Studies were performed on a cytotoxin (CT) from human strains of Campylobacter jejuni isolated in Malaysia. CT was detected by cytopathic effect (CPE) on HeLa cells at titres from 8 to 32, in culture filtrates from 14 (48%) of 29 human isolates. The CPE correlated well with a quantitative 51Cr-release assay where a specific release of 54-68% was noted. CT production was lost after 5-7 subcultures. CT activity was also detected in 5 (26%) of 19 faecal filtrates from which CT-producing isolates were subsequently obtained. The mol. wt of CT was estimated by Sephadex G-50 chromatography to be greater than 30,000. In a suckling-mouse assay, CT consistently failed to demonstrate fluid accumulation after intragastric inoculation of culture filtrate. The Removable Intestinal Tie Adult Rabbit Diarrhoea (RITARD) assay was also used. Rabbits given CT-producing strains of C. jejuni developed bacteraemia and severe watery mucus-containing diarrhoea for the duration of the experiment with death of some animals. Rabbits given CT non-producing strains had less severe disease and none died. Rabbits given partially-purified CT had diarrhoea for 3 days but none died.
1. The major phospholipase A2 (PLA-DE4) of the venom of Trimeresurus purpureomaculatus (shore pit viper) has been purified to electrophoretic homogeneity. 2. The isoelectric point of the purified enzyme was determined to be 4.20, and the mol. wt was 31,700 as estimated by Sephadex G-75 gel filtration chromatography; and 14,000 as estimated by SDS-polyacrylamide gel electrophoresis. The purified enzyme hydrolyzed phosphatidylcholine (PC) faster than phosphatidylethanolamine (PE), whereas phosphatidylserine (PS) was not hydrolyzed at all (PC greater than PE greater than PS =0). However, in reaction system consisted of mixtures of PC and PS, phosphatidylserine was effectively hydrolyzed by the enzyme. 4. The phospholipase A2 exhibited edema-forming activity but not hemolytic, hemorrhagic or anticoagulant activities. It was not lethal to mice at a dosage of 10 micrograms/g by i.v. route.
Trimeresurus wagleri (speckled pit viper) venom exhibited the usual set of enzyme activities occurring in pit viper venoms but the content of alkaline phosphomonoesterase was unusually high, whereas the proportions of protease and arginine ester hydrolase were very low. The venom also exhibited weak thrombin-like activity but did not exhibit hemorrhagic or anticoagulant activity. Analysis of the Sephadex G-200 gel filtration fractions of the venom indicated that the lethal fraction was a low mol.wt protein, and that fractions exhibiting phosphodiesterase, phosphomonoesterase, arginine ester hydrolase, thrombin-like enzyme, L-amino acid oxidase and phospholipase A activities were not lethal. Two lethal toxins, designated as wagleri toxins 1 and 2, were isolated from the venom using Sephadex G-50 gel filtration chromatography followed by SP-Sephadex C-25 ion exchange chromatography. The mol.wts of the two toxins were 8900 by gel filtration. The LD50 (i.v.) values in mice for wagleri toxins 1 and 2 are 0.17 microgram/g and 0.19 microgram/g, respectively.
Some enzymatic activities and toxic properties of four samples of Ophiophagus hannah (king cobra) venom were investigated. There is little intraspecific variation in enzyme contents, protein composition and toxic properties of the venom. The venom does not exhibit hemolytic or edema-inducing activity but is characterized by an exceptionally high alkaline phosphomonoesterase activity. DEAE-Sephacel ion exchange chromatography and Sephadex G-75 gel filtration chromatography of the venom indicate that the major lethal toxins are the low mol.wt, non-enzymatic basic proteins. Venom fractions exhibiting high enzymatic activities apparently do not play an important role in the lethality in mice of Ophiophagus hannah venom.
