Serodiagnosis of wildlife leptospirosis employing recombinant leptospiral immunoglobulin like b protein antigen

In the present study a comparative evaluation microscopic agglutination test with rLigB protein based latex agglutination test was carried out, which is a cross reacting lipoprotein able to detect acute infection caused by any pathogenic leptospiralserovars. It was employed for serodiagnosis of leptospirosis. The 46 KDa 6X His tagged LigB protein, obtained by IPTG induction of recombinant E. coli M15 cells containing the N-terminal region of LigB gee in PQE30 expression vector, was purified by Ni-NTA affinity chromatography and adsorbed on latex bead surface for performing latex agglutination test against Leptospirosis suspected wildlife field sera. A total of 80 wildlife sera samples were collec ted, including 27 wild feline sera samples (18 tigers, 8 lions, and 1 jaguar) obtained from Chhatbir zoo, Chandigarh, 42 sera samples (8 tigers, 4 lions and 6 leopards, 2 cheethals, 1 black buck, 12 buffaloes and 9 zoo staff) were received from Jodhpur zoo Rajasthan, 8 sera samples (4 tigers, 3 leopards, 1 lion) from Van Vihar National park, Bohpal, Madhya Pradesh and 3 sera samples (2 lions and 1 tiger) received from Biwani Mini zoo, Haryana, India. The result showed that sera were tested positive by rLigB based LAT, which were reconfirmed using microscopic agglutination test (MAT). The results from LAT were in concordance with MAT. In conclusion, rLigB based LAT is a rapid, pen site, reliable diagnostic tool of high sensitivity and specificity, under laboratory and field conditions, for the detection of Leptospirosis.
 Asian J. Med. Biol. Res. September 2020, 6(3): 440-448

Humans get leptospirosis by contact with fresh water, damp soil, or vegetation contaminated by the urine of infected animals, swallowing contaminated food or water or while working in contaminated flood plains or at wet agricultural settings. The bacteria enter the body through abrasions in the skin and mucous membranes. In the present study recombinant LigB protein is employed in latex agglutination test, which is a cross reacting lipoprotein able to detect acute infection caused by any pathogenic leptospiral serovars. It was employed for serodiagnosis of leptospirosis. The 46 KDa 6X His tagged LigB protein, obtained by IPTG induction of recombinant E. coli M15 cells containing the N-terminal region of LigB gee in PQE30 expression vector, was purified by Ni-NTA affinity chromatography and adsorbed on latex bead surface for performing latex agglutination test against leptospirosis suspected human sera. A total of 28 human sera samples were received from Post Graduate Institute of Medical Education & Research (PGIMER), Chandigarh India, which tested positive by IgM ELISA test kit were subjected to both rLigB based LAT and MAT. All the 28 sera showed seropositivity by both the tests. Icterohaemorrahigae was the predominant serovar followed by Javanica and Grippotyphosa. Six out seven sera samples received from Indian Veterinary research Institute, Human Hospital and City Hospital, Bareilly were tested positive both by rLAT and MAT. The result showed that sera were tested positive by rLigB based LAT, which were reconfirmed using microscopic agglutination test (MAT). The results from LAT were in concordance with MAT. In conclusion, rLigB based LAT is a rapid, reliable diagnostic tool at resource poor and remote diagnostic centers with high sensitivity and specificity, under laboratory and field conditions, for the detection of leptospirosis.
 Asian J. Med. Biol. Res. June 2020, 6(2): 229-236

