Stray dogs in Bangkok, Thailand: rabies virus infection and rabies antibody prevalence.

In Thailand, the animal most reported rabid is the stray dog. Dog bite related rabies cases in humans account for 70-95% of rabies related deaths. The reported incidence of dog bites is highest in the central part of the country, especially in Bangkok. This epidemiological survey shows that at least five different canine rabies virus types are present in Bangkok. Rabies antigen and antibody prevalence in stray dogs in Bangkok was also investigated. Saliva and serum samples were taken from 3,314 stray dogs, captured between December 2003 and June 2004. One two year-old female was antigen positive by latex agglutination test and the result was confirmed by reverse transcription-polymerase chain reaction (RT-PCR). The overall antibody seroprevalence by enzyme-linked immunosorbent assay was 62% (95% CI: 54, 70%). Antibody seroprevalence was higherfor dogs captured within central Bangkok (86% of 1,208 dogs captured) than in the dogs captured on the outskirts of the greater metropolitan area (49% of 2,106 dogs captured). If our sample of stray dogs is representative, then the seroprevalence achieved from previous vaccination campaigns is insufficient in order to break the rabies transmission cycle among stray and feral dogs.

One or three intradermal injections within one week for rabies pre-exposure immunization.

Pre-exposure rabies vaccination is recommended fortravellers to endemic countries and forselected populations in highly endemic regions. It consists of three injections administered over 3-4 weeks. Travellers often ignore or do not have enough time to receive a complete course prior to departure or leave with only one or two injections. In this study, the secondary antibody response in volunteers who received one or three injections within one week is evaluated. Results are compared to the recommended three injection regimen when administered with two booster vaccinations on days 0 and 3 one year later. Long-lasting immune memory in volunteers who received only one time vaccination of 0.1 ml (1 site) intradermally and then received two booster vaccinations of 0.1 ml (1 site) intradermally on days 0 and 3, one or three years later is also reported. All volunteers developed an accelerated and adequate neutralizing antibody response within seven days after they received two boosters. These data suggest that three injections within one week are as immunogenic as three injections within four weeks. One clinic visit with two-site 0.1 ml ID injections or a one-site single 0.1 ml pre-exposure rabies vaccination is enough to prime the host immune memory for at least one to three years.

Ten-day observation of live rabies suspected dogs.

This study aimed at analyzing a ten-day observation period of rabies suspected dogs and cats according to six criteria. Dogs and cats suspected of being rabid were brought for observation when they had either bitten a person or another animal or when abnormal behaviour or unusual illness was observed. Between 1985 and 2005, retrospective and prospective data from 1,222 dogs and 303 cats was collected during the ten-day observation period. If an animal had died, brain examination using fluorescent antibody testing was routinely performed. If an animal had survived for > or =10 days, it was released to its owner or transferred to the municipal dog shelter. A total of 644 dogs and 58 cats found rabid died within 10 days of observation. In addition, for 208 dogs confirmed rabid with laboratory tests between 1997 and 2005, six criteria were analysed from the day of submission. This experience with the implemented 10-day observation period confirms the WHO recommendation on identifying suspected rabid dogs or cats under veterinary supervision following a human exposure.

Thiazide-induced subtle renal injury not observed in states of equivalent hypokalemia.

Hydrochlorothiazide (HCTZ) is used to manage hypertension and heart failure; however, its side effects include mild hypokalemia, metabolic abnormalities, and volume depletion, which might have deleterious effects on renal and endothelial function. We studied whether HCTZ cause renal injury and/or altered vasoreactivity and if these changes are hypokalemia-dependent. Rats were given a normal diet or a diet moderately low in potassium K+ with or without HCTZ. Animals fed either a low K+ diet alone or HCTZ developed mild hypokalemia. There was no significant difference in systolic blood pressure in the different treatment groups. All three groups with hypokalemia had mild proteinuria; low K(+)-HCTZ rats had reduced creatinine clearance. HCTZ-treated rats displayed hypomagnesemia, hypertriglyceridemia, hyperglycemia, insulin resistance, and hyperaldosteronism. No renal injury was observed in the groups without HCTZ; however, increased kidney weight, glomerular ischemia, medullary injury, and cortical oxidative stress were seen with HCTZ treatment. Endothelium-dependent vasorelaxation was reduced in all hypokalemic groups and correlated with reduced serum K+, serum, and urine nitric oxide. Our results show that HCTZ is associated with greater renal injury for the same degree of hypokalemia as the low K+ diet, suggesting that factors such as chronic ischemia and hyperaldosteronism due to volume depletion may be responsible agents. We also found impaired endothelium-dependent vasorelaxation was linked to mild hypokalemia.

