (11) ◽

pp. 1739-1745 ◽

Cited By ~ 9

Author(s):

Lori D. Wendland ◽

Paul A. Klein ◽

Elliott R. Jacobson ◽

Mary B. Brown

Keyword(s):

Western Blot ◽

Goodness Of Fit ◽

False Negative ◽

Well Being ◽

Management Decision ◽

Enzyme Linked Immunosorbent Assay ◽

Upper Respiratory Tract ◽

Strain Variation ◽

Serum Samples ◽

Mycoplasma Agassizii

ABSTRACT The precarious status of desert (Gopherus agassizii) and gopher (G. polyphemus) tortoises has resulted in conservation efforts that now include health assessment as an important component of management decision-making. Mycoplasmal upper respiratory tract disease (URTD) is one of very few diseases in chelonians for which comprehensive and rigorously validated diagnostic tests exist. In this study, serum samples obtained from eight Gopherus tortoises documented at necropsy to (i) be enzyme-linked immunosorbent assay (ELISA) seropositive using the PS6 antigen, (ii) be infected with Mycoplasma agassizii as indicated by direct isolation of the pathogen from the respiratory surfaces, and (iii) have histological lesions of mycoplasmal URTD were used to evaluate four distinct clinical isolates of M. agassizii as antigens for ELISA and Western blot analyses. Each animal sample reacted in the Western blot with its homologous M. agassizii strain, but recognition of heterologous M. agassizii strains was variable. Further, individual animals varied significantly with respect to the specific proteins recognized by the humoral immune response. An additional 114 Gopherus serum samples were evaluated using ELISA antigens prepared from the four distinct M. agassizii strains; A 405 values were significantly correlated (r 2 goodness of fit range, 0.708 to 0.771; P < 0.0001) for all antigens tested. The results confirm that strain variation is responsible for the observed differences between Western blot binding patterns. Thus, reliance on a single M. agassizii strain as an antigen in Western blot assays may provide false-negative results. This could have adverse consequences for the well-being of these environmentally sensitive hosts if false-negative animals were relocated to sites consisting of true-negative populations.

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Seroprevalence of Toxoplasma gondii and associated risk factors in domestic pigs raised from Cuba

Parasitology Research ◽

10.1007/s00436-021-07245-1 ◽

2021 ◽

Author(s):

Julio César Castillo-Cuenca ◽

Álvaro Martínez-Moreno ◽

José Manuel Diaz-Cao ◽

Angel Entrena-García ◽

Jorge Fraga ◽

...

Keyword(s):

Risk Factors ◽

Toxoplasma Gondii ◽

Meat Products ◽

Control Measures ◽

Health Concern ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Cross Sectional ◽

Weaning Pigs ◽

Associated Risk Factors

AbstractA cross-sectional study was carried out to determine the seroprevalence of Toxoplasma gondii and associated risk factors in pigs in the largest pork-producing region in Cuba. Serum samples from 420 pigs, including 210 sows and 210 post-weaning pigs, were tested for antibodies against T. gondii using a commercial indirect enzyme-linked immunosorbent assay. Anti-T. gondii antibodies were detected in 56 animals (13.3%, 95% CI: 10.1–16.6). A generalized estimating equations model revealed that the risk factors associated with higher seropositivity in pigs were altitude (higher in farm’s location < 250 m above sea level (masl) versus ≥ 250 masl) and age (higher in sows compared to post-weaning pigs). The results indicated that this protozoan parasite is widely distributed on pig farms in the study area, which is a public health concern since the consumption of raw or undercooked pork meat products containing tissue cysts is considered one of the main routes of T. gondii transmission worldwide. Control measures should be implemented to reduce the risk of exposure to T. gondii in pigs in Cuba.

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Serological Investigation of Peste Des Petits Ruminants in East Shewa and Arsi Zones, Oromia Region, Ethiopia

Veterinary Medicine International ◽

10.1155/2017/9769071 ◽

2017 ◽

Vol 2017 ◽

pp. 1-5 ◽

Cited By ~ 5

Author(s):

Getachew Gari ◽

Biressaw Serda ◽

Dejene Negesa ◽

Fethu Lemma ◽

Hagos Asgedom

Keyword(s):

