Immunosuppressive and anti-cancer potential of aqueous extract of Solanum Xanthocarpum

451-457In this study whole plant aqueous extract of Solanum Xanthocarpum (HAESX) was investigated to assess its effect on humoral immune response along with interleukin-2 (IL-2) production and its expression in Wistar albino rats splenocytes culture. Anticancer potential of HAESX was investigated using rat lever hepatoma (N1S1 cancerous cell line). The effect of HAESX over humoral immune response was studied using four groups of five animals each (Group-I as control, Group -II orally fed with 125 mg/kg body weight, Group -III orally fed with 250 mg/kg body weight and Group -IV orally fed with 500 mg/kg body weight of HAESX). Quantification of IL-2 was done by sandwich ELISA and its expression was detected by the real time PCR. SRB assay (Sulforhodamine B) was done for detecting the effect of HAESX on N1S1 cell line. Dose dependent decrease in antibody titer was observed and production of IL-2 was also decreased significantly. Suppression of IL-2 production at 250 µg/mL and 500 µg/mL dose was also confirmed by the Real time PCR. Relative fold change in the expression of IL-2 gene was 592.22 and 10.77 at 250, 500 μg/mL HAESX concentrations respectively with respect to control. Dose dependent suppression of percent growth of N1S1 cells with increasing concentrations (10, 20, 40 and 80 µg/mL) of HAESX was found. It was concluded that S. xanthocarpum have the immunosuppressive, and anti cancer activity that can be further explore in treatment of various inflammatory and autoimmune disease

Immunosuppressive and anti-cancer potential of aqueous extract of Solanum Xanthocarpum

In this study whole plant aqueous extract of Solanum Xanthocarpum (HAESX) was investigated to assess its effect on humoral immune response along with interleukin-2 (IL-2) production and its expression in Wistar albino rats splenocytes culture. Anticancer potential of HAESX was investigated using rat lever hepatoma (N1S1 cancerous cell line). The effect of HAESX over humoral immune response was studied using four groups of five animals each (Group-I as control, Group -II orally fed with 125 mg/kg body weight, Group -III orally fed with 250 mg/kg body weight and Group -IV orally fed with 500 mg/kg body weight of HAESX). Quantification of IL-2 was done by sandwich ELISA and its expression was detected by the real time PCR. SRB assay (Sulforhodamine B) was done for detecting the effect of HAESX on N1S1 cell line. Dose dependent decrease in antibody titer was observed and production of IL-2 was also decreased significantly. Suppression of IL-2 production at 250 µg/mL and 500 µg/mL dose was also confirmed by the Real time PCR. Relative fold change in the expression of IL-2 gene was 592.22 and 10.77 at 250, 500 μg/mL HAESX concentrations respectively with respect to control. Dose dependent suppression of percent growth of N1S1 cells with increasing concentrations (10, 20, 40 and 80 µg/mL) of HAESX was found. It was concluded that S. xanthocarpum have the immunosuppressive, and anti cancer activity that can be further explore in treatment of various inflammatory and autoimmune disease

Detection of anti-Mycobacterium avium subspecies paratuberculosis antibodies in thyroid and type-1 diabetes patients

49-52Mycobacterium avium subspecies paratuberculosis (MAP) causes granulomatous intestinal disease in animals (Johne’s diseases). MAP has also been associated with several autoimmune disorders. In this study, we screened serum samples from confirmed patients of thyroid and type 1 diabetes for the presence of antibody against MAP. We used newly developed 'cocktail ELISA' (based on recombinant secretary proteins) and extensively validated 'indigenous ELISA' (based on whole cell protoplasmic antigen) and both the tests were also compared for their diagnostic potential. A total of 90 serums samples were included of which anti-MAP antibodies was detected in 28.8% and 26.6% of samples by indigenous ELISA (iELISA) and cocktail ELISA (cELISA), respectively. There was almost perfect agreement between the two tests in detecting the anti-MAP antibodies. Study raises concern on high detection of anti-MAP antibodies in human, thus warranting necessary control measure to minimize MAP exposure in human beings

