Development and validation of streptavidin-biotin-based double antibody sandwich ELISA for ricin diagnosis.

BACKGROUND: Ricin is a potential biowarfare agent. It is a phytotoxin isolated from castor seeds. At present there is no antidote available for ricin poisoning, patients only get supportive treatment based on their symptoms. This highlights the importance of early detection to avoid severity of accidents and reduce the risk factor. Considering this, our study aimed to develop a highly sensitive and specific sandwich ELISA for the detection of ricin. METHODS: Ricin was purified from castor seeds. Anti-ricin polyclonal and monoclonal antibodies were generated from rabbit antisera and hybridoma cell (1H6F1) supernatant using a protein A/G column. Antibody titer estimation was done using Indirect ELISA. A streptavidin-biotin-based sandwich ELISA was developed and the limit of detection (LOD), linear range, intra and inter-assay coefficient of variation (CV), and cross-reactivity with other similar toxins were determined. Interference of human plasma samples spiked with ricin was also checked. RESULTS: The LOD of the ELISA was found to be 0.45 ng/ml, with a linear range of 0.90-62 ng/ml, intra and inter-assay CV ranged from 3.34 % to 5 % and 5.17 % to 10.80 % respectively. The assay was not cross-reactive with other similar ribosome-inactivating protein (RIP) toxins. Ricin was detected in spiked plasma samples. CONCLUSION: The developed assay is highly sensitive and specific for detecting ricin and is not cross-reactive with other similar types of toxins. The assay can detect ricin in spiked plasma samples, so it has the potential to be used for the analysis of clinical samples after ricin poisoning.

CRISPR-Cas12a assisted recombinase based strand invading isothermal amplification platform designed for targeted detection of Bacillus anthracis Sterne.

Detection of a pathogen is crucial prior to all prophylaxis and post exposure treatment, as it can prevent further disease manifestation. In this study, we have developed a nucleic acid pre-amplification based CRISPR diagnostic for detection and surveillance of Bacillus anthracis Sterne. Strand Invasion Based isothermal Amplification (SIBA) platform and Cas12a (CRISPR endo-nuclease) was used to develop CRISPR-SIBA, a multifaceted diagnostic platform. SIBA was employed as the isothermal pre-amplification platform. CRISPR-Cas12a based collateral trans-cleavage reaction was used to ensure and enhance the specificity of the system. Efficiency of the detection system was evaluated by detecting Bacillus anthracis Sterne in complex wastewater sample backgrounds. Previously reported, Prophage 3, Cya and Pag genes of Bacillus anthracis were used as targets for this assay. The amplification system provided reliable and specific detection readout, with a sensitivity limit of 100 colony forming units in 40 min. The endpoint fluorescence from CRISPR collateral cleavage reactions gave a detection limit of 105 to 106 CFUs. The experiments conducted in this study provide the evidence for SIBA's applicability and compatibility with CRISPR-Cas system and its efficiency to specifically detect Bacillus anthracis Sterne. CRISPR-SIBA can be translated into developing cost-effective diagnostics for pathogens in resource constrained settings.

Recent Advancements and Novel Approaches Contributing to the Present Arsenal of Prophylaxis and Treatment Strategies Against Category A Bacterial Biothreat Agents.

Bacterial pathogens have always been a part of the ecosystem in which we thrive. Some pathogens have caused deadly outbreaks in the past and have been exploited as an agent of threat. Natural hotspots for these biological pathogens are widely distributed throughout the world and hence they remain clinically important. Technological advancement and change in general lifestyle has driven the evolution of these pathogens into more virulent and resistant variants. There has been a growing concern over the development of multidrug-resistant bacterial strains that could be used as bioweapons. This rapid change in pathogens also propels the field of science to develop and innovate new strategies and methodologies which are superior and safer to the existing ones. Some bacterial agents like-Bacillus anthracis, Yersinia pestis, Francisella tularensis and toxins produced by strains of Clostridium botulinum, have been segregated as Category A substances as they pose imminent threat to public health with a history of life threatening and catastrophic disease. This review highlights some encouraging developments and value additions in the current plan of action for protection against these select biothreat bacterial pathogens.

Designing, synthesis and evaluation of derived analogues of selected small molecule non-peptidic inhibitors against serotype BoNT/ F.

