Diagnostic evaluation of RT-PCR-ELISA for the detection of rabies virus.

Rabies is primarily a disease of terrestrial and airborne mammals. In most cases, rabies is diagnosed primarily on the basis of clinical symptoms and signs, and a corroborative history of or evidence of an animal bite, death of an animal and incomplete or no vaccination following exposure. The facility for laboratory diagnosis and confirmation of rabies is available in only a few institutions in India. Diagnostic tests using conventional assays like fluorescent antibody test (FAT) are unreliable at times, despite the clinical diagnosis. Currently, there are a number of molecular tests that can be used to complement conventional tests in rabies diagnosis. We have developed and evaluated an RT-PCR-ELISA using a panel of brain tissue samples from rabies suspected animals of various species. This assay was able to detect rabies virus genome in all the 43 samples that were previously tested positive for rabies. Moreover this assay was shown to be 100 % sensitive and specific in detecting the rabies virus genome in post-mortem brain tissue samples from different species of animals. Our pilot study shows the potential of this assay as an alternative diagnostic test when the samples are unsuitable for use in FAT and also a supplementary test to FAT. In addition, the region of nucleoprotein gene amplified using this assay can be used for the molecular investigation of geographical origin of the field strains.

Recombinant OmpL1 protein as a diagnostic antigen for the detection of canine leptospirosis.

Microscopic agglutination test (MAT) is the standard method for the diagnosis of leptospirosis, which is laborious and the interpretation of the results is subjective. The present work describes the use of recombinant-based IgG ELISA for the serodiagnosis of leptospirosis. We used recombinant outer membrane protein OmpL1 as an antigen for conducting IgG enzyme-linked immunosorbent assay (ELISA). A total of 475 canine serum samples were subjected to IgG ELISA; 294 sera were positive to ELISA, while 283 were positive to MAT. All samples that were positive to MAT were positive to ELISA also, however, few samples which were negative to MAT were positive to ELISA, which suggested that recombinant-based IgG ELISA showed 100 % sensitivity when compared to MAT. Thus, this present study showed that recombinant OmpL1-based IgG ELISA appears to be a better alternative to MAT for the diagnosis of leptospirosis and rOmpL1 protein could be used as a potential diagnostic antigen in different assay formats for leptospirosis.

Evaluation of rt-PCR assay for routine laboratory diagnosis of rabies in post mortem brain samples from different species of animals.

Rabies in domestic and wild animals continues to be a major public health threat in India. Rapid and accurate diagnosis of rabies in animals is therefore of utmost importance as the individuals who were in contact with the rabid animals are at a greater risk. A significant amount of diagnostic tissue samples submitted to our laboratory are often autolysed and the WHO recommended direct fluorescent antibody test (FAT) for rabies diagnosis cannot be used in such samples. In this pilot study we have evaluated three different diagnostic primer sets for rapid sensitive and specific detection of rabies genome from the brain samples of different species of animals. We have validated a sensitive RT-PCR assay using brain tissue samples from different species of animals such as cat, cattle, dog, mouse and human, for routine diagnosis of rabies. Our results show the potential of this assay as a confirmatory test when the FAT results are unreliable and also as an alternative diagnostic test in circumstances when the diagnostic samples are unsuitable for use in FAT. Furthermore the nucleotide sequence of nucleoprotein gene amplified using this assay can also be used for the molecular epidemiological study of the rabies viruses in India.

Development of rapid flow-through-based dot-immunoassay for serodiagnosis of leptospirosis in dogs.

An IgG-ELISA used recombinant antigen and a rapid flow-through enzyme immunoassay were developed for rapid screening of leptospiral antibodies in dogs using recombinant LipL41, which is one of the conserved outer membrane proteins in pathogenic leptospires as the coating antigen. Results from this study were compared with the standard microscopic agglutination test and found that the sensitivity and specificity of the enzyme-linked immunosorbent assay were 75.46% and 93.29% and whereas that of flow-through-based dot-immunobinding assay were 87.73% and 89.63%, respectively. Relative merits of these tests were also assessed. The flow-through-based dot-immunobinding assay was thus proved to be a valid screening test for canine leptospirosis.

