RESUMEN
BACKGROUND: Chagas disease (CD), a neglected parasitic disease caused by Trypanosoma cruzi, poses a significant health threat in Latin America and has emerged globally because of human migration. Trypanosoma cruzi infects humans and over 100 other mammalian species, including dogs, which are important sentinels for assessing the risk of human infection. Nonetheless, the serodiagnosis of T. cruzi in dogs is still impaired by the absence of commercial tests. In this study, we investigated the diagnostic accuracy of four chimeric recombinant T. cruzi IBMP antigens (IBMP-8.1, IBMP-8.2, IBMP-8.3, and IBMP-8.4) for detecting anti-T. cruzi antibodies in dogs, using latent class analysis (LCA). METHODS: We examined 663 canine serum samples, employing indirect ELISA with the chimeric antigens. LCA was utilized to establish a latent variable as a gold standard for T. cruzi infection, revealing distinct response patterns for each antigen. RESULTS: The IBMP (Portuguese acronym for the Molecular Biology Institute of Paraná) antigens achieved area under the ROC curve (AUC) values ranging from 90.9% to 97.3%. The highest sensitivity was attributed to IBMP-8.2 (89.8%), while IBMP-8.1, IBMP-8.3, and IBMP-8.4 achieved 73.5%, 79.6%, and 85.7%, respectively. The highest specificity was observed for IBMP-8.4 (98.6%), followed by IBMP-8.2, IBMP-8.3, and IBMP-8.1 with specificities of 98.3%, 94.4%, and 92.7%, respectively. Predictive values varied according to prevalence, indicating higher effectiveness in endemic settings. CONCLUSIONS: Our findings underscore the remarkable diagnostic performance of IBMP-8.2 and IBMP-8.4 for the serodiagnosis of Trypanosoma cruzi in dogs, representing a promising tool for the diagnosis of CD in dogs. These chimeric recombinant antigens may not only enhance CD surveillance strategies but also hold broader implications for public health, contributing to the global fight against this neglected tropical disease.
Asunto(s)
Anticuerpos Antiprotozoarios , Antígenos de Protozoos , Enfermedad de Chagas , Enfermedades de los Perros , Ensayo de Inmunoadsorción Enzimática , Sensibilidad y Especificidad , Pruebas Serológicas , Trypanosoma cruzi , Animales , Perros , Enfermedad de Chagas/diagnóstico , Enfermedad de Chagas/veterinaria , Enfermedad de Chagas/parasitología , Trypanosoma cruzi/inmunología , Trypanosoma cruzi/genética , Enfermedades de los Perros/diagnóstico , Enfermedades de los Perros/parasitología , Antígenos de Protozoos/inmunología , Antígenos de Protozoos/genética , Pruebas Serológicas/métodos , Pruebas Serológicas/veterinaria , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/veterinaria , Anticuerpos Antiprotozoarios/sangre , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/genéticaRESUMEN
A simple IgG-speciï¬c ELISA for Leptospira spp. was compared with the microscopic agglutination test (MAT) to detect IgG antibody responses to a commercial vaccine in cattle. We used an enzyme-linked immunosorbent assay (ELISA) with sonicated Leptospira interrogans serovar copenhageni M 20. After initial vaccination, specific antibodies against Leptospira spp. were detected in 90â¯% of the animals by IgG-ELISA and 60â¯% by MAT, while after booster, antibodies were detected in 100â¯% and 80â¯% of the animals by IgG-ELISA and MAT, respectively. Both serological MAT and ELISA tests revealed interferences of vaccine antibodies. Disease diagnosis with ELISA and MAT methods should be made two and a half months and four months, respectively, after vaccination to avoid interference of vaccine antibodies. On the other hand, our results suggest that IgG-ELISA may be a useful method to assess the development of IgG antibodies induced by Leptospira vaccine.
Asunto(s)
Pruebas de Aglutinación , Anticuerpos Antibacterianos , Vacunas Bacterianas , Enfermedades de los Bovinos , Ensayo de Inmunoadsorción Enzimática , Inmunoglobulina G , Leptospirosis , Animales , Leptospirosis/veterinaria , Leptospirosis/diagnóstico , Leptospirosis/inmunología , Bovinos , Enfermedades de los Bovinos/microbiología , Enfermedades de los Bovinos/diagnóstico , Enfermedades de los Bovinos/inmunología , Enfermedades de los Bovinos/sangre , Ensayo de Inmunoadsorción Enzimática/veterinaria , Anticuerpos Antibacterianos/sangre , Vacunas Bacterianas/inmunología , Inmunoglobulina G/sangre , Pruebas de Aglutinación/veterinaria , Leptospira interrogans/inmunología , Leptospira/inmunología , Vacunación/veterinaria , Pruebas Serológicas/veterinariaRESUMEN
Bovine trypanosomosis, caused by Trypanosoma vivax, currently affects cattle and has a significant economic impact in sub-Saharan Africa and South America. The development of new diagnostic antigens is essential to improve and refine existing methods. Our study evaluated the efficacy of two recombinant antigens in detecting specific antibodies in cattle. These antigens are derivatives of an invariant surface glycoprotein (ISG) from T. vivax. A fraction of a previously described antigen (TvY486_0045500), designated TvISGAf, from an African strain was evaluated, and a new ISG antigen from an American isolate, TvISGAm, was identified. The two antigens were expressed as fusion proteins in Escherichia coli: TvISGAf was fused to the MBP-His-tag, and TvISGAm was obtained as a His-tag fused protein. An ELISA evaluation was conducted using these antigens on 149 positive and 63 negative bovine samples. The diagnostic performance was enhanced by the use of a combination of both antigens (referred to as TvISG-based ELISA), achieving a sensitivity of 89.6% and specificity of 93.8%. Following the validation of the TvISG-based ELISA, the seroprevalence of T. vivax infection in 892 field samples from cattle in the central region of Argentina was determined. The mean seroprevalence of T. vivax was 53%, with variation ranging from 21% to 69% among the six departments studied. These results support the use of the TvISG ELISA as a valuable serological tool for the detection and monitoring of T. vivax infection in cattle. Furthermore, we report for the first time the seroprevalence of T. vivax in Argentina, which highlights the widespread endemic nature of the disease in the region. In order to effectively manage the increasing spread of T. vivax in the vast livestock production areas of South America, it is essential to implement consistent surveillance programs and to adopt preventive strategies.