A simple and sensitive double-antibody radioimmunoassay for human growth hormone (HGH) was developed, optimised and validated. The anti-hGH sera raised in 2 rabbits were highly specific with low cross-reactions of 0.19% and 0.3% with human placental lactogen and 0.21% and 0.13% with human prolactin. The mean sensitivity of the assay determined from 28 assays was found to be 0.4 +/- 0.2 mIU/L. Mean recovery of added exogenous hGH was 98.8 +/- 6.8%. Linearity studies of samples diluted at 1:2, 1:4 and 1:8 gave values of 101.3 +/- 5.3%, 109.6 +/- 13.4% and 97.3 +/- 13% respectively of those expected. The reproducibility of the assay was good; within assay coefficient of variation for serum samples with GH concentrations of 2.7, 13.6 and 28.2 mU/l ranged from 5.1 to 8.3% while the inter-assay precision varied from 4.9 to 10.3%. The in-house assay showed good correlation (r = 0.96, p less than 0.001) with a commercial HGH RIA kit (Dainabot, Japan). A reference normal adult fasting GH level of less than 7 mIU/l was established from 95 samples assayed by this method.
Randomly selected samples from different animal colonies from two laboratory animal houses and from the wild-caught monkeys were tested for the presence of anti-rotavirus antibodies to estimate the rates of infection with group A rotavirus. Antibodies to the common group A rotaviral antigen were detected by a competitive enzyme-linked immunosorbent assay (ELISA) using reagents of WHO ELISA rotavirus detection kit. The results of the study showed that white mice, albino rats, and guinea pigs from long-established breeding colonies and resident house rats and house shrews from the animal house had no serological evidence of rotaviral infection. In contrast, one mousedeer from a colony of 19 animals and most of the rabbits from two separate breeding colonies at the same animal house were serologically positive for the infection. Also a significant number of the same species of monkey kept in captivity were found to acquire the infection. Leaf monkeys had no serological evidence of rotaviral infection. The infection rate in wild cynomolgus monkeys did not seem to be influenced by the different ecological environments of their respective habitats. The rate of infection in adults and juveniles was similar.
An extract prepared from the tentacle of Catostylus mosaicus was shown to lyse erythrocytes from rat, rabbit and human to a different extent; those from the rat being most susceptible followed by those from rabbit and human. The haemolytic activity was dependent on the concentration of crude extract protein exhibiting a sigmoidal curve. Only 60% of the haemolytic activity was retained after treament with heat and proteolytic enzyme. The extract was devoid of hydrolytic enzymes normally present in venoms except for phospholipase A activity, which resulted in the hydrolysis of membrane phospholipids with concomittant appearance of their lyso-derivatives.
Hannahtoxin, the major hemorrhagin purified from king cobra (Ophiophagus hannah) venom, elicits hemorrhages in rabbits but not in mice. Two antisera against hannahtoxin were prepared: one raised against purified hannahtoxin, while the other was raised against glutaraldehyde cross-linked and detoxified hannahtoxin. The antisera were refined by pepsin digestion and ammonium sulfate precipitation. They are of approximately equal potency in their ability to neutralize the hemorrhagic activity of king cobra venom in rabbits. The antisera did not form a precipitin line with venom of snakes of the Viperidae family nor neutralize hemorrhages elicited in mice by any of these venoms. However, when the hemorrhagic activity was assayed in rabbits, both antisera were able to abolish the hemorrhages elicited by all of the venoms tested. These results suggest that hannahtoxin displays few epitopes in common with hemorrhagins of viperid venoms, except those involved in the neutralization of hemorrhagic activity in rabbits. The epitopes of viperid venom hemorrhagins involved in the neutralization reaction in rabbits are different from those in mice.
The major hemorrhagin (termed hannahtoxin) of the venom of Ophiophagus hannah (king cobra) was purified to electrophoretic homogeneity by DEAE-Sephacel ion exchange chromatography, Sephadex G-200 gel filtration followed by a second DEAE-Sephacel chromatography. Proteolytic activity was associated with the hemorrhagic activity throughout the purification procedures. Hannahtoxin constituted approximately 2% of the crude venom. It had an isoelectric point of 5.3, a carbohydrate content of 12%, a mol. wt of 66,000 as determined by SDS-polyacrylamide gel electrophoresis and 63,000 as determined by gel filtration. It contains 1 mole of Zn per mole of protein. The minimum hemorrhage doses for hannahtoxin are 0.7 microgram and 75 micrograms, respectively, in rabbits and in mice. Hannahtoxin was not lethal to mice at a dose of 2 mg/kg (i.v.) but killed rabbits at doses above 0.18 mg/kg (i.v.). It liberated protein from rabbit glomerular basement membrane but not rat glomerular basement membrane. Treatment of the hemorrhagin with EDTA and 1,10-phenanthroline eliminated both the proteolytic and hemorrhagic activities completely.
