Identification and Characterization of Pediococcus Species from Piper betle (Betel) Leaves.

Betel vine is an edible creeper used in folk medicine to aid digestion since time immemorial. It is an ideal candidate deemed for the bioprospection of endophytic microorganisms with valuable attributes. This study aimed at the characterization of potential bacteria from fermented betel leaves. We report the presence of Pediococcus species with probiotic properties from betel. The isolated organisms were subjected to preliminary biochemical analysis and exhibited growth at 37°C and pH 6.7 with fermented glucose, sucrose and lactose without the evolution of CO2. Also, the organisms presented tolerance to 6.5% NaCl and 0.3% bile salt. The three isolates assimilated cholesterol dispensed in the medium and when exposed to E. coli evinced antagonism. Based on the 16S rRNA sequencing and phylogenetic tree analysis, the organisms were identified to be Pediococcus acidilactici and Pediococcus pentosaceus. Both the organisms when functionally characterized displayed beta-galactosidase, amylase and esterase activities, but Pediococcus pentosaceus had a substantial effect proving its candidature for probiotic applications.

Expression profile of bio-defense genes in Penaeus monodon gills in response to formalin inactivated white spot syndrome virus vaccine.

White spot syndrome virus (WSSV) is the most devastating pathogen of penaeid shrimp. While developing technology to vaccinate shrimp against WSSV, it is imperative to look into the immune response of the animal at molecular level. However, very little information has been generated in this direction. The present study is an attempt to understand the expression of bio-defense genes in gill tissues of Penaeus monodon in response to formalin inactivated WSSV. A WSSV vaccine with a viral titer of 1×10(9) DNA copies was prepared and orally administered to P. monodon at a rate of 1.75×10(6) DNA copies of inactivated virus preparation (IVP) day(-1) for 7days. The animals were challenged with WSSV on 1st and 5th day post vaccination, and temporal expression of bio-defense genes in gill tissues was studied. Survival of 100% and 50% were observed respectively on 1st and 5th day post vaccination challenge. The humoral immune genes prophenoloxidase (proPO), alpha 2-macroglobulin (α2M), crustin and PmRACK, and the cell mediated immune genes caspase and Rab7 were up regulated in gill tissue upon vaccination and challenge. The expression of humoral gene crustin and cellular gene Rab7 was related to survival in IVP administered shrimp. Results of the study suggest that these genes have roles in protecting shrimp from WSSV on vaccination.

Immune modulations and protection by translationally controlled tumor protein (TCTP) in Fenneropenaeus indicus harboring white spot syndrome virus infection.

Fenneropenaeus indicus translationally controlled tumor protein (Fi-TCTP) was cloned and expressed using pET 100a-D-TOPO in prokaryotic expression system and it exhibited putative antioxidant activity as assessed in vitro by enhanced growth of Escherichia coli (E. coli) in presence of hydrogen peroxide. The protective efficacy of recombinant Fi-TCTP (rFi-TCTP) was evaluated in F. indicus by intramuscular and oral administration. Intramuscular injection of rFi-TCTP to shrimps, on subsequent white spot syndrome virus (WSSV) infection exhibited 42% relative percent survival. To understand the mechanism of protection, immunological parameters such as reactive oxygen species (ROS), phenoloxidase and mitochondrial membrane potential (MMP) were assessed in early (24h) and late (60h) stages of infection. rFi-TCTP pretreatment significantly lowers the WSSV induced ROS generation and respiratory burst during early and late stages of infection. Further, WSSV induced apoptotic changes such as reduced haemocyte count, loss in MMP and DNA fragmentation were significantly reduced during early and late stage of infection upon rFi-TCTP administration. Hence, the immunomodulatory studies suggest that protective effect of rFi-TCTP in treated shrimps, might be due to the reduction in ROS and apoptosis, following decreased mitochondrial damage together with reduced phenoloxidase activity and respiratory burst.

Physicochemical characterization of an aspin (rBm-33) from a filarial parasite Brugia malayi against the important human aspartic proteases.

