Serological Analysis by Enzyme-Linked Immunosorbent Assay Using Recombinant Antigen LipL32 for the Diagnosis of Swine Leptospirosis (original) (raw)
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Bioscience Journal, 2014
The objective of this research was to compare the results obtained by the Microscopic Agglutination Test (MAT) and indirect ELISA using outer membrane proteins (OMP) of the serovar Hardjo as antigen (ELISA/OMP-Hardjo). Ninety-three samples of blood serum from 3 to 6-year-old cattle of both sexes with no history of vaccination for leptospirosis were used. To perform the MAT, live cultures of 14 Leptospira serovars representing 11 serogroups of L. interrogans were used as antigen. Outer membrane proteins (OMP) of the serovar Hardjo (ELISA-OMP/Hardjo) were used as the ELISA antigen. Of the 93 samples tested by MAT, 37 (40%) were positive. None of the samples testing positive in the MAT were negative in the ELISA. Sensitivity was 100% and specificity 64%. A comparison of the MAT and ELISA-OMP/Hardjo tests showed 78% agreement and the Kappa index was 0.58 (p<0.0001). The ELISA-OMP/Hardjo proved to be a sensitive test for the diagnosis of bovine leptospirosis, indicating its potential use as a screening test and for epidemiological studies. The titration results obtained by MAT and by optical density (OD) in the ELISA-OMP/Hardjo test showed a positive correlation, and as the antibody titers in the MAT increased, so did the OD in ELISA, demonstrating the correspondence between the tests evaluated here. microscopic… SOUZA, M. A. et al Biosci. J., Uberlandia, v. 30, supplement 2, p. 833-838, Oct./14 RESUMO: O objetivo desta pesquisa foi comparar os resultados obtidos pela Soroaglutinação Microscópica (SAM) e pelo ELISA indireto empregando proteínas de membrana externa (PME) do sorovar Hardjo como antígeno (ELISA/PME-Hardjo). Utilizou-se 93 amostras de soro sanguíneo de bovinos de ambos os sexos com idade entre três a seis anos e sem histórico de vacinação para leptospirose. Para a realização da SAM utilizou-se como antígeno culturas vivas de 14 sorovares de Leptospira, representando 11 sorogrupos de L. interrogans. Empregou-se como antígeno do ELISA, proteínas de membrana externa (PME) do sorovar Hardjo (ELISA-PME/Hardjo). Das 93 amostras testadas na SAM, 37 (40%) foram positivas. Já o ELISA-PME/Hardjo identificou 57 (62%) positivas e 36 (38%) negativas. Nenhuma amostra positiva na SAM foi negativa no ELISA. A sensibilidade foi de 100% e especificidade 64%. A comparação dos testes de SAM e ELISA-PME/Hardjo demonstrou concordância de 78% e índice Kappa de 0,58 (p<0,0001). O ELISA-PME/Hardjo revelou-se como um teste sensível para o diagnóstico da leptospirose bovina, indicando seu uso potencial como exame de triagem e para estudos epidemiológicos. A correlação dos resultados obtidos pela titulação na SAM e DO no ELISA-PME/Hardjo foi positiva, sendo que a medida que os títulos de anticorpos na SAM aumentaram, a DO no ELISA também aumentou, demonstrando haver correspondência entre os exames avaliados.
Acta Tropica, 2019
Swine leptospirosis poses a major problem in the agricultural sector. The gold standard for serodiagnosis of leptospirosis is Microscopic Agglutination Test (MAT). However, the limitations of this technique make the search for alternative diagnostic methods inevitable. In the present study, ErpY-like recombinant protein (rErpY-like), produced in Escherichia coli and used as antigen in indirect enzyme-linked immunosorbent assay (ELISA), was evaluated for its efficacy as a novel diagnostic tool for swine leptospirosis. For the study, 72 samples of swine sera characterized by microscopic agglutination test (MAT), were evaluated by indirect ELISA. The sensitivity, specificity and accuracy values obtained from the analysis were 96.8%, 100%, and 99%, respectively, thereby suggesting that rErpY-like ELISA being a sensitive and specific method for antibodies detection in swine populations could be used as an alternative for diagnosis of swine leptospirosis.