Significant Variation ◽

Small Ruminants ◽

Control Measures ◽

Economic Losses ◽

Enzyme Linked Immunosorbent Assay ◽

High Seroprevalence ◽

Serum Samples ◽

Male And Female ◽

Significant Difference ◽

Important Disease

Peste des petits ruminant (PPR) is an economically important disease of small ruminants with a rapidly expanding geographical distribution. There are fragmented reports to the occurrence and distribution of the disease in Ethiopia. A total of 700 serum samples were collected from goats and sheep to detect the presence of antibody against PPR virus using Competitive Enzyme-Linked Immunosorbent Assay (C-ELISA). An overall PPR seropositivity was reported to be 48.43% in the area. There is no statistically significant difference in the seroprevalence of the disease between sheep and goats (50.85% and 46.68%), respectively. However, there was statistically significant variation (P<0.05) in the seroprevalence of the disease in young (33.9%) and adult (55.8%) age categories. The seroprevalence in male and female was 42.07% and 50.09%, respectively, where the variation was statistically not significant (P>0.05). High seroprevalence of Peste des petites ruminants in the study area indicated the virus circulation and endemicity of the disease. The disease causes substantial economic losses by affecting the livelihood of the farmers. Therefore, control measures should be put in place to minimize the loss associated with the disease.

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Prevalence of feline coronavirus antibodies in cats in Bursa province, Turkey, by an enzyme-linked immunosorbent assay

Journal of Feline Medicine and Surgery ◽

10.1016/j.jfms.2009.02.008 ◽

2009 ◽

Vol 11 (10) ◽

pp. 881-884 ◽

Cited By ~ 5

Author(s):

Annamaria Pratelli ◽

Kadir Yesilbag ◽

Marcello Siniscalchi ◽

Ebru Yalçm ◽

Zeki Yilmaz

Keyword(s):

Immunosorbent Assay ◽

Predictive Value ◽

Gold Standard ◽

Population Based ◽

Standard Test ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

High Association ◽

Gold Standard Test ◽

Feline Coronavirus

Feline sera from Bursa province (Turkey) were assayed for coronavirus antibody using an enzyme-linked immunosorbent assay (ELISA). The study was performed on 100 sera collected from cats belonging to catteries or community shelters and to households. The serum samples were initially tested with the virus neutralisation (VN) test and the results were then compared with the ELISA. The VN yielded 79 negative and 21 positive sera but the ELISA confirmed only 74 as negative. The ELISA-negative sera were also found to be free of feline coronoviruses-specific antibodies by Western blotting. Using the VN as the gold standard test, ELISA had a sensitivity of 100% and a specificity of 93.6%, with an overall agreement of 95%. The Kappa (κ) test indicated high association between the two tests (κ=0.86, 95% confidence interval (CI) 0.743–0.980). The positive predictive value (PPV) was 0.8, and the negative predictive value (NPV) was 0.93. The prevalence of FCoV II antibodies in the sampled population based on the gold standard was 62% (95% CI 0.44–0.77) among multi-cat environments, and 4% (95% CI 0.01–0.11) among single cat households.

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Development of an Enzyme-Linked Immunosorbent Assay for the Detection of Antibody to Epizootic Hemorrhagic Disease of Deer Virus

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063870001200207 ◽

2000 ◽

Vol 12 (2) ◽

pp. 142-145 ◽

Cited By ~ 18

Author(s):

James O. Mecham ◽

Michael M. Jochim

Keyword(s):

Immunosorbent Assay ◽

Enzyme Linked Immunosorbent Assay ◽

Hemorrhagic Disease ◽

Structural Protein ◽

Serum Samples ◽

Epizootic Hemorrhagic Disease ◽

Diagnostic Potential ◽

The Us ◽

Serotype 1 ◽

Infected Cells

An enzyme-linked immunosorbent assay has been developed to detect antibodies to epizootic hemorrhagic disease of deer virus (EHDV). The assay incorporates a monoclonal antibody to EHDV serotype 2 (EHDV-2) that demonstrates specificity for the viral structural protein, VP7. The assay was evaluated with sequential sera collected from cattle experimentally infected with EHDV serotype 1 (EHDV-1) and EHDV-2, as well as the four serotypes of bluetongue virus (BTV), BTV-10, BTV-11, BTV-13, and BTV-17, that currently circulate in the US. A competitive and a blocking format as well as the use of antigen produced from both EHDV-1-and EHDV-2-infected cells were evaluated. The assay was able to detect specific antibody as early as 7 days after infection and could differentiate animals experimentally infected with EHDV from those experimentally infected with BTV. The diagnostic potential of this assay was demonstrated with field-collected serum samples from cattle, deer, and buffalo.

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Development of a Smartphone-Based Nanozyme-Linked Immunosorbent Assay for Quantitative Detection of SARS-CoV-2 Nucleocapsid Phosphoprotein in Blood

Frontiers in Microbiology ◽

10.3389/fmicb.2021.692831 ◽

2021 ◽

Vol 12 ◽

Author(s):

Bochao Liu ◽

Ze Wu ◽

Chaolan Liang ◽

Jinhui Lu ◽

Jinfeng Li ◽

...