1. An extract prepared from the tentacle of the jellyfish (CE), Catostylus mosaicus exhibited haemolytic, oedema and haemorrhage-inducing activities. 2. Acetone treatment of the tentacle extract produced an acetone soluble extract (AE) which showed an increase in specific haemolytic and haemorrhagic activities by 25- and 120-fold respectively; the minimum oedema dose was reduced by 30-fold. 3. The AE caused a rapid onset of oedema in the mouse foot pad. The effect was long-lasting, reaching a maximum in about 30 min after injection and sustained up to 4 hr. 4. Fractionation of the AE on Q-Sepharose gave 4 bound fractions which induced oedema and haemorrhage; however only 3 of the fractions exhibited haemolytic activity.
1. The hemorrhagic, procoagulant, anticoagulant, protease, arginine ester hydrolase, phosphodiesterase, alkaline phosphomonoesterase, 5'-nucleotidase, hyaluronidase, phospholipase A and L-amino acid oxidase activities of 50 venom samples from 20 taxa of rattlesnake (genera Crotalus and Sistrurus) were examined. 2. The results show that notwithstanding individual variations in the biological activities of Crotalus venoms and the wide ranges of certain biological activities observed, there are some common characteristics at the genus and species levels. 3. The differences in biological activities of the venoms compared can be used for differentiation of the species. Particularly useful for this purpose are the thrombin-like enzyme, protease, arginine ester hydrolase, hemorrhagic and phospholipase A activities and kaolin-cephalin clotting time measurements.
The mechanisms causing inflammation in rheumatoid arthritis (RA) are not yet clearly known. They may be associated with different types of inflammatory cells and probably numerous mediators (SHARMA and MOHSIN 1990). Nowadays, the platelet activating factor (PAF) is discussed as an important mediator in RA.
Schistosoma malayensis Sp N is a putative new species of schistosome discovered in Peninsular Malaysia in 1973. This paper comprises the first report on the detailed gastrointestinal pathology present in rabbits infected with strains of the parasite. Two different strains of schistosome--the Baling and Koyan strains--from two different ecosystems were used to infect inbred rabbits and the resulting pathophysiology was studied. Our results showed that the Baling strain of S. malayensis was more virulent than the Koyan strain and produced nodular, segmental circumferential lesions and large bilharziomas measuring 1-7 cm in diameter in the distal jejunum, ileum and the ileo-caecal junction. The findings indicate that the Baling strain of S. malayensis was more pathogenic for rabbits as compared with the Koyan strain--in relation to the gross pathology of the gut and the tissue egg load. Earlier reports have shown that rabbits infected with S. japonicum induces significant intestinal lesions in rabbits (Cheever et al, 1980 a,b) but these animals are refractory to infection with S. mekongi (Byram and Lichtenberg, 1980). Our studies show that the two strains of S. malayensis adapted well in rabbits. It is also established that in rabbits, the virulence of the Baling strain of S. malayensis is greater than that of S. mekongi and approximates that of S. japonicum.
1. The enzymatic, hemorrhagic, procoagulant and anticoagulant activities of venoms of some animals including snakes, lizards, toads, scorpions, spider, wasps, bees and ants were compared. 2. Snake venom was the richest source of enzymes among the animal venoms. Most other animal venoms were devoid of phosphodiesterase, L-amino acid oxidase, alkaline phosphomonoesterase and acetylcholinesterase activities and only a few exhibited arginine ester hydrolase activity. These venoms, however, exhibited wide ranges of protease, 5'-nucleotidase and hyaluronidase activities. Most of the animal venoms examined exhibited some phospholipase A activity. 3. Other than snake venoms, only venoms of the toad Bufo calamita and the lizards were hemorrhagic, and only venoms of the social wasps, social bees and harvester ant exhibited strong anticoagulant activity. Procoagulant activity occurs only in snake venoms.