The aspartic protease inhibitory efficiency of rBm-33, an aspin from a filarial parasite Brugia malayi was investigated. rBm-33 was found to be thermostable up to 90°C and it forms a stable 'enzyme-product' complex with human pepsin. Aspartic protease inhibitory activity was investigated using UV spectroscopy and isothermal titration calorimetry. Our results suggest that rBm-33 inhibits the activity of important human aspartic proteases that were examined with binding constants (Kb) values between 10.23 × 10(3) and 6.52 × 10(3) M(-1). The binding reactions were enthalpy driven with ΔHb values between -50.99 and -46.07 kJ mol(-1). From kinetic studies, pepsin inhibition by rBm-33 was found to be linear competitive with an inhibition constant (Ki) of 2.5 (±0.8) nM. Because of the inhibitory efficacy of Bm-33 against important human aspartic proteases which play a vital role in immune-regulation along with other functions, Bm-33 can be projected as a drug target for the filariasis.

Molecular cloning, expression analysis and characterization of peroxiredoxin during WSSV infection in shrimp Fenneropenaeus indicus.

Innate immunity is the first line of defense in shrimps against invading pathogenic microorganisms. Peroxiredoxins (PRX) are the family of antioxidant proteins that play a crucial role in reduction of oxidative stress in host during viral infection. Peroxiredoxin from Fenneropenaeus indicus was identified, cloned and expressed in prokaryotic expression system. The Fi-PRX protein consists of two conserved Cys residues and belongs to typical 2-cys PRX family. The phylogenetic analysis revealed a close evolutionary relatedness of Fi-PRX with the PRX from Drosophila melanogaster PRX1 and distant origin with PRX sequences from other shrimp isolates Fenneropenaeus chinensis, Litopenaeus vannamei and Penaeus japonicus. Fi-PRX transcripts are constitutively expressed in hemocytes and tissues (gills, heart and muscle) and down regulated during 12 h, 24 h and 48 h of WSSV challenged shrimps. Fi-PRX protein levels correlated well with the corresponding levels of Fi-PRX transcripts in hemocytes and tissues of WSSV challenged shrimps. Recombinant Fi-PRX reduces insulin only in the presence of DTT suggesting that the antioxidant function of the protein is thiol dependent. These findings suggest that antioxidant activity of Fi-PRX play a significant role in neutralization of excessive free radicals and ROS generated during viral invasion.

Recombinant Wolbachia surface protein (WSP)-induced T cell responses in Wuchereria bancrofti infections.

Human lymphatic filariasis is a debilitating parasitic disease characterized by downregulation of the host's immune response in asymptomatic carriers along with profound hyperreactivity in chronic patients apart from putatively immune endemic normals. The endosymbiont Wolbachia, a bacterium of filarial nematodes has received much attention as possible chemotherapeutic target and its involvement in disease pathogenesis. The role of recombinant Wolbachia surface protein (rWSP), one of the most abundantly expressed proteins of the endosymbiont, in modulating cell-mediated immune responses in patients harboring Wuchereria bancrofti infections was evaluated in the current study. rWSP-induced lymphoproliferation with peripheral blood mononuclear cells suggested an impaired proliferative response in asymptomatic microfilaremic (MF) and symptomatic chronic pathology (CP) patients compared to endemic normals (EN). This was further supported by a significantly diminished expression of CD69 along with elevated levels of CD127 and CD62L in filarial patients (MF and CP) compared to EN. Further, rWSP induced the expression of regulatory T cell markers CTLA-4 and CD25 along with suppressor cytokines IL-10 and TGF-ß in MF and CP patients compared to EN. However, the rWSP-stimulated expression of IFN-γ was diminished significantly in filarial patients compared to endemic normals. Thus, these findings suggest that WSP may also contribute to the suppression of immune responses seen in filarial patients.

Expression, purification and characterization of refolded rBm-33 (pepsin inhibitor homolog) from Brugia malayi: a human Lymphatic Filarial parasite.

Bm-33 (pepsin inhibitor homolog) produced by the human filarial parasite Brugia malayi, was expressed in Escherichia coli. Expression of rBm33 in BL21 (DE3), Rosetta-2 gami (DE3) pLysS and GJ1158 bacterial strains, results in the accumulation of a 33 kDa protein in inclusion bodies. Inactive rBm-33 was purified under the denaturing conditions and refolded by step wise dialysis using buffers of pH ranging from 11 to 7. Size exclusion chromatography of rBm-33 (refolded) reveals that nearly 83% of the recombinant protein exhibits pepsin inhibition activity. Circular dichroism studies indicate that the protein is predominantly composed of 85% α-helix. rBm-33 (refolded) was assessed for its pepsin inhibition activity using casein agar plate method, UV-spectroscopy and zymogram analysis. These findings suggest that rBm-33 (refolded) has affinity for human pepsin and completely inhibits the proteolytic activity with the gradual increase in rBm-33 (refolded) concentration. Size exclusion chromatography reveals the formation of rBm-33-pepsin complex and was cross checked using immunoblot with glutaraldehyde cross linking. These findings reveal that rBm-33 (refolded) is in native fold to exhibit pepsin inhibition.