Botulinum neurotoxins are lethal Biowarfare categorized in group A of selected agents, by CDC USA. The unavailability of counter-measures against these neurotoxins has been a matter of extensive research. The 8-hydroxyquinoline (8-HQ) scaffold is established privileged compound and its potential as drug candidate against BoNTs is recently being explored. We have reported 8-HQ compounds NSC1014 and NSC1011 as potential small molecule inhibitors against BoNT/F. In the present study, analogues of NSC84087 and NSC1014 were designed, synthesized and studied for their inhibitory role against BoNT/F intoxication through in silico study, in vitro and in-vivo assays. ∼25 in-house synthesized small molecule inhibitors were evaluated against rBoNT/F light chain through fluorescence thermal shift (FTS) assay and then further assessed through endopeptidase assay. The binding affinity analysis was done through surface plasmon resonance (SPR) based Proteon™ XPR 36 system. Finally, the in-vivo efficacy of these compounds was evaluated in mice model. Analogues C87.9, C87.10 and C87.12 of compound NSC84087 and C14.10, C14.11 and C14.13 of NSC1014 showed promising results through FTS assay and endopeptidase assay. SPR based protein-small molecule interaction studies showed KD values in sub-micromolar range signifying high affinity interaction. The IC50 of C14.10 was found to be the lowest of 3.016 ± 0.798 µM as determined through endopeptidase assay. Finally, efficacy of selected molecules was evaluated in mice, C14.10 and C14.13 protected 40% animals against 4X LD50 and extended survival time up to 200% at 10X LD50. The present study thus proposes the emergence of NSC84087 and NSC1014 analogues as lead compound against BoNT/F.

Silibinin ameliorates abrin induced hepatotoxicity by attenuating oxidative stress, inflammation and inhibiting Fas pathway.

Abrin is a toxin from the seeds of Abrus precatorius. Abrin is considerably more toxic than ricin and a potent bio-warfare agent. The mechanism of abrin induced hepatotoxicity remains unclear. Silibinin has antioxidant, anti-inflammatory and hepatoprotective activities. But, its therapeutic potential in abrin toxicity is unknown. In view of these facts, the purpose of this study was to delineate the mechanisms and ameliorative role of silibinin against abrin induced hepatotoxicity. Parameters related to liver functions, oxidative stress, inflammation, Fas pathway and histopathology were evaluated in the liver of BALB/c mice after abrin exposure. Abrin intoxication resulted in hepatotoxicity, oxidative stress, inflammation, altered histopathology and increased Fas pathway signaling. Silibinin improves survival of abrin-exposed mice by decreasing serum liver enzymes and reinstating the antioxidant capacity. Silibinin also inhibits abrin-induced inflammation and Fas pathway. Present study for the first time demonstrates the hepatoprotective potential of silibinin against abrin toxicity.

E-N-(2-acetyl-phenyl)-3-phenyl-acrylamide targets abrin and ricin toxicity: Hitting two toxins with one stone.

The efficacy of small molecule inhibitors (SMIs) against the enzymatic activity of Shiga toxin prompted the evaluation of their efficacy on related toxins viz. ricin and abrin. Ricin, like Shiga toxin, is listed as a category B bioweapon and belongs to the type II family of ribosome inactivating proteins (RIPs). Abrin though structurally and functionally similar to ricin, is considerably more toxic. In the present study, 35 compounds were evaluated in A549 cells in in vitro assays, of which 5 offered protection against abrin and 2 against ricin, with IC50 values ranging between 30.5-1379 µM and 300-341 µM, respectively. These findings are substantiated by fluorescence based thermal shift assay. Moreover, the binding of the promising compounds to the toxin components has been validated by Surface Plasmon Resonance assay and in vitro protein synthesis assay. In vivo studies reveal complete protection of mice with compound 4 E-N-(2-acetyl-phenyl)-3-phenyl-acrylamide against orally administered lethal doses of, both, abrin and ricin. The present study thus proposes the emergence of E-N-(2-acetyl-phenyl)-3-phenyl-acrylamide as a lead compound against RIPs.

Characterization of immune response induced against catalytic domain of botulinum neurotoxin type E.