Leptospirosis is one of the worldwide zoonoses. Rodent is the most important reservoir of leptospirosis, and their serovars are related to the ones of human patients. Hence the first part of this study is to investigate the leptospiral sero-prevalence in wild rodents captured during June 2005 and May 2006. Three hundred and twenty-nine rodents were captured from harbors, traditional markets, university campus and the fields in Taiwan. The total prevalence detected by MAT (microscopic agglutination test) was 12.93 % ( 41/317，95 % CI = 11.05 % - 14.82 % ). The Fu-ao habor in Matsu had the highest seropositive rate ( 22.22 % ). The rodent species which found with the highest seropositive rate was Rattus flavipetcus ( 18.18 % ). The main serogroup discovered was Pomona ( 64.29 %，36/56 ), and the second one was Shermani ( 12.50 %，7/56). The highest geometric mean titres were 1: 229.7 of Autumnalis and 1: 181.6 of Pomona. Besides, to avoid the infectious risks of MAT caused by the operation of live pathogens, and to minimize the operation time, labor and material cost, this study evaluated the recombinant LipL32 protein-based enzyme-linked immunosorbent assay ( rLipL32-ELISA ), and heat stable antigen- sensitized ( from non-pathogenic strain, Leptospira biflexa ) latex agglutination test ( LAT ), for the development of simpler and safer serological screening methods with MAT as the gold standard. The sensitivity, specificity and agreement of rLipL32-ELISA were 95.00 %, 91.34 % and 91.80 %, and 33.33 %, 87.80 % and 82.65 % of LAT, respectively. The kappa value compared with MAT were 0.699 ( 95 % CI = 0.59-0.81 ) of rLip32-ELISA and 0.174 ( 95% CI = -0.27-0.62 ) of LAT. This study revealed a higher seroprevalence of leptospirosis in rodents, especially in habor areas. This should be noticed for the workers in such environments. In the meanwhile, the most prevalent serogroup investigated by this study was Pomona, which is different from the previous surveys during the last decade that mainly serogroup was Shermani. In addition, the rLipL32-ELISA has the potential to be a screening tool for leptospirosis, because of its high sensitivity, specificity, and agreement ( up to 91.80 % ).

A One Health approach to investigating the health and prevalence of zoonotic pathogens in snow leopards, sympatric wildlife, domestic animals and humans in the South Gobi Desert in Mongolia

Evaluation of recombinant LigB antigen-based indirect ELISA and latex agglutination test for the serodiagnosis of bovine leptospirosis in India

Early laboratory confirmation is important for the accurate diagnosis and treatment of patient infected by leptospirosis. However, Microscopic agglutination test (MAT) as the gold standard for detection of human leptospirosis has many limitation and only available in reference laboratories. Therefore, many studies suggested LipL32 protein as a good candidate for development of leptospirosis detection kit because it is highly conserved and produced only in pathogenic Leptospira species. In this study, we aim to investigate the performance of our in-house ELISA using recombinant LipL32 to detect leptospirosis in Indonesia. Fourteen human sera were used in this study and the infection status were determine using MAT. The result showed that nine of eleven MAT positive sera were successfully recognized by LipL32 ELISA. The antibody binding to LipL32 was also confirm by immunoblot. There was one of three MAT negative sera has high OD above 0.5 in ELISA, but it showed negative reaction in immunoblot result. Overall, this study demonstrated that recombinant LipL32 protein can recognized antibody from human leptospirosis and can be used as a universal antigen to detect infection by any serovars of pathogenic leptospira.

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 comparison with MAT. A two-sided contingency table was used for analysis of LAT using both the antigens separately against MAT for 390 serum samples. The sensitivity, specificity and positive and negative predictive values of recombinant LigB LAT were found to be 75.65, 91.27, 78.38 and 89.96 %, respectively, and that of heat-killed antigen-based LAT were 72.17, 89.82, 74.77 and 88.53 %, respectively, in comparison with MAT. This developed test will be an alternative/complementary to the existing battery of diagnostic assays/tests for specific detection of pathogenic Leptospira infection in bovine population.

Leptospirosis is a spirochaetal infection that possesses a broad host range affecting almost all mammals. In the present study, the microscopic agglutination test (MAT) was compared with recombinant LigA/B antigen-based point-of-care diagnostics such as the in-house IgM dot ELISA dipstick test (IgM- DEDT) and the latex agglutination test (LAT) for the serodiagnosis of human leptospirosis. The comparison of the MAT with these two point–of-care diagnostics was performed using the MAT as the gold standard test and using Bayesian latent class modelling (BLCM), which considers all diagnostic tests as imperfect. The N-terminal conserved region of the LigA/B protein spanning the first to fifth big tandem repeat domains (rLigA/BCon1-5) was employed as a serodiagnostic marker in both of the bedside assays. A total of 340 serum samples collected from humans involved in high risk occupations were screened using the MAT, IgM DEDT and LAT. During the early phase of leptospirosis, BLCM analysis showed that the IgM DEDT and LAT had similar sensitivities (99.6 (96.0–100)) and (99.5 (95.2–100)), respectively, while the single acute phase MAT had the lowest sensitivity (83.3 (72.8–91.3)). Both the IgM DEDT and the LAT may be superior to the single acute phase MAT in terms of sensitivity during the early phase of infection and may be suitable for the early diagnosis of leptospirosis. However, BLCM analysis revealed that the use of both acute and convalescent samples substantially increased the sensitivity of the final MAT (98.2% (93.0–99.8%)) as a test to diagnose human leptospirosis. Both the IgM DEDT and LAT can be employed as bedside spot tests in remote locations where the MAT is not easily accessible.