Keyword(s):

Immunosorbent Assay ◽

Cross Reactivity ◽

Quantitative Detection ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Primary Screening ◽

Global Pandemic ◽

Novel Coronavirus ◽

Acid Test ◽

Control Samples

Since December 2019, a novel coronavirus (SARS-CoV-2) has resulted in a global pandemic of coronavirus disease (COVID-19). Although viral nucleic acid test (NAT) has been applied predominantly to detect SARS-CoV-2 RNA for confirmation diagnosis of COVID-19, an urgent need for alternative, rapid, and sensitive immunoassays is required for primary screening of virus. In this study, we developed a smartphone-based nanozyme-linked immunosorbent assay (SP-NLISA) for detecting the specific nucleocapsid phosphoprotein (NP) of SARS-CoV-2 in 37 serum samples from 20 COVID-19 patients who were diagnosed by NAT previously. By using SP-NLISA, 28/37 (75.7%) serum samples were detected for NP antigens and no cross-reactivity with blood donors’ control samples collected from different areas of China. In a control assay using the conventional enzyme-linked immunosorbent assay (ELISA), only 7/37 (18.91%) serum samples were detected for NP antigens and no cross-reactivity with control samples. SP-NLISA could be used for rapid detection of SARS-CoV-2 NP antigen in primary screening of SARS-CoV-2 infected individuals.

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Development of an Enzyme-Linked Immunosorbent Assay for Immunoglobulin M Antibodies against Measles Virus

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.10.3.439-442.2003 ◽

2003 ◽

Vol 10 (3) ◽

pp. 439-442 ◽

Cited By ~ 3

Author(s):

F. Roodbari ◽

M. H. Roustai ◽

A. Mostafaie ◽

H. Soleimanjdahi ◽

R. Sarrami Foroshani ◽

...

Keyword(s):

Immunosorbent Assay ◽

Neutralizing Antibodies ◽

Measles Virus ◽

Clinical Symptoms ◽

Maculopapular Rash ◽

Immunoglobulin M ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Vaccination Programs ◽

Elisa System

ABSTRACT Measles is a highly contagious respiratory virus infection, with typical clinical symptoms including maculopapular rash, fever, cough, coryza, and conjunctivitis. Despite implementation of widespread vaccination programs throughout the world, the rates of global morbidity and mortality are still considerable. This study was performed to design a reliable indirect enzyme-linked immunosorbent assay (ELISA) to measure measles-specific immunoglobulin M (IgM). First, human IgM was purified, and then an anti-IgM antibody was produced in rabbits and purified in a multistep process. The rabbit IgG against human IgM was conjugated with peroxidase. Measles virus-infected Vero cells produced viral antigen. One hundred serum samples from infants of 9 to 18 months of age, mostly vaccinated, were evaluated for determining the presence of specific IgM antibodies against measles virus. The samples were also evaluated for neutralizing antibodies against measles virus by a microneutralization test (MNT). By comparing the results of the ELISA with those of MNT, it was demonstrated that ELISA had a sensitivity and specificity of 100 and 92%, respectively. On the other hand, when the results obtained by our ELISA system were compared with those of an imported measles virus IgM ELISA kit (EIAgen; Adaltis Italia SPa, Bologna, Italy), a high level of agreement was shown (k = 0.926).

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Seroprevalence of contagious bovine pleuropneumonia in cattle of Punjab, Pakistan and assessment of risk factors

Indian Journal of Animal Research ◽

10.18805/ijar.b-1130 ◽

2019 ◽

Author(s):

Ahsan Anjum ◽

Asim Aslam ◽

Raheela Akhtar ◽

Tahir Yaqub ◽

Junaid Naseer ◽

...

Keyword(s):

Risk Factors ◽

Cross Sectional Study ◽

Control Measures ◽

Export Market ◽

Economic Losses ◽

Enzyme Linked Immunosorbent Assay ◽

Contagious Bovine Pleuropneumonia ◽

Serum Samples ◽

Cross Sectional ◽

Associated Risk Factors

Contagious bovine pleuropneumonia (CBPP) is an important infectious disease of respiratory system of cattle, caused by Mycoplasma mycoides subspecies mycoides. A cross sectional study was conducted in Lahore, Kasur and Jhang districts of Punjab to determine the prevalence of CBPP. A total of 560 serum samples were examined for the specific antibodies against M. mycoides subspecies mycoides, using a sandwich enzyme-linked immunosorbent assay. Associated risk factors that were evaluated include age, gender and breed. In this study, the overall seroprevalence was 8.75%. The seroprevalence of CBPP in Lahore, Kasur and Jhang were 8.26, 8.20 and 10.06%, respectively, but no significance difference (P less than 0.05) was observed. Among the assessed potential predisposing factors, gender and breed were not significantly associated with the serological status of the animal (P greater than 0.05), in which the age affected significantly the positivity to CBPP (P less than 0.05). The highest seropositivity was observed in animals of more than 7 years of age (32.08%) followed by in 4-7 years of age (8.16%) and lowest in animals of less than 4 years old (0.79%). This study confirms the presence of CBPP in the state which could be a threat to livestock export market and warrants the implementation of control measures to mitigate the economic losses associated with the disease.