Asunto(s)
Antígenos de Protozoos , Enfermedades de los Bovinos , Ensayo de Inmunoadsorción Enzimática , Pruebas Serológicas , Trypanosoma vivax , Animales , Bovinos , Argentina/epidemiología , Trypanosoma vivax/inmunología , Trypanosoma vivax/genética , Trypanosoma vivax/aislamiento & purificación , Pruebas Serológicas/métodos , Pruebas Serológicas/veterinaria , Enfermedades de los Bovinos/diagnóstico , Enfermedades de los Bovinos/epidemiología , Enfermedades de los Bovinos/parasitología , Antígenos de Protozoos/inmunología , Antígenos de Protozoos/genética , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/veterinaria , Anticuerpos Antiprotozoarios/sangre , Sensibilidad y Especificidad , Tripanosomiasis Africana/diagnóstico , Tripanosomiasis Africana/veterinaria , Tripanosomiasis Africana/epidemiología , Ganado/parasitologíaRESUMEN
Hepatitis E virus (HEV) infects roughly 20 million people worldwide, causing self-limiting acute hepatic disease that can evolve into a chronic course. HEV-3, HEV-4, and HEV-7 genotypes are zoonotic and transmitted to humans by consuming raw or undercooked meat. Here, we developed an indirect ELISA based on the recombinant HEV-3 capsid and performed a seroprevalence study on domestic swine in northeastern Brazil. Our in-house ELISA was initially validated using a subset of 79 sera characterized by concordant results for two distinct commercial ELISA kits. Our ELISA exhibited excellent sensitivity (94%) and specificity (100%), with an area under the curve of 0.99 Further testing, including 212 swine sera, revealed a seroprevalence of 57.5% (95% confidence interval, 50.6-64.3%). Our findings indicate that the novel ELISA test could accurately detect specific anti-HEV antibodies in domestic pigs and should be further validated in humans and other mammals.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática , Virus de la Hepatitis E , Hepatitis E , Pruebas Serológicas , Enfermedades de los Porcinos , Animales , Hepatitis E/veterinaria , Hepatitis E/diagnóstico , Hepatitis E/epidemiología , Ensayo de Inmunoadsorción Enzimática/veterinaria , Ensayo de Inmunoadsorción Enzimática/métodos , Porcinos , Virus de la Hepatitis E/inmunología , Virus de la Hepatitis E/genética , Virus de la Hepatitis E/aislamiento & purificación , Enfermedades de los Porcinos/diagnóstico , Enfermedades de los Porcinos/virología , Estudios Seroepidemiológicos , Pruebas Serológicas/veterinaria , Brasil/epidemiología , Anticuerpos Antihepatitis/sangre , Sensibilidad y Especificidad , HumanosRESUMEN
Mycobacterium avium subsp. paratuberculosis (MAP) is the causal agent of paratuberculosis (PTBC), a chronic infectious granulomatous enteritis of ruminants. The PTBC diagnosis with commercial ELISA has limitations in sensitivity and specificity, and its results depend on the state of progress of the disease. This research aimed to evaluate two different ELISAs: (a) an "in-house" ELISA with a sonicated antigen obtained from a MAP I47 strain, and (b) a commercial ELISA. In total, the evaluated sample consisted of 394 bovine serum samples from 12 farms in Argentina with high (5-9%) and low (≤ 0.05%) prevalence of PTBC. The evaluation of the new antigen (2.5 µg/mL) was against a 1:50 dilution of the M. phlei faced sera. The cut-off point, sensitivity, and specificity determinations of both techniques were by ROC curve analysis. The area under the curve for the I47 ELISA was 0.9 (CI 95%, 0.93-0.97). With a cut-off point of 8.8%, the sensitivity was 84.3% and the specificity 96.6%. The agreement between both techniques was 0.7 (CI 95%, 0.6-0.8). These results indicate a high discriminative capacity to differentiate positive and negative bovine sera of MAP infection with the I47 ELISA. This result would represent an advantage to dispense with the imported kit.