Comparison of an in-house latex agglutination test with IgM ELISA and MAT in the diagnosis of leptospirosis

BACKGROUND: The prevalence and incidence of end-stage renal disease are high in Taiwan. Acute leptospirosis infection often causes renal tubulointerstitial disease, but the association of occult leptospirosis and renal disease is unclear. The aim of this study is to define the possible association between occult leptospirosis infection and chronic kidney disease (CKD). METHODS: In this prospective observational cohort study, 371 patients with CKD and 100 residents without CKD in the same community were included. Microscopic agglutination test (MAT) was employed to detect occult leptospirosis. Both renal progression and microalbuminuria were also monitored during a follow-up period of 42 months. RESULTS: Low prevalence of positive MAT is found in normal and CKD subjects. There was no significant difference in amount of microalbuminuria between MAT positive and negative subjects (527.52 ± 125.18 vs. 728.2 ± 216.23, mg/day, P ＞ 0.05). Logistic analysis for CKD progression did not show different CKD progression between MAT positive and negative groups. CONCLUSION: The prevalent rates of positive MAT in normal and CKD subjects were similar. There was also no difference in microalbuminuria and CKD progression between MAT positive and negative subjects. Our findings suggested that occult leptospirosis might not be associated with development and progression of CKD in North Taiwan.

A multiplex polymerase chain reaction (PCR) method using primer sets of G1/G2 and B64-I/B64-II was validated to detect pathogenic leptospira serovars from canine urine samples. The PCR method was found to be specific and sensitive with a detection limit of 100 cells of Leptospira icterohaemorrhagiae per milliliter of urine. The primer set previously designated and erroneously transcribed B64-I/B64-II amplified a DNA fragment of 352 base pairs from Leptospira grippotyphosa and Leptospira sejroe but not from Leptospira autumnalis, Leptospira bratislava, Leptospira canicola, Leptospira hardjo, Leptospira icterohaemorrhagiae, and Leptospira pomona. From 100 diagnostic canine urine samples, 5 were found positive for Leptospira grippotyphosalsejroe with a PCR product of 352 base pairs and 6 were positive for other pathogenic leptospira serovars with a PCR product of 285 base pairs. One 285-base pair product was sequenced and found to be 99.3% homologous to the G1/G2 PCR fragment sequence reported previously. All 352-base pair PCR products of clinical samples and pure cultures of L. grippotyphosa and L. sejroe were sequenced. The 352-base pair fragment sequences of L. grippotyphosa and L sejroe were identical. Only 2 base pairs were found different between the sequences from pure cultures and those from clinical samples. Serum samples from 3 positive cases that generated a PCR product of 352 base pairs were tested by the microscopic agglutination test, and 2 were found to be positive for L. grippotyphosa (1:10,240 and 1:5,120), 1 was positive for L. grippotyphosa (1:320) or L. icterohaemorrhagiae (1:320). The results of this study suggest that the multiplex PCR with the primer set G1/G2 and the erroneously transcribed B64-I/B64-II may be able to differentiate L. grippotyphosa or L. sejroe from other pathogenic leptospira serovars commonly tested for in Canadian diagnostic laboratories.

Leptospirosis is a zoonosis of global distribution caused by infection with pathogenic spirochaetes of the genus Leptospira. Humans are accidental hosts and usually become infected through contact with water or soil contaminated by the urine of infected animals such as rodents, dogs, cattle and pigs. In developing countries such as India, leptospirosis is often underdiagnosed because of its protean clinical manifestations leading to significant morbidity and mortality. It occurs as a self-limited illness in 85% to 90% of the cases and icteric leptospirosis or Weil's syndrome, a more serious, potentially fatal syndrome which occurs in 5% to 10% of the cases. Microbiological diagnosis of leptospirosis aims at demonstrating the leptospires, by culturing them or by demonstrating an appreciable antibody response to them. A definite diagnosis of leptospirosis is based either on isolation of the organism from the patient or on seroconversion or a rise in antibody titre in the MAT. Leptospires may be visualized in clinical material by DGM or by IF or light microscopy after appropriate staining. The sensitivity of blood cultures is low; hence culture is primarily used for retrospective diagnosis. There are numerous serological tests available for diagnosis of leptospirosis like Macroscopic agglutination test (MSAT), Indirect fluorescent antibody test (IFAT), Sensitised erythrocyte lysis test (SEL), Complement fixation test (CFT), Enzyme Linked Sorbent Assay (ELISA), Microcapsule agglutination test (MCAT), Lepto-Dipstick, Latex agglutination test, Dried Latex agglutination test (Lepto Tek Dri-Dot), but they are only genus specific. To identify the specific serovar Microscopic agglutination test (MAT) or culture has to be done. The various available options for a diagnosis of leptospirosis have been explored in this article. A thorough literary search was done in the various published data available- pub med search was done as well as