Cloning and expression of cultural filtrate proteins from novel and native strains of Mycobacterium avium subspecies paratuberculosis and their application in ELISA based sero-diagnosis of Johne's disease

Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP), is endemic in livestock leading to low per animal productivity. MAP as survives pasteurization, poses a public health problem because of high exposure to animals and humans. There is an urgent need for newer diagnostic tests with high specificity and sensitivity as the current ones suffer from lower sensitivity and specificity. In present study, six Mycobacterium avium subspecies paratuberculosis (MAP)-specific culture filtrate proteins (CFPs) were produced and evaluated for sero-diagnosis of MAP infection in goat and cattle herds in India. Genes encoding for six MAP-CFPs were amplified and cloned into easy cloning vector pJET1.2/pTZ57R followed by sub-cloning into expression vector pET28a (+)/pET22b (+) containing C-terminal Histidine. Recombinant CFPs (r-CFPs) expressions were optimized in Escherichia coli (Rosetta cells) and purified using Ni-NTA affinity chromatography. In SDS-PAGE, MAP CFPs viz., 1693c, 2168c, ModD, 85C, Pep AN and Pep AC showed 22, 24, 55, 38, 20 and 25 kDa molecular masses, respectively. Identity of these r-CFPs was further confirmed by immuno-blotting. We developed six different ELISAs using the six individual r-CFPs and one additional ELISA i.e. cocktail ELISA (c-ELISA) was prepared using cocktail of all 6 r-CFPs. The performance of all seven ELISAs were further evaluated against whole cell protoplasmic based indigenous ELISA (i-ELISA). c-ELISA showed almost similar sensitivity as shown by i-ELISA. However, individual r-CFP based ELISA could not reach up to the sensitivity of cocktail of six r-CFPs. None of the r-CFP showed any false positive (as compare to i-ELISA) thereby specificity was 100%. Results of ELISA tests based on cocktail of r-CFPs, ModD and 85C were quite similar to i-ELISA from goat sera whereas in cattle serum c-ELISA was comparable with i-ELISA. Our study showed a comparable specificity of c-ELISA for the diagnosis of JD and it may have applicability in region where disease is endemic. Future validation of c-ELISA against gold standard or confirmatory tests would give a better insight on its diagnostic potential over i-ELISA

Cloning and expression of cultural filtrate proteins from novel and native strains of Mycobacterium avium subspecies paratuberculosis and their application in ELISA based sero-diagnosis of Johne's disease

219-229Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP), is endemic in livestock leading to low per animal productivity. MAP as survives pasteurization, poses a public health problem because of high exposure to animals and humans. There is an urgent need for newer diagnostic tests with high specificity and sensitivity as the current ones suffer from lower sensitivity and specificity. In present study, six Mycobacterium avium subspecies paratuberculosis (MAP)-specific culture filtrate proteins (CFPs) were produced and evaluated for sero-diagnosis of MAP infection in goat and cattle herds in India. Genes encoding for six MAP-CFPs were amplified and cloned into easy cloning vector pJET1.2/pTZ57R followed by sub-cloning into expression vector pET28a (+)/pET22b (+) containing C-terminal Histidine. Recombinant CFPs (r-CFPs) expressions were optimized in Escherichia coli (Rosetta cells) and purified using Ni-NTA affinity chromatography. In SDS-PAGE, MAP CFPs viz., 1693c, 2168c, ModD, 85C, Pep AN and Pep AC showed 22, 24, 55, 38, 20 and 25 kDa molecular masses, respectively. Identity of these r-CFPs was further confirmed by immuno-blotting. We developed six different ELISAs using the six individual r-CFPs and one additional ELISA i.e. cocktail ELISA (c-ELISA) was prepared using cocktail of all 6 r-CFPs. The performance of all seven ELISAs were further evaluated against whole cell protoplasmic based indigenous ELISA (i-ELISA). c-ELISA showed almost similar sensitivity as shown by i-ELISA. However, individual r-CFP based ELISA could not reach up to the sensitivity of cocktail of six r-CFPs. None of the r-CFP showed any false positive (as compare to i-ELISA) thereby specificity was 100%. Results of ELISA tests based on cocktail of r-CFPs, ModD and 85C were quite similar to i-ELISA from goat sera whereas in cattle serum c-ELISA was comparable with i-ELISA. Our study showed a comparable specificity of c-ELISA for the diagnosis of JD and it may have applicability in region where disease is endemic. Future validation of c-ELISA against gold standard or confirmatory tests would give a better insight on its diagnostic potential over i-ELISA