Leptospirosis is an emerging zoonotic disease with worldwide distribution. The diagnostic utility of the recombinant outer membrane protein, LipL 32 was evaluated in Enzyme linked immunosorbent assay (rLipL 32 IgG ELISA) and Latex agglutination Test (rLAT) for serodiagnosis of human and bovine Leptospirosis. A total of 534 sera samples (cattle: 324; Human: 210) suspected for Leptospirosis were subjected to Microscopic agglutination test (MAT), rELISA and rLAT. In MAT 120 (22.5 %) serum samples had positive titres. The rLipL 32 IgG ELISA and rLAT demonstrated that 162 (30.3 %) and 134 (25 %) samples were positive. The sensitivity of IgG ELISA and rLAT relative to MAT was 100 %. The recombinant LipL 32 IgG ELISA demonstrated a specificity of 89.86 % while that of rLAT was 96.61 % relative to MAT. The recombinant LipL 32 ELISA and rLAT differentiated Leptospira positive samples from those which were positive for Brucellosis, Salmonellosis and Pasteurellosis etc. with as 100 % specificity. The recombinant LAT may be an effective screening test for rapid confirmation compared to other conventional tests, during outbreak surveillance. Further, these observations indicate that rLipL 32 IgG ELISA and rLAT could be suitable alternative for the sero diagnosis of bovine and human leptospirosis.
Seroprevalence of Bovine Leptospirosis using Indirect ELISA
Indian Journal of Animal Research
Background: Leptospirosis is a zoonotic bacterial disease causing abortion, stillbirths and loss of milk production in livestock resulting in huge economic losses. Leptospirosis in cattle has been under-reported and under-diagnosed in India due to non-specific symptoms, complex laboratory tests, fastidious nature of bacterium and lack of proper diagnostic facilities. Scanty data pertaining to epidemiological status of leptospirosis is available in land locked states of India including Madhya Pradesh, thus to fulfil the gap, the current work was conducted to investigate seroprevalence of leptospirosis in cattle. Methods: In the present study, cattle with a history of repeat breeding, abortion, haemogalactia and mastitis, were screened from various organized and unorganized farms of Jabalpur. A total of 300 blood samples were collected from suspected cattle and serum was separated. Enzyme linked Immune-sorbent assay was done as per the guidelines. Result: The seroprevalence of leptosp...
Brazilian Journal of Microbiology, 2001
The Counterimmunoelectrophoresis reaction (CIE) was tested as a genus specific test for swine leptospirosis diagnosis using three soluble extracts obtained from Leptospira sp, serovars pomona, icterohaemorrhagiae and patoc. The extracts were treated with hot Triton X-100 and applied to serum samples of animals divided in three groups: Group 1, 10 swines experimentally infected with the Pomona strain; Group 2, 50 naturally infected swines and Group 3, 10 swines control. These animals were serologically evaluated by CIE and by the Microscopic Agglutination Test (MAT), the WHO reference technique. Groups 1 and 3 were monitored during a period of 93 days after infection (a.i.). Group 1 serum convertion took place around the 10 th day a.i. by MAT but ocurred earlier by CIE using any antigen. When CIE was carried out with the homologous antigen to the experimental infection, the results were consistent with MAT, but not when the heterologous antigens were used. Groups 1 and 3 showed distinct results: in Group 3, diferences between results of CIE accomplished with any three antigen extracts were not significant, indicating lack of dependence on the serovar responsible for the outbreak. Although being safe, fast, easy to perform, inexpensive and ideal for analysis of large number of samples, CIE revealed limited genus specificity, which is not convenient for field screening tests. The advantage of CIE is the capability to detect antibodies earlier than MAT.
