Genetic characterization of rabies field isolates from Thailand

We sequenced 512 nucleotides in two variable regions of the N gene of 23 rabies isolates from the northeastern part of Thailand by direct sequencing of PCR-amplified products. The sequencing data revealed two new lineages in these rabies isolates. Based on the results of this study together with the findings of our earlier study, the rabies isolates in Thailand were divided into two genogroups, designated as T1 and T2, which were predominantly localized in the northern and northeastern areas, respectively. Each of these two genogroups consisted of four lineages. There was a correlation between these eight lineages and the geographical origins of the isolates. Two lineages belonging to the T2 genogroup from the northeastern area of Thailand were newly identified in this study. The isolates in both genogroups were also prevalent in the central area of Thailand. Each lineage in the T1 and T2 genogroups was found independently in dogs in the upper and lower southern areas of Thailand, respectively. These genetic data and the historical background suggest that rabies viruses belonging to the T2 genogroups were prevalent many years ago in the central and northeastern areas of Thailand and were later transferred to the lower southern parts of Thailand.     

Effects of multiple-bond ruptures on kinetic parameters extracted from force spectroscopy measurements: revisiting biotin-streptavidin interactions

Force spectroscopy measurements of the rupture of the molecular bond between biotin and streptavidin often results in a wide distribution of rupture forces. We attribute the long tail of high rupture forces to the nearly simultaneous rupture of more than one molecular bond. To decrease the number of possible bonds, we employed hydrophilic polymeric tethers to attach biotin molecules to the atomic force microscope probe. It is shown that the measured distributions of rupture forces still contain high forces that cannot be described by the forced dissociation from a deep potential well. We employed a recently developed analytical model of simultaneous rupture of two bonds connected by polymer tethers with uneven length to fit the measured distributions. The resulting kinetic parameters agree with the energy landscape predicted by molecular dynamics simulations. It is demonstrated that when more than one molecular bond might rupture during the pulling measurements there is a noise-limited range of probe velocities where the kinetic parameters measured by force spectroscopy correspond to the true energy landscape. Outside this range of velocities, the kinetic parameters extracted by using the standard most probable force approach might be interpreted as artificial energy barriers that are not present in the actual energy landscape. Factors that affect the range of useful velocities are discussed.     

Adenosine and Cyclic AMP in Rat Cerebral Cortical Slices: Effects of Adenosine Uptake Inhibitors and Adenosine Deaminase Inhibitors

The endogenous levels of adenosine functionally linked to cyclic AMP systems in rat cerebral cortical slices are regulated by both adenosine deaminase and adenosine uptake systems. 2'-Deoxycoformycin (2'-DCF), an adenosine deaminase inhibitor, slightly increased basal, adenosine, and norepinephrine-elicited accumulations of cyclic AMP, whereas dipyridamole, an uptake inhibitor, had an even greater effect on cyclic AMP accumulations under the same conditions. Combinations of 2'-DCF and dipyridamole elicited a greater effect than either compound alone. Neither 2'-DCF nor dipyridamole significantly augmented accumulations of cyclic AP elicited by a depolarizing agent, veratridine, suggesting that the adenosine "released" during neuronal depolarization of brain slices is not as subject to inactivation by uptake or deamination as endogenous adenosine in control brain slices. The accumulation of cyclic AMP elicited by a combination of norepinephrine and veratridine was greater than additive. The response to a pure beta-adrenergic agonist, isoproterenol, was not potentiated by 2'-DCF, dipyridamole, or veratridine, consonant with minimal interaction of endogenous adenosine with beta-adrenergic systems.     