Canine leptospirosis is a serious public health concern. This study aims to investigate the feasibility of conserved first to fifth domains of recombinant Leptospira immunoglobulin like protein B antigen (rLigBCon1-5) as a serodiagnostic marker for detecting canine leptospirosis. A total of 340 unvaccinated canine serum samples were screened using microscopic agglutination test (MAT) and rLigBCon1-5 based immunoglobulin G (IgG) indirect-enzyme-linked immunosorbent assay (I-ELISA). Further, 60 vaccinated canine sera were screened using MAT and rLigBCon1-5 based latex agglutination test (LAT). Microscopic agglutination test results revealed seropositivity of 28.6%. The relative sensitivity, specificity, and accuracy of IgG I-ELISA in comparison to MAT were 100%, 96.0%, and 97.2%, respectively. Out of 60 vaccinated sera, 46 sera reacted with MAT alone, and eight sera reacted by both tests, while six sera were non-reactive with both tests. Anti-LigB antibodies were detected in eight canine sera by rLigBCon1-5 based LAT. In five LAT reactive sera, agglutinins of locally circulating Leptospira serovars Grippotyphosa (n=4) and Australis (n=1) were detected. In three LAT reactive sera, agglutinins against Icterohaemorrhagiae (n=3) produced due to natural infection were present. Immunoglobulin G based indirect ELISA assay (IgG I-ELISA) can be employed as an alternative test instead of MAT. rLigBCon1-5 based LAT detected anti-LigB antibodies in eight vaccinated sera where the vaccine failure occurred partially or totally due to the limited efficacy spectrum of Nobivac® RL and cold chain breakage. This vaccine could not provide cross-protection against locally circulating Leptospira serovars. The recombinant LigBCon1-5 antigen based LAT possesses capability of differentiating infected from vaccinated individuals (DIVA capability) when employed as a pen-side test for detecting canine leptospirosis.

Farmers in Bali live in close contact to pigs, a setting which favours transduction of leptospirosis to humans. Since little is known about the prevalence of different Leptosipro (L.) serovars in Bali, a serosurvey was initiated to identify Leptospira serovars predominating in domestic pigs. In 1999, a total of 484 sera from pigs on 138 farms in two geographically distinct provinces of Bali (Gianyar and Tabanan), Indonesia, were collected. Agglutinins against 7 serovars of pathogenic Leptospira were determined using the microscopic agglutination test (MAT). The overall percentage of sera giving titres of >= 100 against at least one serovar was 27.1%. Prevalences varied between the provinces Gianyar (30.1%) and Tabanan (22.4%) but didn't differ significantly. Prevalence of Seroreactivity for individual serovars was 1.0% (L. grippotyphoso), 2.1% (L. torossovi), 1.2% (L. copenhageni), 16.3% (L. pomona), 5.4% (L. hardjo), and 9.3% (L. brotislava). Agglutinins against L. conicola could not be detected. High titres (>= 400) were almost exclusively directed against L. pomona, implying that L. pomona is the predominant Leptospira serovar in Balinese pigs

The efficacy of a recombinant leptospiral outer membrane protein LipL41 as an antigen for conducting IgG-Enzyme linked immunosorbent assay (ELISA) and latex agglutination test (LAT) for serodiagnosis of bovine leptospirosis was evaluated. The recombinant LipL41 antigen developed and used for detecting the antibodies was specific in detection of the pathogenic serovars of Leptospira, as the expression of the LipL41 antigen is restricted only to pathogenic leptospires. A total of 430 bovine serum samples were subjected to IgG-ELISA and LAT, and the sensitivity and specificity were assessed in comparison with microscopic agglutination test (MAT). The sensitivity and specificity of IgG-ELISA and LAT were 86.84% and 93.16%, and 95.42% and 98.33% respectively. Both the tests are found to be sensitive, specific and concurred with the standard MAT. The study concluded that the rLipL41 protein could be used as a potential diagnostic antigen in different assay formats for bovine leptospirosis.