Rabies in South Indian Cows: An evidence of Sri Lankan Rabies virus Variant Infection Based on the Analysis of Partial Nucleoprotein Gene.

Rabies is a highly fatal non-suppurative encephalomyelitis, caused by the Rabies virus. Dogs are the major reservoir of rabies in India and are the source of infection to other domestic animals. In this report, laboratory investigation and molecular characterization of isolates from two cows with paralytic rabies is described. Necropsy brain samples from the two cows were tested for the presence of rabies antigen using a fluorescent antibody test and the results were confirmed using RT-PCR. Rabies virus was successfully isolated from both the brain samples in a murine neuroblastoma cell line. The phylogenetic analysis of partial nucleoprotein gene sequences of these isolates showed them to be of a variant of Rabies virus which is closely related to the Sri Lankan Rabies virus lineage as previously reported. In addition, partial nucleoprotein genes of 19 more Rabies virus isolates from southern India were sequenced and of these 11 isolates were found to be closely related to the Sri Lankan lineage. The deduced amino acid sequences of the partial nucleoprotein of the Indian isolates were 96-99% identical to the Sri Lankan isolates. This investigation re-confirms the previous speculations that the Sri Lankan variant of the virus may still be actively transmitted by animals in India.

Serodiagnosis of bovine leptospirosis by IgG-enzyme-linked immunosorbent assay and latex agglutination test.

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.

Diagnosis of leptospirosis by recombinant antigen based single serum dilution ELISA.

BACKGROUND & OBJECTIVES: Leptospirosis, a zoonosis with a worldwide distribution is an acute febrile illness caused by spirochaetes of the pathogenic Leptospira interrogans. Microscopic agglutination test (MAT), the reference method for diagnosis was successively done to evaluate the modified ELISA which was developed with the recombinant LipL32 antigen for the detection of anti-leptospiral antibodies in human serum samples. METHODS: The recombinant LipL32 antigen was developed from the serovar Pomona strain Pomona of the pathogenic L. interrogans species. The predicted titre at a single working dilution was plotted against the observed antiserum titre. Subsequently, predicted antibody activity titres were determined directly from the standard curve by solving the regression line equation. The relative sensitivity, specificity and accuracy of the single dilution ELISA for the detection of anti-leptospiral antibodies were determined in comparison to the MAT. RESULTS: A linear relationship was found between the predicted antibody titres at a single working dilution of 1:250 and the corresponding observed serum titres by the standard serial-dilution method. Regression analysis was used to determine a standard curve from which an equation was derived that allowed demonstration of the mentioned correlation. The equation was then used to convert the corrected absorbance readings of the single working dilution directly into the predicted ELISA antibody titres. A high level of sensitivity of 96 per cent and specificity of 91 per cent between ELISA and MAT titres was found. The kappa value was almost 1.0 indicating perfect agreement. INTERPRETATION & CONCLUSIONS: The r LipL32 ELISA was proved to be sensitive, specific and accurate as compared to the standard MAT and the test could be efficiently utilized as a screening test for a large number of human serum samples for the detection of leptospiral antibodies.

Rapid serodiagnosis of leptospirosis by latex agglutination test and flow-through assay.

PURPOSE: Diagnosis of leptospirosis facilitates patient management and initiation of therapy. The microscopic agglutination test (MAT) is the serological test used in reference laboratories because of its high degree of sensitivity and specificity. But the results are not available quickly for patient management. In the present study, in order to develop a simple, rapid immunodiagnostic assay, one of the outer membrane proteins (OMPs), recombinant LipL41 (rLipL41) has been utilised in latex agglutination test (LAT) and flow-through assay. METHODS: Part of LipL41 gene was expressed in Escherichia coli system and purified. The rLipL41 antigen of pathogenic Leptospira interrogans serovar Icterohaemorrhagiae, which is conserved in all pathogenic Leptospira spp. was used as capture antigen in the LAT and flow-through test. Both tests are very rapid and could be completed within 5 minutes. The sensitivity and specificity of rLipL41 was assessed and evaluated in LAT and flow-through assay in comparison with standard MAT. RESULTS: The sensitivity and specificity of the LAT were 89.70 and 90.45% and flow-through assay were 89.09 and 77.70%, respectively. CONCLUSIONS: The developed LAT and flow-through assays were simple, rapid and economical for the detection of leptospira infection and suitable for large-scale screening of samples in endemic areas without any sophisticated equipment.