Botulinum neurotoxins (BoNTs) represent a family of bacterial toxins responsible for neuroparalytic disease 'botulism' in human and animals. Their potential use as biological weapon led to their classification in category 'A' biowarfare agent by Centers for Disease Control and Prevention (CDC), USA. In present study, gene encoding full length catalytic domain of BoNT/E-LC was cloned, expressed and protein was purified using Ni-NTA chromatography. Humoral immune response was confirmed by Ig isotyping and cell-mediated immunity by cytokine profiling and intracellular staining for enumeration of IFN-γ secreting CD4+ and CD8+ T cells. Increased antibody titer with the predominance of IgG subtype was observed. An interaction between antibodies produced against rBoNT/E-LC was established that showed the specificity against BoNT/E in SPR assay. Animal protection with rBoNT/E-LC was conferred through both humoral and cellular immune responses. These findings were supported by cytokine profiling and flow cytometric analysis. Splenocytes stimulated with rBoNT/E-LC showed a 3.27 and 2.8 times increase in the IFN-γ secreting CD4+ and CD8+ T cells, respectively; in immunized group (P < 0.05). Protection against BoNT/E challenge tended to relate with increase in the percentage of rBoNT/E-LC specific IL-2 in the splenocytes supernatant (P = 0.034) and with IFN-γ-producing CD4+ T cell responses (P = 0.045). We have immunologically evaluated catalytically active rBoNT/E-LC. Our results provide valuable investigational report for immunoprophylactic role of catalytic domain of BoNT/E.

Identification of Inhibitors against Botulinum Neurotoxins: 8-Hydroxyquinolines Hold Promise.

Botulinum neurotoxins (BoNTs) are the most toxic category A biological warfare agents. There is no therapeutics available for BoNT intoxication yet, necessitating the development of a medical countermeasure against these neurotoxins. The discovery of small molecule-based drugs has revolutionized in the last two decades resulting in the identification of several small molecule inhibitors of BoNTs. However, none progressed to clinical trials. 8-Hydroxyquinolines scaffold-based molecules are important 'privileged structures' that can be exploited as inhibitors of a diverse range of targets. In this review, our study of recent reports suggests the development of 8-hydroxyquinoline derived molecules as a potential drug may be on the horizon.

Targeted 8-hydroxyquinoline fragment based small molecule drug discovery against neglected botulinum neurotoxin type F.

OBJECTIVES: Botulinum neurotoxins are highly potent biological warfare agents. The unavailability of countermeasures against these neurotoxins has been a matter of extensive research. However, no clinical therapeutics has come to existence till date. The 8-hydroxyquinoline (8-HQ) scaffold is established privileged compound and its potential as drug candidate against BoNTs is recently being explored. METHODS: In present work, three course studies were performed involving in silico, in vitro and in vivo cascade to screen 8-HQ small molecule inhibitors against BoNT/F intoxication. ~800 molecules obtained from open repositories were screened in silico and commercially obtained twenty-four 8-HQ derived small molecule inhibitors were evaluated against rBoNT/F light chain through fluorescence thermal shift (FTS) assay. Selected compounds were further evaluated through endopeptidase assay. Further binding affinity analysis was done through surface plasmon resonance (SPR) based Proteon™ XPR 36 system. Finally, the in vivo efficacy of these compounds was evaluated in mice model. RESULTS: Three compounds NSC1011, NSC1014 and NSC84094 were found to be highly inhibitory after screening of 8-HQ compounds through FTS assay and endopeptidase assay. SPR based protein-small molecule interaction studies showed highest affinity binding of NSC1014 (KD: 5.58E-06) with BoNT/F-LC. NSC1011, NSC1014, and NSC84094 displayed IC50 of 30.47 ±â€¯6.24, 14.91 ±â€¯2.49 and 17.39 ±â€¯2.74 µM, respectively, in endopeptidase assay. NSC1011 and NSC1014 displayed marked extension of survival time in mice model. CONCLUSION: NSC1011 and NSC1014 have emerged as promising drug candidate against BoNT/F intoxication displaying higher potential than previously reported compounds.

Immunological detection assays for recombinant Shiga toxin & Shigella dysenteriae.

Background & objectives: : Shiga toxin (Stx) is produced by Shigella dysenteriae, a Gram-negative, facultative anaerobic bacillus that causes shigellosis, haemolytic uraemic syndrome (HUS) and Reiter's syndrome. The detection methods for shiga toxin needs to be rapid, accurate, reliable and must be extensively evaluated under field conditions. The aim of this study was to develop rapid, sensitive and specific detection method for Stx. Methods: : Mice and rabbits were immunized with purified recombinant Shiga toxin B (rStxB). Using these antibodies dot ELISA, sandwich ELISA and flow through assay were developed. Results: : The high-titre antibodies specifically reacted with purified rStxB. Dot-ELISA, sandwich ELISA and flow-through assay were developed and standardized that could detect StxB with limit of detection (LOD) of 9.75, 9.7 ng/ml and 0.46 µg/cassette, respectively. Interpretation & conclusions: : The rStxB was used to produce antibodies to avoid handling of pathogen. The Flow through assay 'developed was specific, rapid and field amenable.