Sex- and age-related differences in rabies immunoglobulin hypersensitivity.

We retrospectively reviewed 72132 patients who had received rabies immunoglobulin between 1987 and 2005 at the Queen Saovabha Memorial Institute, Bangkok. Purified equine rabies immunoglobulin (ERIG) was given to 42965 (59.56%) patients and human rabies immunoglobulin (HRIG) to 29167 (40.44%) patients. A total of 812 patients from both groups (1.13%) reported adverse reactions; among those who had received ERIG, 43.13% were male and 56.87% were female, and among those who had received HRIG, 34.62% were male and 65.38% were female. Females were at higher risk of exhibiting ERIG or HRIG hypersensitivity than males (P<0.01). None of the reactions was life-threatening. Serum sickness-like reactions to ERIG and HRIG were rare under the age of 10 years (0.05 and 0.01% among recipients in that age group).

Prevalence of rabies virus infection and rabies antibody in stray dogs: a survey in Bangkok, Thailand.

To investigate the rabies antigen and antibody prevalences among stray dogs in Bangkok, Thailand, we took both a saliva and serum sample from each of 3314 stray dogs captured once each between December 2003 and June 2004. One 2-year-old female was antigen positive in the latex-agglutination test and confirmed by reverse transcription-polymerase chain reaction. The overall antibody seroprevalence from the enzyme-linked immunosorbent assay that we used was 62% (95% CI: 54, 70%). Antibody seroprevalence was greater for dogs captured within central Bangkok (86% of 1208 dogs captured) than in the dogs captured in the outskirts of the greater metropolitan area (49% of 2106 dogs captured). If our samples of stray dogs are representative, then the seroprevalence achieved from previous vaccination campaigns is too low to protect the dog and human populations.

Survival of neutralizing antibody in previously rabies vaccinated subjects: a prospective study showing long lasting immunity.

Physicians dealing with potential rabies exposures and travel medicine are frequently asked how long previous pre- or post-exposure rabies vaccination induced immunity persists. We therefore carried out a prospective study on 118 rabies vaccine recipients who had received pre- or post-exposure regimens with tissue culture rabies vaccines by intramuscular or intradermal schedules 5-21 years previously. Rabies neutralizing antibody was detectable in the sera of all subjects on day 0. They then received one intradermal 0.1 mL booster injection on days 0 and 3. Neutralizing antibody determination was carried out on days 5, 7 and 14. All except one subject showed an accelerated antibody response following the two booster injections. Vaccination with a WHO recognized tissue culture rabies vaccine evokes long lasting immunity. This study supports current recommendations that immunity is long lasting and that boosters without immunoglobulin are sufficient even when prior vaccination was longer than 5 years previously.

Renal function following sea snake venom (Lapemis hardwicki) administration in dogs treated with sodium bicarbonate solution.