The safety of bacterial cells of Clostridium bifermentans serovar malaysia, which is highly toxic to mosquito larvae, was tested on mice, guinea pigs, rabbits, and goldfish. Inoculations of at least 1 x 10(8) cells per animal by routes recommended by World Health Organization (subcutaneous, percutaneous, inhalation, force-feeding, intraperitoneal, intravenous) and tests of subacute toxicity, anaphylactic shock, persistence in heart blood, and virulence by successive passages, were performed on mice, guinea pigs, or both. Growth was monitored for 1 mo before necropsy. Ocular irritation and skin scarification were tested with rabbits. C. bifermentans serovar malaysia did not induce any mortality or abnormal reactions in mammals at a dose 1,000 times higher than the level established by W.H.O. for the demonstration of safety. Bacterial cells are not toxic to goldfish at a dose 1,000 times higher than the LD50 for the target-mosquito larvae. We conclude that C. bifermentans serovar malaysia bacterial cells are safe for laboratory mammals and goldfish.
Two distinct strains of Schistosoma malayensis exist in Malaysia (designated the Baling and Koyan strains). Both these strains show intraspecific variations in pathology (Greer et al, 1988). To evaluate the differences in the pulmonary pathology resulting from infections of the two different strains of Malaysian schistosome, a total of 20 experimental rabbits were infected, 10 each with cercariae of the Koyan strains. Pathological changes were studied over a period of 28 weeks. Granulomas in the lung occurring as a result of infection with the Baling strain were compared with those caused by infection with the Koyan strain. Although both strains produced parenchymatous and alveolar lesions, granulomas caused by the Baling strain of Malaysian schistosome were more numerous and larger (when comparing mean diameter as well as area of granuloma, p < 0.05). In addition, pulmonary vascular hypertensive changes were present in Baling strain infected rabbits. These comprised of pulmonary arteriolar endothelial swelling and damage, intimal elastosis and medial hypertrophy. Angiitis and pulmonary periphlebitis were also noted occasionally. In contrast, Koyan strain infection resulted in fewer and smaller granulomas. Pulmonary vascular changes were minimal.
The biological properties of adult and juvenile inland taipan (Oxyuranus microlepidotus) snake venoms were examined. The enzymatic activities, intravenous median lethal dose and procoagulant activity of the juvenile venom samples were not significantly different from those of the adult venom samples. Also, the juvenile and adult venoms exhibited similar electrophoretic patterns, indicating that they possessed similar protein composition.
Two groups of three rabbits each were infected with 250 cercariae of the Baling and Koyan strain of Schistosoma malayensis. Changes induced by both strains included periportal hepatocellular necrosis and fibrosis. Vascular changes such as portal phlebitis and thrombophlebitis and varying degrees of pericholangitis were also present. Amyloid deposition was noted. A comparative study of the changes induced in rabbits by S. malayensis, S. mekongi and S. japonicum showed that the hepatic lesions induced by the Baling strain of S. malayensis were similar to that induced by S. japonicum, and were more severe than that induced by S. mekongi or the Koyan strain.
A simple, non-isotopic in-house enzyme-linked immunoabsorbant assay (ELISA) for human growth hormone (GH) was developed. The assay involved using in-house polyclonal anti-GH adsorbed onto 96-well microtitre plates, commercially prepared mouse monoclonal anti-GH, and goat anti-mouse IgG horseradish peroxidase detection system. Results of recovery and parallelism studies ranged from 95%-106% and 98%-101% respectively, of the expected values. The detection limit of the assay was 0.008 mIU/well or the equivalent to 0.4 mIU/L of undiluted serum. Intra- and interassay coefficients of variations were 4.8%-7.9% and 6.5%-8.7% respectively. Serum GH levels measured in this assay correlated well with those measured in established in-house radioimmunoassays (r = 0.985, p < 0.001) and immunoradiometric assay from NETRIA (r = 0.984, p < 0.001).