Development of recombinant nucleocapsid protein based IgM-ELISA for the early detection of distemper infection in dogs.

An IgM-ELISA based on a 16-kDa recombinant protein produced for the conserved and functional middle region of nucleocapsid protein of Canine distemper virus was developed. Out of 70 serum samples from distemper-suspected and vaccinated dogs analyzed, 34 serum samples (49%) were positive. The specificity of this ELISA was confirmed by blocking and adsorption experiments. The IgM-ELISA based on the recombinant nucleocapsid protein showed a strong correlation (r=0.857, p<0.0001 at 95% CI) and good agreement (kappa=0.714) with the conventional Vero cell culture distemper antigen based IgM-ELISA. The percent positivity was more in dogs with systemic signs (62%) by recombinant nucleocapsid protein IgM-ELISA. Out of 70 clinical serum samples, 69 samples were used along with 4 control sera used in the IgM-ELISA for the detection of viral RNA by Slot blot hybridization and 26 of them (36%) were positive. Fifty-one percent agreement was observed between the recombinant nucleocapsid protein IgM-ELISA and Slot blot hybridization. The analysis of clinical history of the dogs with systemic signs supported the application of IgM-ELISA over Slot blot hybridization in the early detection of distemper infection.

Recombinant antigen-based dipstick ELISA for the diagnosis of leptospirosis in dogs.

A recombinant LipL 32 antigen-based dipstick ELISA was developed as a screening test for the detection of leptospiral antibodies in serum samples from dogs. The antibodies were detected by a change in the colour of the substrate solution when the recombinant antigen-coated dipsticks were dipped into it. The relative sensitivity, specificity and accuracy of the test, compared with the standard microscopic agglutination test, were 95.9 per cent, 93.8 per cent and 94.8 per cent, respectively.

Evaluation of ELISA based on the conserved and functional middle region of nucleocapsid protein to detect distemper infection in dogs.

A 287bp fragment from the middle region of the nucleocapsid protein of canine distemper virus (CDV) was amplified from the conjunctival samples of distemper-infected dogs and was cloned into pRSET B vector. The recombinant protein was expressed as a 16-kDa-fusion protein with histidine tag in E. coli. Sera of distemper-infected and vaccinated dogs contained IgG antibodies against the purified recombinant protein as observed by enzyme linked immunosorbent assays (ELISA) and showed a strong correlation (r=0.882, p<0.0001 at 95% CI) and good agreement (kappa=0.718) with the conventional tissue culture viral antigen based ELISA. Further, the results of recombinant protein based ELISA and Western blotting with the sera from the infected and vaccinated dogs correlated well (kappa=0.8226). These findings recommend the use of the recombinant protein in the serodiagnosis of canine distemper virus infection in dogs.

Recombinant antigen-based latex agglutination test for rapid serodiagnosis of leptospirosis.

A rapid recombinant antigen-based latex agglutination test (LAT) has been developed to detect specific anti-leptospiral antibodies from human and dog sera. The recombinant LipL32 antigen developed and used for detecting the antibodies is specific in detection of the pathogenic serovars of Leptospira as the expression of the LipL32 antigen is restricted only to the pathogenic leptospires. The sensitized latex beads are stable and could be stored at 4 degrees C for more than three months without showing loss of activity for both weakly and strongly positive samples. The test is found to be sensitive, specific and accurate as compared to the standard microscopic agglutination test (MAT). Moreover, the recombinant antigen-coated latex beads could detect the specific anti-leptospiral antibodies in the acute phase of the illness. The test is simple and inexpensive, and is rapid in the management of large numbers of patients.

Biological and molecular characterization of Indian isolates of Newcastle disease virus from pigeons.