The effects of sea snake venom (SSV) on renal function were studied in two groups of anesthetized experimental dogs pretreated with intravenous infusion of 4.2 gm% NaHCO3 solution. Animals were envenomated by intramuscular injection of SSV at a dosage of 0.34 mg/kg. Systemic hemodynamics showed no significant changes except for a tendency of decrease in cardiac output (CO). The glomerular filtration rate (GFR), the rate of urine flow (V) and effective renal plasma flow (ERPF), and effective renal blood flow (ERBF) significantly decreased, while filtration fraction (FF) significantly increased at 180 min after envenomation. Envenomated animals showed a reduction in renal fraction (RF), while renal vascular resistance (RVR) increased stepwise throughout the experimental periods. Animals pretreated with sodium bicarbonate showed no significant changes of CO, TPR MAP, HR, and packed cell volume (PCV) while receiving sea snake venom. Animals pretreated with sodium bicarbonate showed no changes in GFR, ERPF, ERBF, RF, and RVR after envenomation. The rate of urine flow markedly increased in envenomated animals which received pretreatment with bicarbonate. After envenomation alone, there were no differences in the plasma concentration of sodium (PNa) and chloride (PCl) as compared to the control value, whereas the plasma concentration of potassium (PK) increased at 180 min after envenomation. Animals pre-treated with bicarbonate showed a stepwise increase in both UNaV, FE(NA), U(Cl)V, and FE(Cl) accompanying SSV injection. Neither PNa nor PCl were affected, while PK significantly decreased in animals given SSV with bicarbonate loading. UKV and FEK increased stepwise in envenomated animals treated with bicarbonate throughout the period of study. All groups of animals given SSV, with or without NaHCO3 infusion, showed a marked elevation of the concentration of urinary myoglobin (U(Mb)), plasma lactate dehydrogenase (LDH), and plasma creatine phosphokinase (CPK) throughout experimental periods. The urinary myoglobin excretion markedly increased in animals after SSV injection accompanied by NaHCO3 infusion. It can be concluded that large amounts of myoglobin present in the renal tubules in envenomated animals can precipitate, particularly under acidic conditions, resulting in increased intratubular pressure and subsequently decreased renal hemodynamics including GFR and ERBF. An infusion of NaHCO3 to render urine more alkaline could have a protective role against depression of renal function following sea snake venom administration.

A hemorrhagin as a metalloprotease in the venom of Trimeresurus purpureomaculatus: purification and characterization.

A major hemorrhagin was purified from the venom of the Thai green pit viper (Trimeresurus purpureomaculatus) by gel filtration, ion-exchange and affinity chromatography. A 15-fold purification was achieved with an overall yield of 7% of hemorrhagic activity. The hemorrhagin was homogeneous according to disc- and SDS-PAGE as well as on immunodiffusion. The molecular weight determined by SDS-PAGE was 72kDa. The purified hemorrhagin expresses proteolytic activity with heat-denatured casein and hide powder azure, but it was free of AE-hydrolase and phospholipase activities. Both hemorrhagic and proteolytic activities were inhibited by EDTA, suggesting that the hemorrhagin is a metalloprotease. The hemorrhagin hydrolyzed all gelatin preparations derived from types I, II, III and IV collagen, whereas it hydrolyzed only type IV native collagen. The hemorrhagic activity was neutralized by Thai green pit viper antivenom raised to Trimeresurus albolabris venom.

Isolation of the major lethal toxin in the venom of Bungarus flaviceps.

The major lethal toxin in the venom of Bungarus flaviceps has been isolated by ion-exchange chromatography, absorption chromatography and RP-HPLC with a 14-fold purification and an overall yield of 16.5% of the lethal toxicity contained in crude venom. Its sublethal dose (LD(50)) determined in mice weighing 18-20 g was 0.25 (0.19-0.32) microg per mouse. The lethal toxin was pure according to disc- and SDS-PAGE as well as gel HPLC. Its apparent molecular weight determined by SDS-PAGE was 29 kDa. It is a basic protein consisting of two polypeptide chains having apparent molecular weights of 17 and 8 kDa, respectively. The toxin has PLA activity but is free of ACE activity.

Cross neutralization of the lethal activities of Myanmar and Thai Russell's viper venoms by Thai and Myanmar antivenoms.

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.

Three-year experience with 4-site intradermal booster vaccination with rabies vaccine for postexposure prophylaxis.

For booster vaccination of previously immunized persons with potential exposure to rabies, the World Health Organization recommends 2 doses of cell-culture vaccine administered intramuscularly or intradermally on days 0 and 3. We believe that four 0.1-mL intradermal booster doses given on a single day could be used at no risk to the recipient. We studied use of a single booster vaccination on day 0 followed by four 0.1-mL intradermal doses of cell-culture rabies vaccines, and we determined that this is a safe, convenient, and economical regimen for postexposure treatment of previously vaccinated individuals.

Immunosuppression in rabies virus infection mediated by lymphocyte apoptosis.