Biochemical Characterization of In vitro Reconstituted Biologically Active Recombinant Shiga Toxin.

BACKGROUND: Shiga toxins comprise a family of related proteins produced by bacteria Shigella dysenteriae and some strains of Escherichia coli that cause severe clinical manifestations. Severe Shiga toxin intoxication results in Haemolytic-Uremic Syndrome (HUS), up to 50% of HUS patients manifest some degree of renal failure and ~10% of such cases develop permanent renal failure or death. OBJECTIVE: In present research work production of biologically active rStx from non-toxic rStxA and rStxB subunits were established that can be used in many biomedical applications. METHODS: Purification of Shiga toxin from bacteria is a multistep time consuming process resulting in low yield. To overcome this problem, the rStxA and rStxB protein were separately cloned and expressed in E. coli host and purified through affinity chromatography. GST pull-down assay was performed for interaction study between rStxA and pentameric rStxB. The affinity between A and B subunits of reconstituted recombinant Shiga toxin (AB5) was determined by SPR. The biological activity of the toxin was confirmed in Vero cells and mouse lethality assay. RESULTS: The yield of GST-StxA and His6X-StxB obtained after affinity chromatography was estimated to 2 and 5 mg/l, respectively. Samples analyzed in pull down assay revealed two bands of ~58 kDa (rStxA) and ~7.7 kDa (rStxB) on SDS-PAGE. Affinity was confirmed through SPR with KD of 0.85 pM. This rStx produced from 1:5 molar ratio found to be cytotoxic in Vero cell line and resulted lethality in mouse. CONCLUSIONS: Large scale production of rStx using the method can facilitate screening and evaluation of small molecule inhibitors for therapeutics development.

High level expression and immunochemical characterization of botulinum neurotoxin type F light chain.

Botulinum neurotoxins (BoNTs) are the most toxic biological substances known. Their potential use as biological warfare agent results in their classification as category A biowarfare agent by Centers for Disease Control and Prevention (CDC), USA. Presently, there are no approved detection system and pharmacological treatments for BoNT intoxication. Although a toxoid vaccine is available for immuno-prophylaxis, vaccines cannot reverse the effect of pre-translocated toxin. Direct handling of the live BoNTs for developing detection and therapeutics may pose fatal danger. This concern was addressed by purifying the recombinant catalytically active light chain of BoNT/F. BoNT/F-LC gene was amplified from the genomic DNA using specifically designed primers and expressed in Escherichia coli. Expression and purification profile were optimized under different conditions for biologically active light chain production. Specific polyclonal antibodies generated against type F illustrates in vivo neutralization in mice and rabbit. These antibodies play key role in conceiving the development of high throughput SPR based detection system which is a highly precise label free technique for protein interaction analysis. The presented work is first of its kind, signifying the production of highly stable and active rBoNT/F-LC and its immunochemical characterization. The study aids in paving the path towards developing a persistent detection system as well as in presenting comprehended scheme for in vitro small molecule therapeutics analysis.

In Silico Discovery and Validation of Amide Based Small Molecule Targeting the Enzymatic Site of Shiga Toxin.

Shiga toxin (Stx), a category B biothreat agent, is a ribosome inactivating protein and toxic to human and animals. Here, we designed and synthesized small molecules that block the active site of the Stx A subunit. On the basis of binding energy, 20 molecules were selected for synthesis and evaluation. These molecules were primarily screened using fluorescence-based thermal shift assay and in vitro in Vero cells. Among 32 molecules (including 12 reported), six molecules offered protection with IC50 of 2.60-23.90 µM. 4-Nitro-N-[2-(2-phenylsulfanylethylamino)ethyl]benzamide hydrochloride is the most potent inhibitor with IC50 at 7.96 µM and selectivity index of 22.23 and is better than any known small molecule inhibitor of Stx. Preincubation with Stx offered full protection against Shiga toxin in mice. Surface plasmon resonance assay further confirmed that these molecules bind specifically to Stx A subunit. Further optimization is continued to identify a potential candidate which will be in vivo effective.

Small-molecule quinolinol inhibitor identified provides protection against BoNT/A in mice.