Inhibitor of Sarco/Endoplasmic Reticulum Calcium-ATPase Impairs Multiple Steps of Paramyxovirus Replication

Sarco/endoplasmic reticulum calcium-ATPase (SERCA) is a membrane-bound cytosolic enzyme which is known to regulate the uptake of calcium into the sarco/endoplasmic reticulum. Herein, we demonstrate for the first time that SERCA can also regulate virus replication. Treatment of Vero cells with SERCA-specific inhibitor (Thapsigargin) at a concentration that is nontoxic to the cells significantly reduced Peste des petits ruminants virus (PPRV) and Newcastle disease virus (NDV) replication. Conversely, overexpression of SERCA rescued the inhibitory effect of Thapsigargin on virus replication. PPRV and NDV infection induced SERCA expression in Vero cells, which could be blocked by Thapsigargin. Besides inducing enhanced formation of cytoplasmic foci, Thapsigargin was shown to block viral entry into the target cells as well as synthesis of viral proteins. Furthermore, NDV was shown to acquire significant resistance to Thapsigargin upon long-term passage (P) in Vero cells. As compared to the P0 and P70-Control, the fusion (F) protein of P70-Thapsigargin virus exhibited a unique mutation at amino acid residue 104 (E104K), whereas no Thapsigargin-associated mutations were observed in HN gene. To the best of our knowledge, this is the first report describing the virus-supportive role of SERCA and a rare report suggesting that viruses may acquire resistance even in the presence of an inhibitor that targets a cellular factor

Recombinant secretory proteins based new ‘Cocktail ELISA’ as a marker assay to differentiate infected and vaccinated cows for Mycobacterium avium subspecies paratuberculosis infection

967-972Johne's disease is endemic in the domestic livestock population of India. Recently, we developed highly effective 'Indigenous vaccine' to control Johne’s disease in animals. In order to gain disease free status as per World Organization for Animal Health, it is essential to have a marker assay to differentiate between infected and vaccinated animals before vaccine can be used in the field. We have developed a new marker assay ‘Cocktail ELISA’ using six ‘recombinant secretary proteins’ (MAP 1693c, MAP 2168c, MAP Mod D, MAP 85c, MAP Pep AN and MAP Pep AC) and evaluated for diagnosis of Johne’s disease along with 'Indigenous ELISA kit'. This ‘Cocktail ELISA’ successfully differentiated the infected, vaccinated and healthy (non-infected) cows and will facilitate the use of Johne’s disease vaccine to control the disease in cows at national levels

‘Indigenous’ and ‘Ethanol Vortex’ ELISA kits for diagnosis of <em>Mycobacterium avium</em> subsp. <em>paratuberculosis </em>infection in cattle: Is there a ‘globally relevant kit’ in the ‘Reverse Ice-burg’ environment?

279-286Highly versatile and robust 'Indigenous ELISA kit' for the diagnosis of Mycobacterium avium subsp. paratuberculosis infection in cattle herds was compared with 'Ethanol Vortex (EV) ELISA kit' of USA. Of 160 (118 vaccinated and 42 non-vaccinated) cattle screened, 129 and 35 were positive in 'Indigenous' and 'Ethanol Vortex' ELISA kits, respectively. 'I-ELISA', using 'semi-purified protoplasmic antigen' from native highly prevalent biotype ('Indian Bison type') of MAP of goat origin, was highly sensitivitive (91.4%) as compared to the 'EV-ELISA. 'I-ELISA kit using whole cell sonicate from native 'S 5' (‘Indian Bison type’) strain of MAP as 'antigen source' was significantly superior than EV-ELISA kit using surface antigens from 'Linda' strain ('cattle type') of cattle origin in USA. Therefore, 'i-ELISA kit' may be recommended for the screening of domestic cattle herds against MAP infection in India. The present study has demonstrated that in the diagnosis of chronic infections and diseases, such as Johne's disease, 'Indigenous kits' are significantly superior to kits made in other countries, EV-ELISA, in the present case, particularly screening of native cattle herds endemically infected with MAP