Previously, we demonstrated a depression of cell-mediated immunity in mice by street rabies virus infection. In the present study, we investigated several events during the course of infection and looked for alterations in the host lymphoid cells for evidence of apoptosis. Total cellular RNA was extracted from muscle tissues at the inoculation site of peripherally infected mice at different intervals after infection. Rabies virus mRNA was monitored by reverse transcription-PCR. The length of virus localization at the site of exposure in the muscle was as long as 5 days post-inoculation before the virus entered the central nervous system. At this inoculation site, the virus disappeared transiently between days 7 and 9 after infection but then was restored thereafter until death. Annexin V-fluorescein isothiocyanate staining of splenocytes and thymocytes from mice revealed apoptotic changes in these cells with a marked increase after day 6 of infection. Rabies virus antigen in the brain became detectable 6 days after infection; this occurred parallel to the appearance of apoptosis in the lymphoid cells. There was atrophy of the spleen and thymus, with no evidence of infection. Our results suggest that the interaction between the rabies virus and infected neurons triggers the process of lymphoid cell apoptosis, which reflects the defective operation of the immune system.

Failure of multiple-site intradermal postexposure rabies vaccination in patients with human immunodeficiency virus with low CD4+ T lymphocyte counts.

Human immunodeficiency virus (HIV)-infected patients with low CD4(+) T lymphocyte counts had a poor neutralizing antibody response to pre- and postexposure rabies vaccination. This study of HIV-infected patients with CD4(+) T lymphocyte counts < 200/microL indicated that patients had a poor response after 4-site intradermal vaccinations (4-4-4-0-2-2, doubling the intradermal doses of cell-culture rabies vaccine).

Potassium status of Northeast Thai constructors in three different geographic locations.

Sudden and unexpected death of young adults during sleep is a phenomenon among Southeast Asians and particularly young Northeast (NE) Thailand constructors in Singapore. Survivor of sudden unexplained death syndrome (SUDS) without structural heart disease with idopathic ventricular fibrillation (VF) has been documented. Low plasma potassium (K) and depletion of K can occur simply through a reduction of K intake and are associated with increased risk of VF. The K-status of the populations was evaluated in the NE (Group 1, n=30), Bangkok (Group 2, n=48) and Singapore (Group 3, n=46). Groups 2 and 3 were further subdivided into Group 2A (worked in Bangkok < or = 1 year, n=8), Group 2B (worked in Bangkok > 1 year, n=40), Group 3A (consumed self-prepared or ready-to-buy meals, n=25) and Group 3B (regularly consumed foods provided free-of-charge by construction companies, n=21). Thirty-four male healthy university personnels from the NE and Bangkok served as the control--Group 4. Two 24-h urine samples and a fasting blood sample were collected from each subject. Dietary-K from food was determined by duplicated meal analysis. All these samples were then analyzed for their K-content. Group 3A had the lowest K-status: their K-intake, serum-K, and urinary-K level were 29 +/- 5.8 mmol/day (% low K-intake=100), 3.43 +/- 0.34 mmol/L (% hypokalemia=48) and 19.23 +/- 8.2 mmol/day (% hypokaliuria=87.5), respectively. Among the construction workers, average K-intake, serum-K and urinary-K levels were 45.5 +/- 6.1 mmol/day (% low K-intake = 37.5), 3.93 +/- 0.2 mmol/L (% hypokalemia = 2.5) and 39.6 +/- 9.2 mmol/day (% hypokaliuria = 12.5), respectively. The values of Group 2B were similar to Group 4. In addition, when the data from all of the groups were compared, there was a positive correlation between dietary-K (intake) and urinary-K (excretion) (r=0.881, p<0.001). In conclusion, NE Thailand constructors from various locations exhibited low K status with low dietary-K, high incidence of hypokalemia, and low urinary-K. From the present study, this low K status may be an important trigger factor for VF in construction workers and associated with increase risk of SUDS.

Effects of Russell's viper venom on mediator production in cultured human umbilical vein endothelial cells.