Botulinum neurotoxins (BoNTs), etiological agents of the life threatening neuroparalytic disease botulism, are the most toxic substances currently known. The potential for the use as bioweapon makes the development of small-molecule inhibitor against these deadly toxins is a top priority. Currently, there are no approved pharmacological treatments for BoNT intoxication. Although an effective vaccine/immunotherapy is available for immuno-prophylaxis but this cannot reverse the effects of toxin inside neurons. A small-molecule pharmacological intervention, especially one that would be effective against the light chain protease, would be highly desirable. Similarity search was carried out from ChemBridge and NSC libraries to the hit (7-(phenyl(8-quinolinylamino)methyl)-8-quinolinol; NSC 84096) to mine its analogs. Several hits obtained were screened for in silico inhibition using AutoDock 4.1 and 19 new molecules selected based on binding energy and Ki. Among these, eleven quinolinol derivatives potently inhibited in vitro endopeptidase activity of botulinum neurotoxin type A light chain (rBoNT/A-LC) on synaptosomes isolated from rat brain which simulate the in vivo system. Five of these inhibitor molecules exhibited IC(50) values ranging from 3.0 nM to 10.0 µM. NSC 84087 is the most potent inhibitor reported so far, found to be a promising lead for therapeutic development, as it exhibits no toxicity, and is able to protect animals from pre and post challenge of botulinum neurotoxin type A (BoNT/A).

Deletion mutant comprising 198 residues of BoNT/A toxin receptor binding domain retained gt1b binding property but failed to induce protective antibody response in a mouse model.

The most effective protection against toxin is inducing protective immune response through vaccination that will produce neutralizing antibodies. Antibodies will bind to and clear toxin from the circulation before it can enter nerve cells and block neurotransmission and can also be used for development of detection system. In the present study we constructed a deletion mutant of the binding domain (1098-1296) to produce smallest toxin fragment as vaccine candidate against BoNT/A. The BoNT/A-HCC protein was highly expressed in Escherichia coli SG13009 and found to form inclusion bodies. The purified inclusion bodies were solubilized in 6 M guanidine-HCl containing 10 mM ß-mercaptoethanol and 20 mM imidazole and the rBoNT/A-HCC was purified and refolded in a single step on Ni2+ affinity column. The purified protein was ∼98 % pure as assessed by SDS-polyacrylamide gel with the yield of 8 mg/L and showed binding to polysialoganglioside (GT1b). The rBoNT/A-HCC at dose of 40 µg/mouse generated high IgG antibody titre with predominance of IgG1 subtype, but failed to protect animals against BoNT/A challenge. Antibody titre in serum was determined by enzyme linked immunosorbent assay and specific binding to rBoNT/A-HCC was demonstrated by surface plasmon resonance (SPR), with a dissociation constant of 0.8 pM.

Recombinant Shiga toxin B subunit elicits protection against Shiga toxin via mixed Th type immune response in mice.

Shigella dysenteriae is the causative agent of the third commonest bacterial disease for childhood diarrhoea and responsible for millions of deaths per year. It produces potent toxin termed Shiga toxin which is listed in category B biological warfare agent of CDC, USA. Earlier we have reported production of recombinant Shiga toxin B subunit that produced antibodies which neutralized Shiga toxin toxicity in HeLa cells. In the present study, we have evaluated the immunomodulatory potential of rStxB protein in Balb/c mice using Freunds adjuvants. Animal protection with recombinant StxB was conferred through both humoral and cellular immune responses as indicated by an increased antibody titre with predominance of IgG2a and IgG2b isotypes along with elevated levels of IgG1 subtype. Cytokine profile of the mice antiserum and splenocyte also indicates Th2 and Th1 type of immune responses where former dominates in early stage of immunization. Histopathology study of kidneys of vaccinated mice showed remarkable differences when compared to the animals infected with Shigella dysenteriae type1. The present study indicates that recombinant StxB is a promising vaccine candidate and can be used for production of therapeutic antibodies to counter Shiga intoxication.

Prevention of aggregation and autocatalysis for sustaining biological activity of recombinant BoNT/A-LC upon long-term storage.

