Background: Bacillus Calmette-Guerin (BCG) Tokyo 172-1 strain has been used as a parent strain since 1988 by Queen Saovabha Memorial Institute, Thai Red Cross Society to manufacture BCG vaccine in Thailand. In general, the BCG Tokyo strain includes 2 BCG type populations: types I and II. Several studies have found that the type I population is higher than type II. Polymerase chain reaction has shown the presence of 2 types of subpopulations in the BCG Thai Red Cross Society (TRCS) strain. However, the characteristics of all genes from BCG TRCS have not been studied in depth, including whether or not the original features of the BCG Tokyo 172-1 strain are still retained. Objectives: To compare BCG TRCS and BCG Tokyo 172-1 for differences between their genomes. Methods: The whole genome of BCG TRCS was analyzed by next-generation sequencing. Results: BCG TRCS contained 23 different points and was 4 bases less in length than BCG Tokyo 172-1. However, its main features remain similar to those of the original BCG Tokyo 172-1 strain. Conclusions: The changes in the genome may be caused by a variety of factors including the strain of Tokyo 172, vaccine processing, storage, and natural changes in the genome.

This study evaluates the in vitro anti-snake venom potential of Peristrophe bivalvis (PB) extracts against Naja kaouthia (NK) and Trimeresurus albolabris (TA) venoms, including inhibition of cytotoxic effects and enzymatic activities, and the binding-precipitation of extracts and venom proteins analysis. In addition, the antioxidant, cytotoxic and in vivo acute oral toxic activities of PB extracts are also reported. The in vitro cytotoxic and enzymatic analysis reveals that the ethanol extracts of stems and leaves of PB showed good anti-snake venom activity against NK and TA venoms. In addition, the antioxidant result indicated that only the ethanol extract of leaves exhibited weak DPPH radical-scavenging activity. The ethanol whole-plant extract of PB also showed no cytotoxicity against four cell lines. Moreover, the in vivo acute oral toxicity result of the ethanol whole-plant extract showed that all treated rats did not exhibit abnormal toxic signs or deaths.

Detection of Mycobacterium tuberculosis (MTB) infection by delayed-type hypersensitivity skin test with purified derivative (PPD) antigen is still used in many countries including Thailand. However, it is less specificity due to significant cross-reactivity with non-tuberculosis mycobacteria. Antigen such as early secretory antigenic target (ESAT6) is secreted in the early phase of infection and elicits cell mediated immune responses. Moreover it is found only in M. tuberculosis, M. bovis and few other mycobacterial species. ESAT-6 protein can be a good candidate for MTB specific diagnosis. Here, we report the cloning, expression, purification of ESAT-6 protein and its utility as specific antigen for MTB diagnosis by skin testing in guinea pig sensitized with killed M. tuberculosis and live BCG. Our results show that 200 mg of the purified ESAT-6 protein elicits a positive skin reactivity only in the guinea pigs sensitized with killed M. tuberculosis and not in the animals sensitized with live BCG. These data in this study support the use of ESAT-6 protein in specific detection of MTB infection.

The ability of monovalent IgG and F(ab')2 antivenoms to neutralize lethality, phospholipase A2, and coagulant activities induced by Daboia siamensis venom was studied.Both antivenoms were produced from the same batch of hyperimmune horse plasma and were adjusted to the same potency against the lethal effect of D. siamensis venom in experiments involving preincubation of venom and antivenom.Intact neutralization experiments involving independent injection of venom and antivenoms showed that the F(ab')2 antivenom was slightly more effective.Significant differences in favour of F(ab')2 antivenom were observed with respect to neutralization of phospholipase A2 and coagulant activities.Both IgG and F(ab')2 antivenoms were able to activate human complement in vitro.IgG antivenom had a significantly higher anticomplementary activity than F(ab')2 antivenom.

The protein concentration and protein pattern of crude venoms of three major haematotoxic snakes of Thailand, Cryptelytrops albolabris (green pit viper), Calloselasma rhodostoma (Malayan pit viper), and Daboia russelii siamensis (Russell's viper), were studied. The protein concentrations of all lots of venoms studied were comparable. The chromatograms, from reversed phase high performance liquid chromatography, of C. albolabris venom and C. rhodostoma venom were similar but they were different from the chromatogram of D. r siamensis venom. C. rhodostoma venom showed the highest number of protein spots on 2-dimensional gel electrophoresis (pH gradient 3-10), followed by C. albolabris venom and D. r siamensis venom, respectively. The protein spots of C. rhodostoma venom were used as reference proteins in matching for similar proteins of haematotoxic snakes. C. albolabris venom showed more similar protein spots to C. rhodostoma venom than D. r siamensis venom. The minimum coagulant dose could not be determined in D. r. siamensis venom.