Asunto(s)
Enfermedades de los Bovinos , Ensayo de Inmunoadsorción Enzimática , Mycobacterium avium subsp. paratuberculosis , Paratuberculosis , Sensibilidad y Especificidad , Bovinos , Animales , Paratuberculosis/diagnóstico , Paratuberculosis/sangre , Paratuberculosis/microbiología , Enfermedades de los Bovinos/diagnóstico , Enfermedades de los Bovinos/sangre , Enfermedades de los Bovinos/microbiología , Ensayo de Inmunoadsorción Enzimática/veterinaria , Ensayo de Inmunoadsorción Enzimática/métodos , Mycobacterium avium subsp. paratuberculosis/inmunología , Mycobacterium avium subsp. paratuberculosis/aislamiento & purificación , Pruebas Serológicas/veterinaria , Pruebas Serológicas/métodos , ArgentinaRESUMEN
Cystic echinococcosis (CE) is an emergent neglected disease affecting human and animals in Egypt with a wide distribution and incidence. This study aimed to evaluate the use of a polyclonal antibody-based sandwich ELISA in the detection of Echinococcus granulosus antigen in human and camel sera. Hydatid cyst protoscoleces antigen (PsAg) was isolated from hydatid cysts collected from naturally infected camel livers and lungs. PsAg was used for immunization of rabbits to raise IgG polyclonal antibodies (IgG PsAb). IgG PsAb were then precipitated, purified using Protein-A Sepharose gel and labeled with horseradish peroxidase enzyme. We assayed the purity of the IgG PsAb, and the two prepared E. granulosus antigens CPsAg from camel cysts and HPsAg from human cysts by Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The resulted protein bands of the prepared CPsAg appeared at different molecular weights: 180, 90, 68, 54, 42 and 22 kDa while, HPsAg shared with it in 4 common bands at 68, 54, 42, and 22 kDa. The purified IgG PsAb had been resolved at two bands at 52 kDa and at 32 kDa. Sandwich ELISA were performed for the detection of circulating E. granulosus antigens in sera of human (n = 183) and camels (n = 190). The purified IgG PsAb showed strong reactivity against E. granulosus infected human and camel samples and no cross reactivity neither with free-healthy negative sera nor with others parasitic diseases (Schistosomiasis, Fascioliasis, Toxoplasmosis, Ancylostomiasis for human samples and Fascioliasis, ticks' infestation, Eimeriosis, Cryptosporidiosis, Nasal myiasis, Toxoplasmosis for camel samples). The sensitivity of the assay was 98.25% (56/57) and 96.9% (31/32) against human and camel samples, respectively. Specificity was 100% in both human and camel samples. Sandwich ELISA detected CE in 33.3% (24/72) and 55.6% (50/90) random human and camel samples, respectively. Indirect ELISA, using CPsAg, was used for detection of antibodies in positive human and camels' sera and detected 96.5% (55/57) and 93.8% (30/32) of human and camel samples, respectively. In our study, Genomic DNA was extracted from protoscoleces fluid of human liver hydatid cysts to identify the Echinococcus sp. isolate based on NADH dehydrogenase subunit 1 (NAD1) gene by Polymerase Chain Reaction (PCR) and the isolate (GenBank: OP785689.1) were identified as E. granulosus sensu lato genotype. In conclusion, Sandwich ELISA technique was found to be a potent and sensitive assay for detection of hydatid antigen in both human and camel samples.
Asunto(s)
Camelus , Equinococosis , Echinococcus granulosus , Ensayo de Inmunoadsorción Enzimática , Pruebas Serológicas , Animales , Camelus/parasitología , Ensayo de Inmunoadsorción Enzimática/veterinaria , Ensayo de Inmunoadsorción Enzimática/métodos , Equinococosis/diagnóstico , Equinococosis/veterinaria , Equinococosis/parasitología , Equinococosis/sangre , Humanos , Echinococcus granulosus/inmunología , Pruebas Serológicas/veterinaria , Pruebas Serológicas/métodos , Anticuerpos Antihelmínticos/sangre , Antígenos Helmínticos/inmunología , Conejos , Sensibilidad y EspecificidadRESUMEN
Coprological and serological diagnostic tests were compared to define the status of a pig farm with regard to Ascaris suum. On each of the 100 farms in France visited for the study, 10 blood samples were taken from pigs at the end of fattening (at least 22 weeks old) and 20 to 30 faecal samples were taken, depending on the category of animals present on the farm (10 sows, 10 piglets aged 10 to 12 weeks and 10 pigs at the end of fattening, aged at least 22 weeks). A SERASCA® ELISA test (Laboratory of Parasitology, Ghent University) was performed on each blood sample (cut-off 0.5) and a coprological analysis on each faecal sample. A Bayesian approach was used to estimate the sensitivity and specificity of the coprological and serological tests. A farm was considered positive if at least one A. suum egg was observed in the faecal samples. With regard to the serological test, various hypotheses were tested in order to define the number of seropositive animals required to consider a farm positive for A. suum. The coprological test has very good specificity in the search for A. suum, whether 20 or 30 samples are taken per farm. However, even with an increase in the number of samples, the sensitivity of this diagnostic approach is very low (less than 30%). On the other hand, the serological diagnostic method, which consists of taking blood samples from 10 animals at the end of fattening, has good sensitivity and seems better suited to defining the status of a farm with regard to A. suum, provided that a farm is considered seropositive only if two out of 10 samples are positive.