Five Newcastle disease virus (NDV) isolates from pigeons were characterized by biological and molecular methods. Four of the five isolates were found to be velogenic with high intracerebral pathogenicity indices (ICPI). The fusion protein cleavage site (FPCS) sequences of these isolates had multiple basic amino acids RRQKRF at positions 112-116 and a phenyl alanine at position 117 characteristic of velogenic isolates. Three of these velogenic isolates were phylogenetically related to mesogenic vaccine virus strain and the fourth one to a few exotic velogenic isolates. The lentogenic isolate obtained in this study was identical with the LaSota strain.

Recombinant LipL32 antigen-based single serum dilution ELISA for detection of canine leptospirosis.

A recombinant antigen-based single serum dilution enzyme-linked immunosorbent assay (ELISA) was developed to measure the specific antibody activity in sera of dogs with leptospirosis. The recombinant antigen developed and used in the assay was specific for the pathogenic serovars of Leptospira. A linear relationship was found to exist between the predicted antibody titres at a single working dilution of 1:1000 and the corresponding observed serum titres as determined by the standard serial-dilution method. Regression analysis was used to determine a standard curve from which an equation can be derived that allows demonstration of the mentioned correlation. The equation was then used to convert the corrected absorbance readings of the single working dilution directly into the predicted ELISA antibody titres. The assay was proved to be sensitive, specific and accurate as compared to the standard microscopic agglutination test (MAT).

Sequence analysis of infectious bursal disease virus isolates from India: phylogenetic relationships.

Prevalence of infectious bursal disease (IBD) among chickens in different parts of Tamil Nadu, India, has been studied by collection of bursal samples from suspected flocks and by performing reverse transcription-polymerase chain reaction (RT-PCR) for amplification of a specific product of 474 bp from the variable region of the VP2 gene. Among 53 bursal samples examined by RT-PCR, 40 showed a positive reaction. The amplified products were subjected to nucleotide sequencing and the obtained sequences were compared with those of IBD virus (IBDV) vaccine strain Georgia, the classical virulent strain 52/70 and the very virulent Japanese OKYM strain. Nucleotide homology data indicated that all the Tamil Nadu isolates showed homology ranging from 91 to 99.6% among themselves. When compared with the very virulent Japanese OKYM strain, four isolates grouped with that strain. Majority of the isolates clustered with the very the virulent OKYM strain as evident from phylogenetic analysis performed using the MEGA program. Comparison of the deduced amino acid sequences of IBDV isolates with those of the vaccine strain Georgia, the classical virulent strain 52/70 and the very virulent strain OKYM also revealed the presence of conserved serine-rich heptapeptide sequence in most of the isolates. Results of this study indicate that majority of the IBDV isolates are very virulent, which is evident from heavy mortality that has been reported in few flocks of poultry in spite of regular vaccination.

Arbitrarily primed PCR- a rapid and simple method for typing of leptospiral serovars.

PURPOSE: To investigate the use of arbitrarily primed polymerase chain reaction (AP-PCR) for typing of leptospiral serovars. METHODS: AP-PCR was adopted for identification of laboratory strains of leptospires and leptospiral cultures at serovar level. A primer of 12 bp was used for amplifying DNA of 13 laboratory strains of leptospires as well as culture pellets of leptospires. RESULTS: Each serovar produced distinct DNA fingerprint which was characteristic for each serovar. These patterns were used for typing of 81 serum culture samples obtained from human leptospiral cases. Of these samples, 39 could be typed based on AP-PCR fingerprints belonging to serovars autumnalis, pomona, canicola, javanica, icterohaemorrhagiae, patoc and pyrogenes. These results were confirmed by RAPD fingerprinting of the DNA samples of the respective leptospiral serovars after culturing -*them in EMJH media. One of the important findings of this work was that straight culture sample could be used for AP-PCR assay, without purification of DNA. By having more number of AP-PCR reference fingerprints, more serovars could be typed. CONCLUSIONS: AP-PCR technique provides great potential for simple and rapid identification of leptospires at serovar level, which could be useful in molecular epidemiological studies of leptospirosis.

Identification of leptospiral isolates by bacterial restriction endonuclease analysis (Brenda).