Immunological and Molecular Diagnostic Techniques for Leptospirosis: An Update
A number of laboratory techniques are employed to establish the diagnosis of microbial diseases that cause significant morbidity as well as mortality in humans and animals throughout the world. Leptospirosis is an emerging and re-emerging enigmatic zoonotic disease of public health and economic importance. Currently, over 600,000 human deaths are attributed due to leptosprirosis annually in the world. However, there is a lack of information on Leptospira strains in remote parts of the world. The diagnosis of leptospirosis is challenging due to non-specific clinical feature. Thus, laboratory tests are necessary to confirm the diagnosis of disease due to its varied symptomatology. The microscopic agglutination test (MAT), which determines agglutinating antibodies in sera for various serovars of Leptospira species is considered the gold standard for the diagnosis of leptospirosis. Enzyme linked immunosorbent assay (ELISA) usually detects only the antibodies reacting with a broadly reactive genus specific antigen and thus gives no indication of the causative serovar or serogroup, which limits its application. Polymerase chain reaction on the other hand is considered sensitive and specific for the rapid detection of Leptospira in clinical samples. It is imperative to employ immunological and molecular techniques in order to make an unequivocal diagnosis of leptospirosis to institute immediate therapy to mitigate the suffering of the patients.
scienceline, 2021
Leptospira spp. is a pathogenic bacteria that causes leptospirosis in humans and cattle. The World Health Organization (WHO) recommends the microscopic agglutination test (MAT) as the laboratory gold standard in the detection of leptospirosis. However, the limitation of MAT triggers the laboratory technicians to develop alternative laboratory tests against leptospirosis. The current study aimed to compare the sensitivity and specificity of histopathology special stain using modified Gram staining (MGS) and molecular test using reverse transcriptasepolymerase chain reaction (RT-PCR), compared to the MAT for Leptospira spp. detection in cattle. This study used a total of 38 serum and 38 kidney samples from the cattle slaughtered in the Sidoarjo slaughterhouse, Indonesia. The collected serum samples were tested against MAT and RT-PCR. The kidneys were processed for histopathology using MGS. The result indicated that 16 (42.10%) of the tested samples were positive against MAT, 6 (15.78%) were positive against MGS, and 18 (47.36%) were positive against RT-PCR. The RT-PCR indicated better sensitivity and lower specificity, compared to MAT and MGS. The findings revealed that the RT-PCR is an appropriate laboratory test for detecting cattle leptospirosis with better sensitivity and specificity. Therefore, this method can be suggested to substitute MAT and overcome its limitations.
International Journal of Current Microbiology and Applied Sciences, 2017
The present study was undertaken to identify the presence of anti-leptospiral antibodies in the bovine serum samples using two serological tests, a genus specific indirect ELISA and a serovar specific microagglutination test (MAT). A total of 250 serum samples from bovine leptospirosis suspected cases were sampled in and around Pondicherry (Southern India). The anit-leptospiral antibodies were detected by indirect ELISA using outer membrane proteins (OMP's) of Leptospira interrogans serovar pomona as antigen and compared with MAT using six infecting serovars (hardjo, icterohaemorrhagiae, grippotyphosa, pomona, canicola and australis) as antigen. Statistical analysis of the data was performed using chi-square test. Out of 250 sera samples, 91 (36.4 %) and 62 (24.8%) were positive by indirect ELISA and MAT respectively. The indirect ELISA employed in this study revealed a very high sensitivity of 98.38 % in comparison to 67.03 % in MAT. Chi-square analysis revealed a significant difference (i.e. P<0.001) between the two serological tests. This study revealed that indirect ELISA was found to be highly sensitive, rapid and easy to perform for detection of bovine anti-leptospiral antibodies in comparison with MAT.
Tropical animal health and production, 2015
This study aimed to develop latex agglutination test (LAT) using recombinant leptospiral immunoglobulin-like protein (LigB) (rLigB) antigen and compare its diagnostic efficacy with LAT using conventional heat-killed leptospiral antigen and microscopic agglutination test (MAT) in diagnosing bovine leptospirosis. The PCR-amplified 1053-bp ligB gene sequences from Leptospira borgpetersenii Hardjo serovar were cloned in pET 32 (a) vector at EcoRI and NotI sites and expressed in BL21 E. coli cells as fusion protein with thioredoxin (-57 kDa) and characterized by SDS-PAGE and immunoblot. Out of 390 serum samples [cattle (n = 214), buffaloes (n = 176)] subjected to MAT, 115 samples showed reciprocal titre≥100 up to 1600 against one or more serovars. For recombinant LigB protein/antigen-based LAT, agglutination was observed in the positive sample, while no agglutination was observed in the negative sample. Similarly, heat-killed leptospiral antigen was prepared from and used in LAT for comp...