Asunto(s)
Ascariasis , Ascaris suum , Teorema de Bayes , Heces , Sensibilidad y Especificidad , Enfermedades de los Porcinos , Animales , Ascaris suum/aislamiento & purificación , Ascaris suum/inmunología , Enfermedades de los Porcinos/diagnóstico , Enfermedades de los Porcinos/parasitología , Heces/parasitología , Heces/química , Porcinos , Ascariasis/diagnóstico , Ascariasis/veterinaria , Ascariasis/parasitología , Pruebas Serológicas/veterinaria , Pruebas Serológicas/métodos , Ensayo de Inmunoadsorción Enzimática/veterinaria , Ensayo de Inmunoadsorción Enzimática/métodos , Francia , Granjas , Anticuerpos Antihelmínticos/sangreRESUMEN
Bovine fasciolosis is a parasitic disease with a global reach. Coprological based on egg detection in fecal samples and liver inspection to evaluate the presence of the parasite is currently the gold standard for diagnosing chronic fasciolosis in cattle. However, these techniques are labor-intensive and ineffective during the acute phase of the disease. Serodiagnosis using native and recombinant antigens has become an interesting alternative in efforts to identify cattle fasciolosis. We evaluated cattle from abattoir (n = 139) and farms (n = 500) through liver inspection and coprological examination, respectively. Our laboratory team optimized and validated enzyme-linked immunosorbent assay tests based on somatic antigen, excretory/secretory proteins, and the recombinant antigen cathepsin L-1 to detect serum antibodies against fasciolosis in cattle. For animals from abattoir, 10 were positive for fasciolosis according to liver inspection. Both FhES and FhrCL-1 presented an area under the receiver operating characteristic (AUROC) curve of 0.80, with a sensitivity of 0.80 (95% CI: 0.46-0.95) and 0.70 (95% CI: 0.38-0.90) and specificity of 0.81 (95% CI: 0.73-0.87) and 0.87 (95% CI: 0.80-0.92), respectively. For those cattle from farms, 28 were positive only for fasciolosis according to coprological examination. In this scenario, FhES gave the best performance, with an AUROC of 0.84, sensitivity of 0.79 (95% CI: 0.60-0.90), and specificity of 0.86 (95% CI: 0.82-0.89). In conclusion, our study highlights the potential of serodiagnosis for accurately screening cattle fasciolosis. The promising sensitivity and specificity values of FhES when compared to liver inspection or coprological examination enhance its importance for cattle fasciolosis diagnosis. IMPORTANCE: The aim of this article was to identify antibodies against fasciolosis in cattle in Brazil. The methodology was reproduced in our laboratory and applied for the first time to the Brazilian cattle herd. The antigens tested can be used as a screening test and thus speed up the diagnosis of bovine fascioliasis.
Asunto(s)
Anticuerpos Antihelmínticos , Antígenos Helmínticos , Enfermedades de los Bovinos , Ensayo de Inmunoadsorción Enzimática , Fascioliasis , Sensibilidad y Especificidad , Animales , Bovinos , Fascioliasis/diagnóstico , Fascioliasis/veterinaria , Fascioliasis/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/veterinaria , Enfermedades de los Bovinos/diagnóstico , Enfermedades de los Bovinos/parasitología , Antígenos Helmínticos/inmunología , Brasil , Anticuerpos Antihelmínticos/sangre , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/genética , Heces/parasitología , Pruebas Serológicas/métodos , Pruebas Serológicas/veterinaria , Fasciola hepatica/inmunología , Mataderos , Curva ROC , Hígado/parasitologíaRESUMEN
Toxoplasma gondii is a paradigmatic zoonotic parasite from the One Health perspective, since it is broadly distributed and virtually infects all warm-blooded species. A wide variety of serological techniques have been developed to detect T. gondii infection in humans and animals. Our aim was to describe and compare the main characteristics of these serological tests and validation processes and to critically analyze whether these tests meet the standards required to ensure an accurate serological diagnosis. The current systematic review and meta-analysis included 134 studies that were published from 2013 to 2023. QUADAS 2 tool was used to evaluate the quality of the included studies. A total of 52 variables related to the characteristics of the techniques and analytical and diagnostic validation parameters were studied. A wider panel of tests was developed for humans, including techniques exclusively developed for humans that involve costly equipment and the measurement of different Ig isotypes that are considered biomarkers of congenital toxoplasmosis. Studies conducted in humans frequently employed commercial techniques as reference tests, measured different immunoglobulin isotypes with a predominance for IgG (>50%) and discriminated between acute and chronic infections. In animals, the most commonly used reference techniques were in-house tests, which almost exclusively detected IgG. Common limitations identified in a large number of studies were some misunderstandings of the terms "gold standard" and "reference test" and the absence of information about the negative and positive control sera used or the exact cutoff employed, which were independent of the quality of the study. There is a lack of analytical validation, with few evaluations of cross-reactivity with other pathogens. Diagnostic odds ratio values showed that indirect ELISA based on native or chimeric antigens performed better than other tests. The reproducibility of serological test results in both humans and animals is not guaranteed due to a lack of relevant information and analytical validation. Thus, several key issues should be considered in the future, including interlaboratory ring trials.
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Anticuerpos Antiprotozoarios , Pruebas Serológicas , Toxoplasma , Toxoplasmosis Animal , Toxoplasmosis , Animales , Humanos , Anticuerpos Antiprotozoarios/sangre , Reproducibilidad de los Resultados , Pruebas Serológicas/veterinaria , Pruebas Serológicas/normas , Pruebas Serológicas/métodos , Toxoplasma/inmunología , Toxoplasmosis/diagnóstico , Toxoplasmosis/inmunología , Toxoplasmosis/sangre , Toxoplasmosis Animal/diagnóstico , Toxoplasmosis Animal/inmunología , Toxoplasmosis Animal/sangreRESUMEN
Objectives: Lyme borreliosis incidence is increasing in several areas; moreover, it has recently gained the public's attention. Apart from erythema migrans, Lyme disease diagnosis relies (among others) on serology test; however, the prevalence of positive enzyme-linked immunosorbent assay (ELISA) and western blot (WB) assay has been poorly studied in the general population. We aimed to approach the seroprevalence of infection by Borrelia species responsible for Lyme disease in the French Isere department using city laboratories data. Patients and Methods: We retrieved all serological tests for Borrelia species responsible for Lyme disease performed in the two main networks of city laboratories between 2015 and 2020. All patients with both ELISA and WB IgG were considered seropositive. Results: We analyzed 27,360 tests (ELISA/ELISA+WB). Mean age was 50.9 ± 20.3 years (ranges: 0-101), with 57.1% females. Overall, 11.7% had IgG detected by ELISA, and 4.7% had IgG detected by both ELISA and WB assay. Seropositive status was more frequent in males (7.0% vs. 2.9%, p < 0.001). Seropositivity rate increased with age after a first peak in childhood; men aged 61-70 years had the highest seropositivity rate (10.3%). In addition, seropositivity rate was higher in persons from a rural area. In multivariate analysis, older age, male gender and living in a rural area were independently associated with seropositivity. Seropositivity rate was stable on the 2017-2020 period. Conclusion: The seroprevalence of infection by Borrelia species responsible for Lyme disease is high in Isere; this probably reduces the predictive positive value for Lyme disease of ELISA and WB IgG, suggesting that this serological test should not be performed for nonspecific symptoms.
