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

Risk factors associated with sero-positivity to Johne’s disease in Indian dairy herds

Johne’s disease of domestic livestock has high economic significance. Environmental factors and farm level management practices are associated with the incidence and occurrence of disease in farm and farmers herds/ flocks. A cross-sectional study was conducted in the dairy herds (315) maintained in different geographical regions and management practices in the Punjab state to determine ‘herd level’ risk factors associated with Johne’s disease. Of 16 factors studied, univariate analysis showed that 6 factors were significantly associated with sero-positivity. Multivariate analysis showed contamination of feed and water with adult manure (OR=3.97) and history of chronic diarrhoea in the herd (OR=2.04) as the factors significantly associated with positive status of animals in the herd. It is the first report on ‘risk factors’ analysis for Johne’s disease in India

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

Control of paratuberculosis: who, why and how. A review of 48 countries

Paratuberculosis, a chronic disease affecting ruminant livestock, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). It has direct and indirect economic costs, impacts animal welfare and arouses public health concerns. In a survey of 48 countries we found paratuberculosis to be very common in livestock. In about half the countries more than 20% of herds and flocks were infected with MAP. Most countries had large ruminant populations (millions), several types of farmed ruminants, multiple husbandry systems and tens of thousands of individual farms, creating challenges for disease control. In addition, numerous species of free-living wildlife were infected. Paratuberculosis was notifiable in most countries, but formal control programs were present in only 22 countries. Generally, these were the more highly developed countries with advanced veterinary services. Of the countries without a formal control program for paratuberculosis, 76% were in South and Central America, Asia and Africa while 20% were in Europe. Control programs were justified most commonly on animal health grounds, but protecting market access and public health were other factors. Prevalence reduction was the major objective in most countries, but Norway and Sweden aimed to eradicate the disease, so surveillance and response were their major objectives. Government funding was involved in about two thirds of countries, but operations tended to be funded by farmers and their organizations and not by government alone. The majority of countries (60%) had voluntary control programs. Generally, programs were supported by incentives for joining, financial compensation and/or penalties for non-participation. Performance indicators, structure, leadership, practices and tools used in control programs are also presented. Securing funding for long-term control activities was a widespread problem. Control programs were reported to be successful in 16 (73%) of the 22 countries. Recommendations are made for future control programs, including a primary goal of establishing an international code for paratuberculosis, leading to universal acknowledgment of the principles and methods of control in relation to endemic and transboundary disease. An holistic approach across all ruminant livestock industries and long-term commitment is required for control of paratuberculosis

Revisited immune reactivity between native semi-purified protoplasmic (caprine) versus commercial purified protoplasmic (bovine) antigens for the screening of goatherds endemic for Johne’s disease

22-29The present study compared the immune-reactivity of 3 antigens of Mycobacterium avium subspecies paratuberculosis (MAP) sourced from different geographical regions and livestock species for the diagnosis of Johne’s disease (JD) in goats. Screening of 360 faecal and serum samplesof goats by microscopy, i-ELISA, b-ELISA and r-ELISA gave 56.9, 40.0, 34.4 and5.2% positive samples, respectively. Considering all the 4 tests (microscopy, i-ELISA, b-ELISA & r-ELISA kits), 3.0 and 35.2% goats were found common positive and negative, respectively. Of 3 ELISA tests, i-ELISA had the highest sensitivity, followed by b-ELISA and r-ELISA kit. ‘i-ELISA’ based on ‘indigenous antigen’ recovered from native (‘Indian Bison Type’) MAP strain of goat origin had superior immune reactivity as compared to imported commercial PPAs(protoplasmic antigens) of bovine origin (b-ELISA from Allied Monitor Inc., USA) and commercial ELISA kit for ruminant species (ID Vet, France). Lower immune-reactivity of commercial antigens as compared to ‘indigenous antigen’ indicated that search for universally acceptable ‘ELISA kit’ is not practically possible

Application of IS1311 locus 2 PCR-REA assay for the specific detection of ′Bison type′ Mycobacterium avium subspecies paratuberculosis isolates of Indian origin

Background & objectives: Of the three major genotypes of Mycobacterium avium subspecies paratuberculosis (MAP), ′Bison type′ is most prevalent genotype in the domestic livestock species of the country, and has also been recovered from patients suffering from Crohn′s disease. Recently, a new assay based on IS1311 locus 2 PCR- restriction endonuclease analysis (REA) was designed to distinguish between ′Indian Bison type′ and non-Indian genotypes. The present study investigated discriminatory potential of this new assay while screening of a panel of MAP isolates of diverse genotypes and from different geographical regions. Methods: A total of 53 mycobacterial isolates (41 MAP and 12 mycobacterium other than MAP), three MAP genomic DNA and 36 MAP positive faecal DNA samples from different livestock species (cattle, buffaloes, goat, sheep and bison) and geographical regions (India, Canada, USA, Spain and Portugal) were included in the study. The extracted DNA samples (n=92) were analyzed for the presence of MAP specific sequences (IS900, ISMav 2 and HspX) using PCR. DNA samples were further subjected to genotype differentiation using IS1311 PCR-REA and IS1311 L2 PCR-REA methods. Results: All the DNA samples (except DNA from non-MAP mycobacterial isolates) were positive for all the three MAP specific sequences based PCRs. IS1311 PCR-REA showed that MAP DNA samples of Indian origin belonged to ′Bison type′. Whereas, of the total 19 non-Indian MAP DNA samples, 2, 15 and 2 were genotyped as ′Bison type′, ′Cattle type′ and ′Sheep type′, respectively. IS1311 L2 PCR-REA method showed different restriction profiles of ′Bison type′ genotype as compared to non-Indian DNA samples. Interpretation & conclusions: IS1311 L2 PCR-REA method successfully discriminated ′Indian Bison type′ from other non-Indian genotypes and showed potential to be future epidemiological tool and for genotyping of MAP isolates