DNA samples from 19 reference serovars belonging to 19 different serogroups of Leptospira interrogans and two serovars belonging to Leptospira biflexa were examined by bacterial restriction endonuclease analysis using EcoR I and Hae III enzymes. All the serovars gave unique restriction patterns that differed from each other. DNA from 10 local isolates digested with these enzymes produced patterns which on comparison with the standard patterns produced by reference strains could be identified to serovar level.

Fatty acid-mediated activation of vascular endothelial cells.

Vascular endothelial cell activation and dysfunction are critical early events in atherosclerosis. Selected dietary lipids (eg, fatty acids) may be atherogenic by activating endothelial cells and by potentiating an inflammatory response. Due to their prooxidant property, unsaturated fatty acids may play a critical role in endothelial cell activation and injury. To test this hypothesis, porcine endothelial cells were exposed to 18-carbon fatty acids differing in the degree of unsaturation, ie, 90 micromol/L stearic (18:0), oleic (18:1n-9), linoleic (18:2n-6), or linolenic acid (18:3n-3) for 6 to 24 hours and/or tumor necrosis factor alpha ([TNF-alpha] 500 U/L) for up to 3 hours. Compared with control cultures, treatment with 18:0 and 18:2 decreased glutathione levels, suggesting an increase in cellular oxidative stress. Both 18:2 and 18:0 activated the transcription factor nuclear factor kappaB (NF-kappaB) the most and 18:1 the least. This NF-kappaB-dependent transcription was confirmed in endothelial cells by luciferase reporter gene assay. The fatty acid-mediated activation of NF-kappaB was blocked by preenrichment of the cultures with 25 micromol/L vitamin E. All fatty acids except 18:1 and 18:3 increased transendothelial albumin transfer, and 18:2 caused the most marked disruption of endothelial integrity. Preenrichment of endothelial cells with 18:2 followed by exposure to TNF-alpha resulted in a 100% increase in interleukin-6 (IL-6) production compared with TNF-alpha exposure alone. In contrast, cellular preenrichment with 18:0, 18:1, or 18:3 had no effect on TNF-alpha-mediated production of IL-6. Cellular release of radiolabeled arachidonic acid (20:4) was markedly increased only by cell exposure to 18:2 and 18:3, and the release of 20:4 appeared to be mainly from the phosphatidylethanolamine fraction. These data suggest that oleic acid does not activate endothelial cells. Furthermore, linoleic acid and other omega-6 fatty acids appear to be the most proinflammatory and possibly atherogenic fatty acids.

Zinc protects against apoptosis of endothelial cells induced by linoleic acid and tumor necrosis factor alpha.

BACKGROUND: Zinc requirements of the vascular endothelium may be increased in inflammatory conditions, ie, atherosclerosis, in which apoptotic cell death is prevalent. OBJECTIVE: We hypothesized that zinc deficiency may potentiate disruption of endothelial cell integrity mediated by fatty acids and inflammatory cytokines by enhancing pathways that lead to apoptosis and up-regulation of caspase genes. DESIGN: Endothelial cells were maintained in low-serum medium or grown in culture media containing selected chelators, ie, diethylenetriaminepentaacetate or N,N,N', N'-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN), with or without zinc supplementation. Subsequently, cells were treated with linoleic acid, tumor necrosis factor alpha (TNF-alpha), or both. We studied the effect of zinc deficiency and supplementation on the induction of apoptosis by measuring caspase-3 activity, cell binding of annexin V, and DNA fragmentation. RESULTS: Our results indicated that linoleic acid and TNF-alpha independently, but more markedly in concert, up-regulated caspase-3 activity and induced annexin V binding and DNA fragmentation. Zinc deficiency, especially when induced by TPEN, dramatically increased apoptotic cell death induced by cytokines and lipids compared with control cultures. Supplementation of low-serum- or chelator-treated endothelial cells with physiologic amounts of zinc caused a marked attenuation of apoptosis induced by linoleic acid and TNF-alpha. Morphologic changes of cells observed during zinc deficiency were prevented by zinc supplementation. Media supplementation with other divalent cations (eg, calcium and magnesium) did not mimic the protective role of zinc against apoptosis. CONCLUSIONS: Our data indicate that zinc is vital to vascular endothelial cell integrity, possibly by regulating signaling events to inhibit apoptotic cell death.