Protein aggregation during expression, purification, storage, or transfer into requisite assay buffers hampers the use of proteins for in vitro studies. The formation of these aggregates represents a major obstacle in the study of biological activity and also restricts the spectrum of protein products being available for the biomedical applications. The catalytic light chain of botulinum neurotoxin type A undergoes autocatalysis and aggregation after purification upon long-term storage and freeze-thawing. In present study the conditions for the high level expression and purification of biologically active light chain protein of botulinum neurotoxin were optimized from a synthetic gene. Several co-solvents were screened in order to prevent autocatalysis and aggregation of rBoNT/A-LC. The effect of the co-solvents is studied on endopeptidase activity during long term storage of the recombinant protein. The purified rBoNT/A-LC was also evaluated for its immunogenicity.

Characterization of LC-HCC fusion protein of botulinum neurotoxin type A.

Botulinum neurotoxins (BoNTs) are highly potent toxins that inhibit neurotransmitter release from peripheral cholinergic synapses. The gene for encoding the full length light chain with H(CC) (binding) domain of Clostridium botulinum neurotoxin A was synthesized and cloned into a bacterial expression vector pQE30-UA and produced as an N-terminally six-histidine-tagged fusion protein (rBoNT/A LC-H(CC)). This protein was expressed in two different strains of Escherichia coli namely BL21(DE3) and SG13009. Expression at 37 °C revealed localization of rBoNT/A LC- H(CC) in inclusion body whereas it was expressed in soluble form at 21°C. The recombinant fusion protein was purified by nickel affinity gel column chromatography and identified by monoclonal antibody and peptide mass fingerprinting. The recombinant protein was shown to bind with synaptic vesicles and gangliosides (GT1b) using enzyme-linked immunosorbent assay. The rBoNT/A LC-H(CC) was also found to be highly active on its substrate (SNAP-25) from rat brain, indicating that the expressed and purified rBoNT/A LC-H(CC) protein retains a functionally active conformation. Biologically active recombinant fusion protein was also evaluated for its immunological potential.

Botulinum toxin: bioweapon & magic drug.

Botulinum neurotoxins, causative agents of botulism in humans, are produced by Clostridium botulinum, an anaerobic spore-former Gram positive bacillus. Botulinum neurotoxin poses a major bioweapon threat because of its extreme potency and lethality; its ease of production, transport, and misuse; and the need for prolonged intensive care among affected persons. A single gram of crystalline toxin, evenly dispersed and inhaled, can kill more than one million people. The basis of the phenomenal potency of botulinum toxin is enzymatic; the toxin is a zinc proteinase that cleaves neuronal vesicle associated proteins responsible for acetylcholine release into the neuromuscular junction. As a military or terrorist weapon, botulinum toxin could be disseminated via aerosol or by contamination of water or food supplies, causing widespread casualties. A fascinating aspect of botulinum toxin research in recent years has been development of the most potent toxin into a molecule of significant therapeutic utility . It is the first biological toxin which is licensed for treatment of human diseases. In the late 1980s, Canada approved use of the toxin to treat strabismus, in 2001 in the removal of facial wrinkles and in 2002, the FDA in the United States followed suit. The present review focuses on both warfare potential and medical uses of botulinum neurotoxin.

Biomethanation under psychrophilic conditions.

The biomethanation of organic matter represents a long-standing, well-established technology. Although at mesophilic and thermophilic temperatures the process is well understood, current knowledge on psychrophilic biomethanation is somewhat scarce. Methanogenesis is particularly sensitive to temperature, which not only affects the activity and structure of the microbial community, but also results in a change in the degradation pathway of organic matter. There is evidence of psychrophilic methanogenesis in natural environments, and a number of methanogenic archaea have been isolated with optimum growth temperatures of 15-25 °C. At psychrophilic temperatures, large amounts of heat are needed to operate reactors, thus resulting in a marginal or negative overall energy yield. Biomethanation at ambient temperature can alleviate this requirement, but for stable biogas production, a microbial consortium adapted to low temperatures or a psychrophilic consortium is required. Single-step or two-step high rate anaerobic reactors [expanded granular sludge bed (EGSB) and up flow anaerobic sludge bed (UASB)] have been used for the treatment of low strength wastewater. Simplified versions of these reactors, such as anaerobic sequencing batch reactors (ASBR) and anaerobic migrating blanket reactor (AMBR) have also been developed with the aim of reducing volume and cost. This technology has been further simplified and extended for the disposal of night soil in high altitude, low temperature areas of the Himalayas, where the hilly terrain, non-availability of conventional energy, harsh climate and space constraints limit the application of complicated reactors. Biomethanation at psychrophilic temperatures and the contribution made to night-soil degradation in the Himalayas are reviewed in this article.