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Borrelia burgdorferi , Borrelia , Enfermedad de Lyme , Femenino , Humanos , Masculino , Adulto , Persona de Mediana Edad , Anciano , Estudios Seroepidemiológicos , Anticuerpos Antibacterianos , Enfermedad de Lyme/diagnóstico , Enfermedad de Lyme/epidemiología , Enfermedad de Lyme/veterinaria , Ensayo de Inmunoadsorción Enzimática/veterinaria , Pruebas Serológicas/veterinaria , Inmunoglobulina GRESUMEN
Competition and indirect ELISAs are currently being used to monitor rabbit haemorrhagic disease viruses (RHDV1 and RHDV2) in rabbits worldwide. Temporal changes in the sensitivity (Se) and specificity (Sp) of RHDV1 competition-ELISA (cELISA1), RHDV2 competition-ELISA (cELISA2), and RHDV1 Immunoglobulin G (IgG1) ELISA, were investigated using Bayesian Latent Class models (BCLM) in the Australian wild rabbit population where both viruses circulate simultaneously and a long-term serological dataset exists. When cELISA1 was compared to IgG1 ELISA, the Se of cELISA1 improved while the Sp of IgG1 ELISA declined over the 2011-21. This corresponded with a decline in the true RHDV1 prevalence in 2018-21, suggesting that a large proportion of RHDV1 exposed rabbits survived the introduction and dominance of RHDV2 up to approximately 2017/2018, after which they died and were not replaced. The Se and Sp estimates for 2014-15 for both cELISA1 and IgG1 ELISA, and the true prevalence when analysing all three tests together were similar to those obtained from the analysis of cELISA1/IgG1 ELISA. The same was also true for the Se and Sp of cELISA2 and IgG1 ELISA estimates from 2018 onwards. This suggests that RHDV1 was the dominant infection type in 2014-15, but RHDV2 was the dominant infection type in 2018-21. Further, the increase in Se of cELISA2 and the low Sp of IgG1 ELISA in the cELISA2/IgG1 ELISA analysis, compared to the Se of cELISA2 and Sp of IgG1 ELISA when analysing all three tests together suggests that the underlying infection status was more influenced by RHDV2 and that the higher Se of IgG1 ELISA is due to cross-reaction of RHDV2 antibodies on IgG1 ELISA. The true prevalence data suggest that RHDV2 exposure peaked in 2017. Our findings show that test characteristics changed in response to the changing virus prevalences over time. IgG1 ELISA, currently having a high Se, should be used to monitor both viruses and will perform better than both cELISAs.
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Infecciones por Caliciviridae , Virus de la Enfermedad Hemorrágica del Conejo , Animales , Conejos , Teorema de Bayes , Infecciones por Caliciviridae/diagnóstico , Infecciones por Caliciviridae/epidemiología , Infecciones por Caliciviridae/veterinaria , Australia/epidemiología , Inmunoglobulina G , Sensibilidad y Especificidad , Pruebas Serológicas/veterinariaRESUMEN
Onchocerca lupi is a zoonotic filarioid parasite of dogs and cats with widespread distribution. A specific non-invasive diagnostic assay for the detection of O. lupi infections remains unavailable. This study aimed to assess the accuracy, specificity, and sensitivity of an ELISA test designed using nine peptides from two O. lupi proteins. Sera (n = 54) collected from O. lupi infected dogs from endemic areas (Portugal and USA), alongside sera from dogs positive for Dirofilaria immitis, D. repens, Cercopithifilaria bainae, and Acanthocheilonema reconditum (n = 53) from a non-endemic area for O. lupi, as well as from helminth-free dogs (n = 60), were tested. The checkerboard titration method was applied for the optimization of peptide concentrations and conjugate anti-dog dilutions. Sensitivity, specificity, and optimal cut-off values were calculated using ROC curve analysis. All peptides reacted against sera of O. lupi, with no correlation between optic density (OD) values and microfilariae (mfs) loads. Sensitivity and specificity values ranging from 85.45 to 100%, and 88.89% to 100%, respectively, were recorded for all peptides examined, with 100% specificity and sensitivity observed for peptides 40_3, 40_5, 130_3, 120_3 and 40_1, 130_5, respectively. The maximum cut-off value was observed for peptides 40_5 (0.765) and 40_3 (0.708). Testing of sera from dogs positive for other filarioids resulted in lower OD values (up to 1.565) for peptides 40_3 and 40_5 when compared with O. lupi (up to 2.929). The availability of this assay will be of value in epidemiological studies of canine O. lupi infection in both endemic and non-endemic areas, and in assessing the risk for zoonotic transmission.