Hemodynamic alterations in Russell's viper envenomation are the result of interactions of various vasoactive mediators and perhaps proinflammatory cytokines. Since vascular endothelium is likely to be exposed to high concentrations of the venom and the endothelial cell itself not only plays an important role in the physiologic control of the circulation, but also play a role in inflammation with the synthesis and secretion of proinflammatory cytokines. It was therefore, the objective of this study to determine the effects of Russell's viper venom (RVV) on proinflammatory cytokine production by cultured human umbilical vein endothelial cells (HUVEC) and the release of endothelium-derived substances. Endothelial cells were isolated from freshly obtained human umbilical cord vein and grown in tissue culture to confluence as a homogeneous population. Cells were then incubated at 37 degrees C under 5 per cent CO2 with RVV (0.2, 1.0, 5.0, and 25.0 microg/ml) or lipopolysaccharide (LPS, 10 microg/ml) for 3, 6, 12 and 24 hours. After an indicated time, the levels of endothelin-1 (ET-1); 6-keto-PGF1alpha (a stable metabolite of PGI2) tumor necrosis factor-alpha (TNF-alpha); interleukin-1beta (IL-1beta); and interleukin-6 (IL-6) in supernatants were measured by using ELISA or EIA. The effect of RVV or LPS on cell viability was also measured using MIT assay. The results showed copious amounts of ET-1 production irrespectively with the presence of RVV or LPS. Whereas, production of PGI2 (measured as 6-keto-PGF1alpha, a stable metabolite) was increased significantly higher in the RVV- and LPS-treated EC than in the control EC. However, TNF-alpha and IL-6 productions were not different among these groups. The levels of IL-1beta were very low, although IL-1beta was detectable in the group treated with RVV at a concentration of 25.0 microg/ml. In conclusion, RVV upto 25 microg/ml stimulated PGI2 production by cultured HUVEC. This effect was unlikely related to production of proinflammatory cytokines since LPS or RVV is not sufficient per se to elevate a substantial amount of EC-derived cytokines. The higher amount of IL-6 compared to TNF-alpha and IL-1beta may be produced through other pathways apart from production via a cascade of cytokines. This is the first report showing that RVV up to 25 microg/ml has no effect on prominent proinflammatory cytokine production by HUVEC. However, in blood circulation, the major source of cytokines production is monocyte-macrophage lineage cell. Thus, RVV in blood circulation may activate the production of proinflammatory cytokines mainly from those cells and subsequently induce toxicity.

Molecular cloning of phospholipase A2 from a Thai Russell's viper venom gland cDNA library.

Snake venom contains several toxins. Russell's viper (D. russellii, RV) is a venomous snake prevalent in northern and central Thailand. RV bites can cause disseminated coagulation, hemolysis, and edema of the bitten limbs. To identify protein components of RV venom, we made a cDNA library from RV venom glands, and randomly sequenced cloned cDNA. We were able to clone a cDNA encoding RV phospholipase A2 (PLA2). PLA2 is an active enzyme found in several species of snake venom worldwide. PLA2 is thought to be toxic to cell membrane, thereby, can cause local cell and tissue damage, as well as systemic effects in snake bite victims. This PLA2 cDNA clone would facilitate in vivo studies of the pathophysiology of RV bite.

Effectiveness of Thai cobra (Naja kaouthia) antivenom against sea snake (Lapemis hardwickii) venom: verification by affinity purified F(AB')2 fragments.

Commercial Thai cobra (Naja kaouthia) antivenom was found to be effective in neutralizing sea snake (Lapemis hardwickii) venom. Neurotoxin specific F(ab')2 fragments obtained from the antivenom by chromatography using Thai cobra neurotoxin or sea snake venom affinity columns were able to neutralize both venoms.

Decreased parasympathetic activities in Malayan krait (Bungarus candidus) envenoming.

Three patients were bitten by the Malayan krait (Bungarus candidus). The patients developed ptosis and generalized muscle weakness which later progressed to respiratory paralysis. All patients showed evidence of decreased parasympathetic activity manifested by mydriasis, hypertension and tachycardia. No specific antivenom was available. All patients received assisted ventilation and supportive treatment. The other forms of treatment included administration of neostigmine, the banded krait (Bungarus fasciatus) antivenom (Thai Red Cross) and plasmapheresis without beneficial response. Two patients recovered. The other patient had permanent brain damage due to anoxia from two episodes of cardiac arrest. While hypertension resolved 6-60 days after admission, mydriasis and tachycardia persisted after discharge in all patients for between 7 days and 2 years. One patient had constipation and defect in micturition which still persisted 2 years after the bite. Decreased parasympathetic activities in Malayan krait bite are perhaps not uncommon and should be examined.