Venom phospholipases A2 (PLA(2)) are associated with neurotoxic, myotoxic, cardiotoxic, platelet aggregation, and edema activities. A PLA(2) (Drs-PLA(2)) was purified from Daboia russelii siamensis venom by a two-step purification procedure consisting of size-exclusion, followed by anion exchange high performance liquid chromatography (HPLC). The molecular weight of the Drs-PLA(2) was 13,679Da, which was determined by MALDI-TOF mass spectrometry. Its N-terminal amino acid sequence was homologous to basic PLA(2)s of viperid snake venoms. The Drs-PLA(2) had indirect hemolytic and anticoagulant activities, cytotoxic activity with a CC(50) of 65.8nM, and inhibited SK-MEL-28 cell migration with an IC(50) of 25.6nM. In addition, the Drs-PLA(2) inhibited the colonization of B16F10 cells in lungs of BALB/c mice by ∼65%.

Malayan pit viper (Calloselasma rhodostoma) envenomation is a major health problem in South East Asia. During envenomation, venom components mainly affect the hemostatic system. The sera from the North American Virginia opossums (Didelphis virginiana) were able to neutralize the venom of the Malayan pit viper. These natural inhibitors could be explored as potential therapeutics against envenomations of a variety of venomous snake species in different geographical habitats.

The Malayan pit viper (Calloselasma rhodostoma) is a snake found in most of Southeast Asia. The snake's venom contains proteins with various biological effects. In this study, proteins from Malayan pit viper venom were analysed by electrophoresis titration (ET) and two dimensional gel electrophoresis (2-D gel). In addition, venom proteins were separated by high performance liquid chromatography (HPLC) connected to a hydrophobic interactive chromatography (HIC) column. Fractions collected from HPLC were tested for biological activities. As the result, the ET profile showed that crude venom consisted of both positively and negatively charged proteins. Most of the 191 protein spots found on 2-D gel of crude venom have an isoelectric point in the range 4.5-5.5. After HPLC, eighteen fractions were eluted from HIC column. Each fraction was tested for fibrinolytic, haemorrhagic, gelatinase, and disintegrin activities. Both fibrinolytic and haemorrhagic fractions showed gelatinase activity as well, while the fibrinolytic fraction had no haemorrhagic activity. Our results are valuable to venom research and drug discovery.

Green pit viper bite is a common public health problem in Southeast Asia. Although most patients experience only local swelling, some may suffer from severe systemic bleeding that can be delayed. Venom antigenaemia was measured by enzyme-linked immunosorbent assay and correlated with clinical findings in 42 patients. Initial venom antigenaemia was not predictive enough for clinical uses. A kinetic study (n = 27) showed highest levels at presentation and, then, progressive decline. The average half-life was 27.5 h during the first three days and over 50 h on days 5-7 after bite. Two small subsets (7.4% each) showed persistently detectable venom on day 14 and a subsequent rise in venom antigenaemia. They were associated with prolonged thrombocytopaenia and coagulopathy, respectively. These data demonstrated the long half-life of the venom, suggesting that waiting for spontaneous resolution of coagulopathy is not preferable. In addition, the delayed venom disappearance, not the initial values, was correlated with haemostatic disorders.

The in vitro venom neutralizing capacity of tannic acid against the activities of Naja kaouthia (Naja naja kaouthia Lesson [Elapidae]) venom was investigated. Tannic acid was found to be effective in neutralizing the activities of Naja kaouthia (NK) venom. The lethal effect of four-times the LD50 in mice and the necrotizing effect of one minimum necrotizing dose (I MND) in rats of NK venom were fully inhibited by tannic acid at >= 431 mu g/mouse, with the median effective dose (ED50) of 334 mu g/mouse and >= 30 mu g/rat, respectively. The acetylcholinesterase activity of NK venom was almost completely neutralized (>= 99%) by tannic acid at >= 1% w/v. It was evident that tannic acid was nontoxic and did not cause either a lethal effect in mice (at its maximum tested dose of 845 mu g/mouse) or necrotic lesions in rats (at doses between 7.5 and 60 mu g/rat).

The ethyl acetate extract of Eclipta prostrata L. (Asteraceae) was evaluated for its antivenom potential against Calloselasma rhodostoma Kuhl (Viperidae) (Malayan pit viper; MPV) venom. The partially purified ethyl acetate extract (PEE) was found to contain 47% wedelolactone as its major constituent. PEE and wedelolactone demonstrated strong antiproteolytic and antihemorrhagic activities against MPV venom in a dose-dependent manner. The extract, at 5 mg/mL, could inhibit proteolytic activity of 100 tg of the venom and hemorrhagic activity of 3 minimum hemorrhagic doses (MHD) to 95% and 68%, respectively. At the same concentration, wedelolactone could neutralize the proteolytic activity at around 76% and, at doses of 0.25-1 mg/mL, offered protection against hemorrhagic activity of the venom in the range 3-35%. Both PEE and wedelolactone displayed partial anti-phospholipase A(2) activity (21% for PEE and 7% for wedelolactone) and could not neutralize the lethal effect of either 2LD(50) or 4LD(50) of MPV venom.