Asunto(s)
Enfermedades de los Gatos , Enfermedades de los Perros , Animales , Perros , Gatos , Onchocerca , Enfermedades de los Perros/diagnóstico , Enfermedades de los Perros/parasitología , Ensayo de Inmunoadsorción Enzimática/veterinaria , Pruebas Serológicas/veterinaria , PéptidosRESUMEN
Bovine tuberculosis (bTB), which is caused by Mycobacterium bovis, is a single health concern, which causes economic losses, is a sanitary barrier and is a zoonotic concern. The golden-pattern intradermic tests have low sensitivity of about 50%. To fix this sensitivity problem, immunoassays could be a powerful tool. However, few studies produced antigens for bTB immunoassays, which needs improvements. Aim of this study was to produce multiepitope chimeric antigens (MCA) to use for bTB diagnosis. To achieve MCA design and development, extensive bibliographic search, antigenic epitope prediction, specificity, hydrophobicity, and 3D structure modeling analyses were performed, as well as cloning, expression and purification. Seven epitopes from four different target proteins (MPB-70, MPB-83, ESAT-6 and GroEL) were combined in five chimeras containing five repetitions of each epitope to enhance antibodies affinity. 3D predicted models revealed that all chimeras have a high percentage of disorder, which could enhance antibody recognition, although taking to protein instability. Each chimera was cloned into pET28a (+) expression plasmids and expressed in six Escherichia coli expression strains. Chimeras 3, 4 and 5 could be solubilized in 8â¯M urea and purified by ion exchange affinity chromatography. Against bTB positive and negative sera, purified chimera 5 had the best results in indirect dot blot and ELISA, as well as in lateral flow dot blot immunoassay. In conclusion, chimera 5, an MPB-83 containing MCA, could be used for further studies, aimed to develop a serologic or rapid test for bTB diagnosis.
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Enfermedades de los Bovinos , Tuberculosis Bovina , Animales , Bovinos , Tuberculosis Bovina/diagnóstico , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Pruebas Serológicas/veterinaria , Ensayo de Inmunoadsorción Enzimática/veterinaria , Ensayo de Inmunoadsorción Enzimática/métodos , Epítopos/genética , Biología Computacional , Sensibilidad y Especificidad , Proteínas RecombinantesRESUMEN
Bovine brucellosis, primarily caused by Brucella abortus, severely affects both animal health and human well-being. Accurate diagnosis is crucial for designing informed control and prevention measures. Lacking a gold standard test makes it challenging to determine optimal cut-off values and evaluate the diagnostic performance of tests. In this study, we developed a novel Bayesian Latent Class Model that integrates both binary and continuous testing outcomes, incorporating additional fixed (parity) and random (farm) effects, to calibrate optimal cut-off values by maximizing Youden Index. We tested 651 serum samples collected from six dairy farms in two regions of Henan Province, China with four serological tests: Rose Bengal Test, Serum Agglutination Test, Fluorescence Polarization Assay, and Competitive Enzyme-Linked Immunosorbent Assay. Our analysis revealed that the optimal cut-off values for FPA and C-ELISA were 94.2 mP and 0.403 PI, respectively. Sensitivity estimates for the four tests ranged from 69.7% to 89.9%, while specificity estimates varied between 97.1% and 99.6%. The true prevalences in the two study regions in Henan province were 4.7% and 30.3%. Parity-specific odds ratios for positive serological status ranged from 1.2 to 2.2 for different parity groups compared to primiparous cows. This approach provides a robust framework for validating diagnostic tests for both continuous and discrete tests in the absence of a gold standard test. Our findings can enhance our ability to design targeted disease detection strategies and implement effective control measures for brucellosis in Chinese dairy farms.
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Brucelosis Bovina , Brucelosis , Enfermedades de los Bovinos , Femenino , Humanos , Bovinos , Animales , Brucella abortus , Teorema de Bayes , Análisis de Clases Latentes , Sensibilidad y Especificidad , Pruebas de Aglutinación/veterinaria , Brucelosis/epidemiología , Brucelosis/veterinaria , Ensayo de Inmunoadsorción Enzimática/veterinaria , Brucelosis Bovina/diagnóstico , Brucelosis Bovina/epidemiología , Anticuerpos Antibacterianos , Pruebas Serológicas/veterinariaRESUMEN
The direct methods for diagnosis of bovine brucellosis have several limitations, therefore serological tests are the basis for the diagnosis of the disease. However, a meta-analysis estimating the diagnostic sensitivity (DSe) and diagnostic specificity (DSp) on the main tests used in bovine brucellosis control programs worldwide has not been performed. This systematic review and meta-analysis aimed to estimate the DSe, DSp and thereby accuracy of serological tests individually used in the diagnosis of bovine brucellosis. The databases CABI, Cochrane Library, PubMed/MEDLINE, SciELO, Scopus and Web of Science were used to select articles. The search resulted in 5308 studies, of which 71 were selected for systematic review using quality assessment tools and 65 studies were included in the meta-analysis. For the meta-analysis, 178 assays and 11 different serological tests were considered. To estimate DSe and DSp of the tests, studies were divided according to animal selection for the studies: (1) studies that carried out a random or consecutive selection of participants (noncasecontrol studies) and (2) all studies, including casecontrol studies. Considering only the non-case-control studies to estimate the DSe, the tests that exhibited the best and worst performance were the iELISA test (indirect enzyme immunoassay - bacterial suspension as antigen - BS) (96.5%, 95% CI: 94.1-97.9%) and 2ME (2- mercaptoethanol test) (85.0%, 95% CI: 79.6-89.1%), respectively; while for DSp, the FPA (fluorescence polarization assay) (99, 7%, 95% CI: 99.5-99.8%) and PCFIA tests (protein concentration fluorescence immunoassay) (78.5%, 95% CI: 70.0-85.1%) showed better and worse performance, respectively. Overall, our results showed an overestimation in the DSe and DSp of the eleven serological tests assessed when casecontrol studies were included in the meta-analysis, which is a concern considering its impacts on the time and costs associated with populational diagnosis of the diseases, since several of these tests are routinely used in the control and eradication programs of bovine brucellosis worldwide. Furthermore, the tests that exhibited the best DSe and DSp, iELISA (BS) and FPA, respectively, are relatively easy to perform and interpret and the test which showed the best overall accuracy was FPA.