Immune responses to recombinant Brugia malayi pepsin inhibitor homolog (Bm-33) in patients with human lymphatic filariaisis.

Immune responses to recombinant Brugia malayi pepsin inhibitor homolog (rBm-33) were investigated in patients with human lymphatic filariasis (microfilaremics (MF) and chronic pathology (CP)) along with endemic normals (EN). Flow cytometric analysis (24 h) revealed CD4(+) T cell activation in patients (MF and CP) compared to normals (EN), with increased expression of CD69 and diminished levels of CD62L and CD127. This was associated with an elevated expression of CD154 but not CD28 and CTLA4 in CP patients. However, Bm-33-induced cytokine expression profile (IL-1ß, IL-12, IL-8, IFN-γ, IL-10 and TGF-ß) did not exhibit any significant difference between normals and patients at the same time point. Although CD4(+) T cell activation was observed initially in filarial patients (24 h), lymphoproliferation studies (96 h) suggested diminished proliferation compared to normals, indicating functional inactivation in the former upon prolonged antigen exposure. This indicates that rBm-33 induces an early T cell activation in MF and CP patients followed by a decreased lymphoproliferation that might contribute to immune suppression in these individuals.

Wuchereria bancrofti: diminished platelet activation in filarial patients.

Blood platelets are the innate immune elements that have not been investigated in human filarial infections. Platelet activation status in the endemic normals (EN), microfilaria positive individuals (MF) and patients with chronic pathology (CP) was evaluated in whole blood, under unstimulated as well as antigen exposed (BmA, E. coli) conditions for PAC-1 expression by Flow cytometry. A diminished PAC-1 expression was observed in MF compared to CP and EN spontaneously as well as upon antigen exposure. Besides this, PAC-1 expression within the groups did not exhibit any significant difference under all the experimental conditions. However in CP patients, E. coli antigen exposure resulted in a significantly reduced PAC-1 expression compared to the spontaneous expression levels. NO release in platelet culture supernatants from EN was inversely proportional to platelet aggregation. Collagen stimulated platelets from EN, exposed to sera and immune complexes from CP and MF patients resulted in elevated Nitric Oxide (NO) release, compared to those exposed to autologous sera and fetal calf serum. In addition, under similar conditions, collagen stimulated platelets from EN, exposed to filarial antigen (BmA) exhibited increased NO compared to the E. coli antigen exposed ones and light microscopic observations of cultured platelets supported the above findings. Thus it appears from the results of the present study that filarial antigen may play a role in the loss of platelet aggregation, leading to platelet inactivation.

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.

Honey as a natural preservative of milk.

The anti-bacterial property and preservative nature of honey has been studied by evaluating the role of hydrogen peroxide in these properties, against bacterial strains isolated and identified from pasteurized milk samples. The antibacterial property of honey examined by agar incorporation assay and turbidometry, indicated a concentration dependent inhibition of bacterial growth in all catalase negative strains in comparison with catalase positive strains, highlighting a probable role of hydrogen peroxide. Samples of commercial milk stored at 40C in presence of honey were shown to inhibit opportunistic bacterial growth better compared to samples stored without honey. Due to the bactericidal property of hydrogen peroxide and its preservative nature, honey which is chiefly a combination of various sugars and hydrogen peroxide, can be used a preservative of milk samples.

Brugia malayi Wolbachia hsp60 IgG antibody and isotype reactivity in different clinical groups infected or exposed to human bancroftian lymphatic filariasis.