Evaluation of goat based ‘Indigenous vaccine’ against Bovine Johne’s Disease in endemically infected native cattle herds

16-24‘Indigenous vaccine’ prepared from ‘Indian Bison Type’ a native bio-type of Mycobacterium avium subspecies paratuberculosis strain ‘S5’ of goat origin (goat based) was evaluated in indigenous cattle herds located in gaushalas (cow shelters), endemic for Bovine Johne’s disease. Cows (893) were randomly divided into vaccinated (702 = 626 adults + 76 calves) and control (191 = 173 adults + 18 calves) groups. Response to vaccination was evaluated on the basis of health (mortality, morbidity), productivity (growth rate, reproductive performance, total milk yield), immunological parameters (LTT, ELISA titer), survivability of animals naturally infected with MAP, bacterimia (by specific blood PCR), sero-conversion (by indigenous ELISA) and status of shedding of MAP in feces (by microscopy) in the two groups before and after vaccination. Reduction in MAP shedding [to the extent of 100% in Herd A; and from 82.1% (0 DPV) to 10.7% (270 DPV) in Herd C] was the major finding in vaccinated cows. Whereas, the control group cows have shown no improvement. As the first indicator of vaccine efficacy, MAP bacilli disappeared from the blood circulation as early as 15 days post vaccination, however, peak titers were achieved around 90 DPV. Peak titers initially declined slightly but were maintained later throughout the study period. Control animals did not show any pattern in antibody titers. Mortality was low in vaccinated as compared to the control groups. Vaccination of endemically infected native cattle herds with inactivated whole-cell bacterin of novel ‘Indian Bison Type’ bio-type of goat origin strain ‘S5’ effectively restored health and productivity and reduced clinical BJD. Application of goat based ‘indigenous vaccine’ for therapeutic management of BJD in native cattle herds (gaushalas) is the first of its kind. </span

Novel recombinant Mce-truncated protein based ELISA for the diagnosis of Mycobacterium avium subsp. paratuberculosis infection in domestic livestock.

Johne's disease (JD) is an infectious wasting condition of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP) in domestic livestock of every country that has been investigated. Controlling JD is problematic due to the lack of sensitive, specific, efficient, and cost-effective diagnostic tests. A major challenge in the development of diagnostics like ELISA is the selection of an ideal antigen/(s) that is pathogen-specific and allows sensitive recognition. Therefore, the purpose of this study was to identify and use Mce-truncated protein-based ELISA assay for the diagnosis of MAP infection with high sensitivity and specificity. In silico epitope prediction by epitope mapping throughout the whole length of MAP2191 protein revealed that C-terminal portion of this protein presented potential T- and B-cell epitopes. Therefore, a novel Mce-truncated protein encoded by the selected region of MAP2191 gene was expressed, purified with Ni-NTA gel matrix and confirmed by SDS PAGE and western blot. A profiling ELISA assay was developed to evaluate sera from MAP infected and non-infected ruminant species for antibodies against Mce-truncated protein to infer the immunogenicity of this protein in the host. Using this Mce protein-based ELISA, 251 goats, 53 sheep, 117 buffaloes, and 33 cattle serum samples were screened and 49.4, 51.0, 69.2, and 54.6% animals, respectively, were found positive. Comparing with i-ELISA, the new Mce-based ELISA kit showed a relatively higher specificity but suffered from slightly reduced sensitivity. Mce-based ELISA excluded apparently false positive results of i-ELISA. Mce protein was found to be antigenic and Mce-ELISA test could be employed as a diagnostic test for JD in domestic livestock in view of the a relatively higher specificity and accuracy. The antigenic potential of Mce antigen can also be exploited for the development of a new vaccine for the control of MAP infection