Although systemic administration of antivenom can promptly reverse coagulopathy, efficacy on local effects of viper venom remains to be determined. Currently, there has been no proven specific treatment for snakebite patients with severe local effects. This study is a randomized, double-blind, placebo-controlled trial. Patients bitten by green pit vipers (Trimeresurus albolabris or T. macrops) with marked limb swelling, but no severe coagulopathy requiring antivenom, were randomized to receive either equine F(ab′)2 antivenom, or placebo. Twenty-eight cases were included, 14 in each group, and they had their limb circumferences measured on days 1, 2, 4 and 6 after interventions. The percentage reduction in limb circumference was significantly better in the antivenom group compared with the placebo group (ANOVA, P=0.03), especially in the first 24h (1.14 vs. 3.62%, in placebo and antivenom group, respectively, P=0.014). The reduction in pain score was similar. The plasma venom levels were not different at presentation but lower in the antivenom group 24h after intervention (P=0.033). These data suggest that intravenous antivenom could accelerate local oedema resolution in humans. However, the degree is not clinically significant, and, therefore, general use is not recommended.

Plant polyphenols from the aqueous extracts of Pentace burmanica, Pithecellobium dulce, Areca catechu and Quercus infectoria were tested for their inhibitory activities against Naja kaouthia (NK) venom by in vitro neutralization method. The first three extracts could completely inhibit the lethality of the venom at 4 LD50 concentration and the venom necrotizing activity at the minimum necrotizing dose while also inhibited up to 90% of the acetylcholinesterase activity of NK venom at much lower tannin concentrations than that of Quercus infectoria. The ED50 of plant tannins in inhibiting NK venom activities varied according to condensed tannins and their content in the extracts. Molecular docking of the complexes between alpha-cobratoxin and either hydrolysable or condensed tannins at their lowest energetic conformations were proposed. The anti-venom activities of these plant polyphenols by selectively blocking the nicotinic acetylcholine receptor and non-selectively by precipitation of the venom proteins were suggested.

The butanolic and purified butanolic extracts (PBEs) of Eclipta prostrata were evaluated for their anti-venom potential. Inhibition of lethal, hemorrhagic, proteolytic, and phospholipase A2 activities of Calloselasma rhodostoma (Malayan pit viper (MPV)) venom by these extracts were determined. Demethylwedelolactone was identified as their major constituent. The butanolic extract, at 2.5 mg per mouse, was able to completely neutralize the lethal activity of 2LD50 of MPV venom, but increasing the dose diminished the effect. The PBE, at 1.5-4.5 mg per mouse, was able to neutralize the lethality of the venom at around 50-58%. Both extracts partially inhibited the hemorrhagic activity but displayed very low anti-phospholipase A2 activity and did not inhibit proteolytic activity of MPV venom.

Antivenom cross‐reactions have been studied using immunologic techniques, immunodiffusion, immunoelectrophoresis, immunoblotting and ELISA. Cross precipitation does not necessarily mean that there is cross protection. However, a cross protection is generally observed between closely related species. Cross‐reactivity of monovalent antivenoms and polyvalent antivenoms among various venoms are described. The results strongly suggest the presence of genus specificity in each antivenom. Cross‐reactions of antivenoms are difficult to foretell and they necessitate individual verification. Knowledge of cross‐reactivity of antivenoms is a very important tool to identify for phylogenic relationships of snake species and variation in venoms, and to be a guide for effective treatment of snake bite.

The efficacy of homologous neutralization of the lethal activity of Myanmar Russell's viper venom (MRV) and Thai Russell's viper venom (TRV) by Myanmar antivenom (MAV) and Thai antivenom (TAV), respectively, were studied and compared with the heterologous neutralization of the lethal activity of MRV and TRV by TAV and MAV, respectively, in experimental mice. Although MRV and TRV were the same subspecies, their lethal activities and protective efficacy of MAV and TAV were apparently different from each other. However, there was some extent of cross reactivities between MRV and TAV, and TRV and MAV. The findings of this study suggested that it is necessary to administer heterologous antivenom in dosage of 1.7-4 times greater than that of homologous antivenom to achieve an equal potency.

A PCR technique was used in this study to identify and distinguish monocellate cobra snake bites using snake venoms and swab specimens from snake bite-sites in mice from bites by other common Thai snakes. The sequences of nucleotide primers were selected for the cobrotoxin-encoding gene from the Chinese cobra (Naja atra) since the sequences of monocellate cobra (Naja kaouthia) venom are still unknown. However, the 113-bp fragment of cDNA of the cobrotoxin-encoding gene was detected in the monocellate cobra venom using RT-PCR. This gene was not found in the venoms of Ophiophagus hannah (king cobra), Bungarus fasciatus (banded krait), Daboia russelii siamensis (Siamese Russell's Viper, and Calloselasma rhodostoma (Malayan pit viper). Moreover, direct PCR could detect a 665-bp fragment of the cobrotoxin-encoding gene in the monocellate cobra venom but not the other snake venoms. Likewise, this gene was only observed in swab specimens from cobra snake bite-sites in mice. This is the first report demonstrating the ability of PCR to detect the cobrotoxin-encoding gene from snake venoms and swab specimens. Further studies are required for identification of this and other snakes from the bite-sites on human skin.