Wolbachia, an endosymbiotic bacterium in filarial parasites, comes into contact with the host immune system upon parasite death. Here, we analyzed, total IgG and isotype antibody responses to Wolbachia hsp60 in individuals from an area endemic for Wuchereria bancrofti. Wolbachia derived hsp60 gene was cloned and the recombinant protein was used to determine the IgG and isotype reactivity by Western blotting and ELISA. All individuals from the endemic area generated antibody responses to Brugia malayi Wolbachia hsp60, which were elevated in the group with chronic pathology. Isotype analysis showed that, all clinical groups mounted IgG1-IgG4 responses with higher levels of B. malayi Wolbachia hsp60 specific IgG1 observed in the sera of patients with chronic pathology compared to microfilaraemics and endemic normals. These findings suggests that Wolbachia-derived hsp60 generates antibody responses in individuals infected or exposed to W. bancrofti and an elevated IgG and IgG1 reactivity is observed in people with filarial pathology.

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.

Setaria digitata: identification and characterization of a hypodermally expressed SXP/RAL2 protein.

SXP-1, an immunodominant filarial protein identified from Wuchereria bancrofti from our centre and previously exploited for diagnosis of human lymphatic filariasis, has been shown to be well conserved across several filarial species. In the present study, we describe the identification of SXP protein from the cattle filarid Setaria digitata using antiserum raised against recombinant WbSXP-1, and were able to detect 34 and 66kDa proteins from the crude protein extracts of S. digitata. These reactive proteins were found to be sheath proteins localized to the hypodermal region of the parasite.

Diminished monocyte function in microfilaremic patients with lymphatic filariasis and its relationship to altered lymphoproliferative responses.

Antigen-specific hyporesponsiveness to filarial antigens is a phenomenon observed in patent infection with lymph-dwelling filarial parasites of humans. This phenomenon has been attributed to a multitude of factors, one of which is altered monocyte function. To examine the role played by monocytes in filarial infection, we assessed the responses of monocytes obtained from normal and filarial parasite-infected individuals to both crude filarial antigen and purified recombinant filarial antigen WbSXP-1 and attempted to relate these to the altered lymphoproliferative responses seen in filarial infection. Monocytes from microfilaremic (MF) patients demonstrated an inability to respond to lipopolysaccharide compared to monocytes from endemic normal persons or from lymphedema patients. Indeed, interleukin 1beta (IL-1beta) production was severely limited, a finding that did not extend to monocyte responses to filarial antigens. Serum from MF patients reduced adherence and spreading of normal monocytes, a finding not seen with serum from the other clinical groups. Interestingly, there was a significant correlation between the production of IL-1beta and adherence. Moreover, the levels of spontaneous production of IL-1beta correlated with high levels of spontaneous secretion of IL-10. The effects observed were not a result of diminished viability or alteration in the expression of the cell surface markers CD14 and HLA-DR. These data suggest that monocyte function is dampened in MF patients, a finding which could alter lymphoproliferative responses during patent infection.

Production of polyclonal antiserum against recombinant VP28 protein and its application for the detection of white spot syndrome virus in crustaceans.

The VP28 gene of white spot syndrome virus (WSSV) was cloned into pRSET B expression vector. The VP28 protein was expressed as a protein with a 6-histidine taq in Escherichia coli GJ1158 with NaCl induction. Antiserum was raised against this recombinant-VP28 protein in rabbits and it recognized VP28 protein in naturally and experimentally WSSV-infected shrimp, marine crabs, freshwater prawns and freshwater crabs. The antiserum did not recognize any of the other known WSSV structural proteins. Various organs such as eyestalks, head muscle, gill tissue, heart tissue, haemolymph, tail tissue and appendages were found to be good materials for detection of WSSV using the antiserum and detection of WSSV was successful in experimentally infected Penaeus monodon and P. indicus at 12 and 24 h post-infection (p.i.), respectively. The antiserum was capable of detecting WSSV in 5 ng of total haemolymph protein from WSSV-infected shrimp.

Wuchereria bancrofti: cloning and characterization of heat shock protein 70 from the human lymphatic filarial parasite.