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Brucelosis Bovina , Brucelosis , Enfermedades de los Bovinos , Bovinos , Animales , Sensibilidad y Especificidad , Brucelosis Bovina/diagnóstico , Inmunoensayo de Polarización Fluorescente/métodos , Inmunoensayo de Polarización Fluorescente/veterinaria , Ensayo de Inmunoadsorción Enzimática/veterinaria , Ensayo de Inmunoadsorción Enzimática/métodos , Pruebas Serológicas/veterinaria , Brucelosis/diagnóstico , Brucelosis/veterinaria , Anticuerpos AntibacterianosRESUMEN
Toxoplasma gondii is a major foodborne zoonotic pathogen that can be transmitted through the consumption of raw or undercooked meat of small ruminants, among others. Serology has been suggested as an epidemiological indicator and several tests are available nowadays. However, there is no comparative study with the most used ones. Therefore, the objective of this study was to develop and validate two in-house tests (Western blot -TgSALUVET WB- and ELISA -TgSALUVET ELISA 2.0-) and perform a comparative study including such tests and four commercial ELISA kits (IDScreen®, PrioCHECK®, Pigtype® and IDEXX). First, a specific pattern of recognition of immunodominant antigens by TgSALUVET WB was determined with serum panels of noninfected sheep and sheep infected with T. gondii or Neospora caninum. Next, TgSALUVET WB was used as a reference to preliminary validate TgSALUVET ELISA 2.0 using sera from sheep and goats naturally infected with T. gondii. Then, the abovementioned sheep serum panels were analyzed by all tests and subjected to TG-ROC analyses and agreement tests, and cross-reactivity with the anti-N. caninum IgGs was studied. All the techniques were accurate enough for the cutoff values initially suggested with all serum panels (Se and Sp ≥ 94%), except for PrioCHECK®, which showed 83% Sp. However, a cutoff readjustment improved their diagnostic performance. Additionally, cross-reactions between anti-N. caninum antibodies and T. gondii antigens were detected with all tests. Thus, a second cutoff readjustment was carried out and the use of both readjusted cutoff values is recommended to obtain comparable data and avoid false-positive results.
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Coccidiosis , Enfermedades de las Cabras , Neospora , Toxoplasma , Toxoplasmosis Animal , Ovinos , Animales , Toxoplasmosis Animal/diagnóstico , Anticuerpos Antiprotozoarios , Rumiantes , Cabras , Ensayo de Inmunoadsorción Enzimática/veterinaria , Pruebas Serológicas/veterinaria , Estudios Seroepidemiológicos , Coccidiosis/diagnóstico , Coccidiosis/veterinaria , Enfermedades de las Cabras/diagnósticoRESUMEN
BACKGROUND: Enzyme-linked immunosorbent assays (ELISAs) are the most widely used diagnostic tools in bovine paratuberculosis (bPTB) control. However, their diagnostic accuracy may be compromised by bovine tuberculosis (bTB) infection, as both diseases share diagnostic targets. METHODS: The bPTB and bTB infection status of 228 animals was determined using microbiological tissue culture as a reference test. The diagnostic performance (sensitivity, specificity, likelihood ratios and predictive values) of the bPTB-ELISA on blood serum samples, taking into account the bPTB animal-level prevalence of the area and the bTB status of the animals, was evaluated. RESULTS: A sensitivity of 40.7% (95% confidence interval [CI]: 27.5%-53.9%) and a specificity of 94.7% (95% CI: 91.4%-98.0%) were obtained for bPTB-ELISA in all animals. A bPTB-ELISA-positive animal would have a post-test probability of 70% or more of being infected in areas with a bPTB prevalence of 23% or more. A negative bPTB-ELISA result, in areas with a bPTB prevalence of 41% or less, would rule out the disease with more than 70% certainty. In bTB-positive animals, sensitivity increased (94.4% [95% CI: 81.4%-100%] vs. 25.1% [95% CI: 11.8%-38.4%]) and specificity decreased (82.6% [95% CI: 71.8%-93.4%] vs. 99.4% [95% CI: 98.0%-99.9%]). The bPTB-ELISA is a good tool to rule out bPTB co-infection in bTB-positive animals, while in bTB-negative animals, it allows confirmation of disease with more than 70% probability if disease prevalence is 6% or more. LIMITATIONS: The observed differences could be enhanced by the effect of frequent application of the intradermal tuberculin test, which was unknown in the animals studied. CONCLUSIONS: These results provide useful guidance for the application and interpretation of ELISA as a tool for bPTB disease control.