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Seroprevalence of avian metapneumovirus infection in broiler and broiler breeder chickens in Iran

Veterinární Medicína ◽

10.17221/1554-vetmed ◽

2011 ◽

Vol 56 (No. 8) ◽

pp. 395-399 ◽

Cited By ~ 3

Author(s):

M. Rahimi

Keyword(s):

Broiler Chickens ◽

Economic Losses ◽

Enzyme Linked Immunosorbent Assay ◽

Upper Respiratory Tract ◽

Serum Samples ◽

Broiler Breeder ◽

Blood Samples ◽

Avian Metapneumovirus ◽

Commercial Chicken ◽

Future Work

Avian metapneumovirus causes an acute highly contagious upper respiratory tract infection primarily of turkeys and chickens. The disease can cause significant economic losses in turkey and chicken flocks, particularly when exacerbated by secondary pathogens. The purpose of this study was to determine the prevalence of avian metapneumovirus antibodies in broiler and broiler breeder flocks in Kermanshah province, west of Iran. All the flocks had not been vaccinated against avian metapneumovirus. The province were divided into four geographic areas; southwest, southeast, northwest, and northeast. Flocks in each area, and 14–15 birds in each flock, were randomly sampled. The blood samples were taken regardless of the presence of any signs of respiratory or any other clinical disease in the flocks. A total of 435 blood samples were collected from 30 commercial chicken flocks (24 broiler flocks, aged between six and eight weeks, and six broiler breeder flocks, aged between 56 and 72 weeks). The presence of antibodies against avian metapneumovirus in each serum sample was tested twice by enzyme-linked immunosorbent assay using a commercial kit which was able to determine antibodies against A, B and C subtypes of avian metapneumovirus. Out of 347 serum samples obtained from broiler chickens, 167 (48.1%) were positive to avian metapneumovirus antibodies, which represented 20 (83.3%) of 24 examined broiler flocks. Out of 88 samples obtained from broiler breeder chickens, 82 (93.2%) were positive to avian metapneumovirus antibodies, which belonged to six (100%) of examined broiler breeder flocks. Detection of anti-avian metapneumovirus antibodies among broiler breeder (100%) was higher than broiler (83.3%) flocks. A higher rate of seropositivity (83.3% of samples and 100% of broiler flocks) was observed in northwest. The results of this study may indicate the possible involvement of avian metapneumovirus in the respiratory disease we are seeing in chickens in Iran. Its prevalence has to be investigated in other parts of Iran. Future work may and should include the use of molecular methods and isolation of the virus. Isolation of avian metapneumovirus will allow the possibility of making autogenous vaccines.

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An enzyme-linked immunosorbent assay for the detection of IgG antibodies against Babesia equi in horses

Ciência Rural ◽

10.1590/s0103-84782004000500031 ◽

2004 ◽

Vol 34 (5) ◽

pp. 1525-1529 ◽

Cited By ~ 12

Author(s):

Cristiane Divan Baldani ◽

Rosangela Zacarias Machado ◽

Paulo de Tarso Landgraf Botteon ◽

Felipe Santoro Takakura ◽

Carlos Luiz Massard

Keyword(s):

Immunosorbent Assay ◽

Serological Test ◽

Epidemiological Studies ◽

Sao Paulo ◽

Enzyme Linked Immunosorbent Assay ◽

São Paulo ◽

Igg Antibodies ◽

Serum Samples ◽

Babesia Equi ◽

Specificity And Sensitivity

A crude antigenic preparation of Babesia equi was used to develop and establish the suitability of an enzyme-linked immunosorbent assay (ELISA) for the detection of parasite carriers. Optimal dilutions of the antigen, using positive and negative reference sera, were determined by checkboard titrations. The specificity and sensitivity of the ELISA were 100 %. A total of 90 serum samples were taken from horses from the Northeast region of São Paulo State and examined for diagnosis of equine B. equi infection by ELISA. Approximately 75% (n=67) of all the horses tested were found serologically positive for B. equi. These results suggest that the ELISA described may prove to be an appropriate serological test for epidemiological studies on B. equi infections in the field and that equine piroplasmosis is a cause for serious concern in the State of São Paulo, Brazil.

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