The role of small ruminants in the epidemiology of leptospirosis
Scientific Reports, 2022
Leptospirosis is a common global zoonotic disease of man and all farm animals. Although most leptospiral infections in sheep and goats are asymptomatic, they may play a role in the epidemiology of the disease by the spread of Leptospira through the urine. This study was carried out to evaluate the role of sheep and goats in the epidemiology of leptospirosis. Blood and urine samples were taken from 210 goats and 246 sheep. To detect antibodies, sera samples were tested with 8 live serovars of L. interrogans (Hardjo, Pomona, Grippotyphosa, Canicola, Ballum, Icterhemorrhagiae, Tarasovi, and Australis) by MAT. Then, urine samples were tested by Nested PCR targeting 16S rRNA gene for detection of pathogenic Leptospira. Results of MAT showed that 10.95% of goats and 8.53% of sheep had antibodies against at least one examined serovars. In both species, the highest reacting was L. i. Pomona with a rate of 68.18% and 56% in sheep and goats, respectively. Moreover, in PCR, 2 (0.95%) urine samples of goat and 12 (4.87%) urine samples of sheep were positive. All of the MAT positive studied animals were PCR negative and, statistical analysis showed that there was no relationship and agreement between the results of PCR and MAT in sheep (kappa = − 0.07, p > 0.05) and goats (kappa = − 0.02, p > 0.05). Finally, it is concluded that sheep and goats can excrete L. interrogans in the urine and thus transmit them to other animals and humans. Leptospires, spirochetes belonging to the family of leptospiraceae, are the causative agents of leptospirosis which is a common global zoonotic disease of both man and all farm animal species especially in subtropical and tropical regions of the world. Currently, over 250 pathogenic serovars and 24 pathogenic serogroups are recognized 1. Leptospirosis is an emerging infectious disease of humans with a marked increase in the number of cases and frequent outbreaks in SouthEast Asia and Latin America. Humans are most commonly infected through occupational, recreational, or domestic contact with the urine of carrier animals, either directly or via contaminated water or soil 2-4. Although this disease is rare in sheep and goats that good descriptions of the naturally occurring disease in them are lacking 1 , but the clinical signs related to leptospiral infection in these animals are as follows: high fever, abortion, stillbirth, agalactiae, and prenatal death. Lambs and kids especially those in poor condition, are most susceptible and consequence leptospiral infection may manifest fever, haemoglobinuria and jaundice which may result in death 1,5,6. Because of the leptospiral fragile nature, the cost and complexity of the isolation method, and the long incubation period, most diagnostic laboratories do not attempt to isolate leptospires. Therefore, diagnosis of leptospiral infection has been based generally on serological methods. Among serological methods developed for the recognition of leptospirosis, the Microscopic Agglutination Test (MAT) is the most commonly used one that provides more information about the serovars in each area 1,7,8. Unfortunately, it is less useful in the diagnosis of the early stage and chronic disease. In addition, the main challenge of the MAT is the maintenance of live Leptospira cultures, which are difficult to grow in a laboratory 8 , and the major concern is the failure of the MAT to differentiate antibody response to natural infection and those after vaccination 1. The available serological techniques for the diagnosis of leptospirosis have low sensitivity during the early stage of the disease. Therefore, early diagnosis of leptospirosis is important because the severe leptospiral infection can have a fulminant course. Efforts for solving this problem resulted in developing simpler, effective, efficient, and inexpensive diagnostic methods, e.g. polymerase chain reaction (PCR) which has also been used to detect leptospirosis in farm animals. In fact, PCR is faster and more sensitive than conventional methods. PCR assay has been used on various clinical specimens such as urine, blood, semen, and aborted fetus in order to detect leptospiral DNA. Also, PCR may be used for differentiation of pathogenic and saprophytic leptospires 9-17 .