Heat shock protein 70 (HSP70) was identified as an immunodominant antigen by screening a Wuchereria bancrofti (Wb) microfilarial cDNA library with pooled Wb-infected sera, with 28% of the immunopositive clones coding for Wb-HSP70. The deduced amino acid sequence showed greater than 97 and 85% identity with HSP70 from filarial nematodes and humans, respectively. Recombinant HSP70 (74 kDa) and a recombinant protein from the C-terminal portion (43 kDa) also reacted with pooled Wb-infected sera, suggesting that the C-terminal region of HSP70 contains at least one antibody epitope. Brugia malayi L3 larvae showed increasing levels of HSP70 with increasing temperatures. Further, a polyclonal mouse anti-Wb-HSP70 antibody had reactivity to the HSP70 of cattle filarial parasite Settaria digitata and to human HSP70 derived from a Hep-2 cell line. Immune reactivity to Wb-HSP70 was strong, with uninfected non-endemic normal sera showing significantly greater reactions than sera from filaria-infected individuals. Both immunodominant self-HSP70 and HSP70 from other microbial infections may be primary targets for developing autoantibodies naturally.

Larvae and early post-larvae of Penaeus monodon (Fabricius) experimentally infected with white spot syndrome virus (WSSV) show no significant mortality.

The pathogenicity of white spot syndrome virus (WSSV) was tested with different developmental stages of Penaeus monodon, i.e. nauplius, protozoeae, mysis, early post-larvae (PL1-10), late post-larvae (PL11-20) and juveniles. WSSV challenge was done by immersion and oral routes. No disease occurred in the larvae and early post-larvae but they were positive for WSSV by nested polymerase chain reaction (PCR) assay. Significant mortality was observed in late post-larvae and juveniles and both single and nested PCR assays gave positive results with these samples. The results demonstrated that WSSV virulence in P. monodon increases with advancing stages of development and that WSSV infection does not result in disease for larvae and post-larvae younger than PL10.

Thioredoxin from Brugia malayi: defining a 16-kilodalton class of thioredoxins from nematodes.

Thioredoxins are a family of small redox proteins that undergo NADPH-dependent reduction by thioredoxin reductase. This results in a supply of reducing equivalents that cells use in a wide variety of biological reactions, which include maintaining reduced forms of the enzymes important for protection against damage from high-energy oxygen radicals, the regulation of transcription factor activity, and the inhibition of apoptosis. Here we report on a new member of the thioredoxin family of proteins from the filarial nematode Brugia malayi, Bm-TRX-1, which defines a new subclass of 16-kDa thioredoxins that occur widely in nematodes, including Caenorhabditis elegans. In addition to being larger than the thioredoxins found in mammalian and bacterial species, the putative active site sequence of Bm-TRX-1, WCPPC, does not conform to the highly conserved WCGPC reported for thioredoxins from mammals to bacteria. Interestingly, an allelic form of Bm-TRX-1 was identified with an active site sequence WCPQC, which appears to be unique to the thioredoxins from filarial species. Bm-TRX-1 was between 98% and 35% identical to thioredoxins from other nematodes and approximately 20% identical to the thioredoxins from mammals and Escherichia coli. Bm-TRX-1 was constitutively transcribed throughout the B. malayi life cycle, and Bm-TRX protein was detectable in somatic extracts and excretory-secretory products from adults and microfilariae. Recombinant Bm-TRX-1 had thiodisulfide reductase activity, as measured by the reduction of insulin, and protected DNA from the nicking activity of oxygen radicals. Overexpression of Bm-TRX-1 in a human monocyte cell line negatively regulated tumor necrosis factor alpha-induced p38 mitogen-activated protein kinase activity, suggesting a possible role of the 16-kDa Bm-TRX-1 in immunomodulation.

Comparison between the skin snip test and simple dot blot assay as potential rapid assessment tools for Onchocerciasis in the postcontrol era in Ghana.

Successful control of onchocerciasis through mass distribution of ivermectin needs to be coupled with reliable, sensitive, specific, yet affordable diagnostic methods to monitor and ensure the efficacy of such measures. The effort put into the development of diagnostic methods for onchocerciasis that can substitute for or work in combination with the present "gold standard," the skin snip test, has resulted in the discovery of a number of immunogenic proteins with potential use as diagnostic tools in the postcontrol era. Most of these proteins have now been produced through recombinant DNA techniques. However, when costs are not a trivial issue, none of them have yet found their way into the areas where the disease still exists. In the present study, we have evaluated the performance of a simple dot blot assay which uses a mixture of native proteins designated PakF as a serious contender in the quest for a less invasive and more sensitive method to detect Onchocerca volvulus infection in areas with diverse endemicities. Our results indicate that the assay we propose is more sensitive than the skin snip test and shows high specificity, both characteristics required for a suitable tool for the monitoring of onchocerciasis in the postcontrol era.