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Enfermedades de los Bovinos , Paratuberculosis , Tuberculosis Bovina , Bovinos , Animales , Paratuberculosis/diagnóstico , Paratuberculosis/epidemiología , Sensibilidad y Especificidad , Tuberculosis Bovina/diagnóstico , Tuberculosis Bovina/epidemiología , Enfermedades de los Bovinos/diagnóstico , Enfermedades de los Bovinos/epidemiología , Ensayo de Inmunoadsorción Enzimática/veterinaria , Pruebas Serológicas/veterinariaRESUMEN
Leishmaniasis is an anthropozoonosis with vector transmission, and knowledge regarding the occurrence of this parasitosis in sentinels can contribute to infection and disease control measures in humans. The objectives of this study were to evaluate the occurrence of Leishmania exposure and infection in dogs from urban and rural areas in the North Pioneer Mesoregion of the state of Paraná, to evaluate possible risk factors, and to analyze the statistical agreement between the serological techniques that were used. Using a convenience sampling, serum and whole blood samples were collected to perform serological and molecular assays, respectively. The enzyme-linked immunosorbent assay (ELISA) and indirect fluorescent antibody test (IFAT) identified 29/204 (14.2%) and 20/204 (9.8%) seropositive dogs, respectively. Five dogs (2.4%) were seropositive for both serological tests, and four dogs presented high titers in the IFAT. None of the samples tested positive for Leishmania spp. DNA according to polymerase chain reaction analysis. No factors were significantly associated with infection. Leishmania parasites circulate in urban and rural dogs in the North Pioneer Mesoregion of the state of Paraná. Despite the absence of clinical cases, seropositive animals with high antibody titers should serve as a warning to the local population that should be properly informed regarding the prevention.
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Enfermedades de los Perros , Leishmania infantum , Leishmania , Leishmaniasis Visceral , Leishmaniasis , Humanos , Animales , Perros , Brasil/epidemiología , Leishmaniasis/veterinaria , Leishmaniasis/epidemiología , Pruebas Serológicas/veterinaria , Ensayo de Inmunoadsorción Enzimática/veterinaria , Enfermedades de los Perros/parasitología , Leishmaniasis Visceral/epidemiología , Leishmaniasis Visceral/veterinaria , Leishmaniasis Visceral/parasitología , Anticuerpos AntiprotozoariosRESUMEN
Fasciolosis caused by Fasciola hepatica is a disease of zoonotic importance that is common worldwide and can cause serious problems in farm animals, some wild animals and humans. The development of diagnostic kits for the correct diagnosis of fasciolosis in sheep is important in terms of preventing yield losses. With this study, it is aimed to clone and express the enolase gene to be isolated from adult F. hepatica and to determine the effectiveness of the recombinant antigen in the serodiagnosis of sheep fasciolosis. For this aim, primers were designed to amplify the enolase gene from the F. hepatica enolase sequence, mRNA was isolated from F. hepatica adult fluke obtained from an infected sheep followed by cDNA was obtained. Enolase gene was amplified by PCR and the product was cloned and then expressed. The efficiency of the purified recombinant protein was displayed by Western blot (WB) and ELISA using positive and negative sheep sera. As a result, the sensitivity and specificity rates of the recombinant FhENO antigen were 85% and %82.8 by WB while the rates were 90% and 97.14% by ELISA, respectively. At the same time, in sheep blood sera samples collected from the Elazig and Siirt provinces of Turkey, 100 (50%) of 200 sera were found to be positive by WB and 46 (23%) were found to be positive by ELISA. The most important problem in ELISA was the high cross-reaction rate of the recombinant antigen used, as in WB. In order to prevent the cross-reactions, it will be useful to compare the genes encoding the enolase protein of parasites from the closely related parasite family, and select the regions where there are no common epitopes, and clone them and test the purified protein.
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Fasciola hepatica , Fascioliasis , Enfermedades de las Ovejas , Humanos , Animales , Ovinos , Fosfopiruvato Hidratasa/genética , Fascioliasis/diagnóstico , Fascioliasis/veterinaria , Fascioliasis/parasitología , Pruebas Serológicas/veterinaria , Ensayo de Inmunoadsorción Enzimática/veterinaria , Proteínas Recombinantes/genética , Clonación Molecular , Enfermedades de las Ovejas/diagnóstico , Enfermedades de las Ovejas/parasitologíaRESUMEN
Equine infectious anemia (EIA) is an important viral disease characterized by persistent infection in equids worldwide. Most EIA cases are life-long virus carriers with low antibody reactions and without the appearance of clinical symptoms. A serological test with high sensitivity and specificity is required to detect inapparent infection. In this study, a B-cell common epitope-based blocking ELISA (bELISA) was developed using a monoclonal antibody together with the EIAV p26 protein labelled with HRP. The test has been evaluated against the standard and with field serum samples globally. This bELISA test can be completed within 75 min, and the sensitivity is higher than those of either the AGID or one commercial cELISA kit. This bELISA assay was 8-16 times more analytically sensitive than AGID, and 2 to 4 times more analytically sensitive than one cELISA kit by testing three sera from the USA, Argentina, and China, respectively. The 353 serum samples from Argentina were tested, in comparison with AGID, the diagnostic sensitivity and specificity of our bELISA assay were 100% (154/154) and 97.0% (193/199), respectively, and the accuracy of the bELISA test was 98.3%. The bELISA test developed in this study is a rapid, sensitive, specific method for the detection of EIAV infection, and could be a promising candidate for use in the monitoring of the EIA epidemic worldwide. KEY POINTS: ⢠A universal epitope-based blocking enzyme-linked immunosorbent assay (bELISA) was developed for detection of antibodies to EIAV. ⢠The bELISA assay can be used to test EIAV serum samples from different regions of the world including North America, South America, Europe, and Asia. ⢠The bELISA assay was evaluated in three different international labs and showed a better performance than other commercial kits.