Evaluation of IgG4 Subclass Antibody Detection by Peptide-Based ELISA for the Diagnosis of Human Paragonimiasis Heterotrema

A synthetic peptide was prepared based on the antigenic region of Paragonimus westermani pre-procathepsin L, and its applicability for immunodiagnosis for human paragonimiasis (due to Paragonimus heterotremus) was tested using an ELISA to detect IgG4 antibodies in the sera of patients. Sera from other helminthiases, tuberculosis, and healthy volunteers were used as the references. This peptide-based assay system gave sensitivity, specificity, accuracy, and positive and negative predictive values of 100%, 94.6%, 96.2%, 100%, and 88.9%, respectively. Cross reactivity was frequently seen against the sera of fascioliasis (75%) and hookworm infections (50%). Since differential diagnosis between paragonimiasis and fascioliasis can be easily done by clinical presentation and fascioliasis serology, this cross reaction is not a serious problem. Sera from patients with other parasitoses (0-25%) rarely responded to this synthetic antigen. This synthetic peptide antigen seems to be useful for development of a standardized diagnostic system for paragonimiasis.     

Susceptibility of Laboratory Rodents to Trichinella papuae

Members of the genus Trichinella are small nematodes that can infect a wide range of animal hosts. However, their infectivity varies depending on the parasite and host species combination. In this study, we examined the susceptibility of 4 species of laboratory rodents, i.e., mice, rats, hamsters, and gerbils to Trichinella papuae, an emerging non-encapsulated Trichinella species. Trichinella spiralis and Trichinella pseudospiralis were also included in this study for comparison. Fifteen animals of each rodent species were infected orally with 100 muscle larvae of each Trichinella species. Intestinal worm burden was determined at day 6 and 10 post-inoculation (PI). The numbers of muscle larvae were examined at day 45 PI. The reproductive capacity index (RCI) of the 3 Trichinella species in different rodent hosts was determined. By day 6 PI, 33.2-69.6% of the inoculated larvae of the 3 Trichinella species became adult worms in the small intestines of the host animals. However, in rats, more than 96% of adult worms of all 3 Trichinella species were expelled from the gut by day 10 PI. In gerbils, only 4.8-18.1% of adult worms were expelled by day 10 PI. In accordance with the intestinal worm burden and the persistence of adults, the RCI was the highest in gerbils with values of 241.5±41.0 for T. papuae, 432.6±48 for T. pseudospiralis, and 528.6±20.6 for T. spiralis. Hamsters ranked second and mice ranked third in susceptibility in terms of the RCI, Rats yielded the lowest parasite RCI for all 3 Trichinella species. Gerbils may be an alternative laboratory animal for isolation and maintenance of Trichinella spp.     

Cell-Based Multi-Parametric Model of Cleft Progression during Submandibular Salivary Gland Branching Morphogenesis

Cleft formation during submandibular salivary gland branching morphogenesis is the critical step initiating the growth and development of the complex adult organ. Previous experimental studies indicated requirements for several epithelial cellular processes, such as proliferation, migration, cell-cell adhesion, cell-extracellular matrix (matrix) adhesion, and cellular contraction in cleft formation; however, the relative contribution of each of these processes is not fully understood since it is not possible to experimentally manipulate each factor independently. We present here a comprehensive analysis of several cellular parameters regulating cleft progression during branching morphogenesis in the epithelial tissue of an early embryonic salivary gland at a local scale using an on lattice Monte-Carlo simulation model, the Glazier-Graner-Hogeweg model. We utilized measurements from time-lapse images of mouse submandibular gland organ explants to construct a temporally and spatially relevant cell-based 2D model. Our model simulates the effect of cellular proliferation, actomyosin contractility, cell-cell and cell-matrix adhesions on cleft progression, and it was used to test specific hypotheses regarding the function of these parameters in branching morphogenesis. We use innovative features capturing several aspects of cleft morphology and quantitatively analyze clefts formed during functional modification of the cellular parameters. Our simulations predict that a low epithelial mitosis rate and moderate level of actomyosin contractility in the cleft cells promote cleft progression. Raising or lowering levels of contractility and mitosis rate resulted in non-progressive clefts. We also show that lowered cell-cell adhesion in the cleft region and increased cleft cell-matrix adhesions are required for cleft progression. Using a classifier-based analysis, the relative importance of these four contributing cellular factors for effective cleft progression was determined as follows: cleft cell contractility, cleft region cell-cell adhesion strength, epithelial cell mitosis rate, and cell-matrix adhesion strength.     

The binding of fluorocatecholamines to adrenergic and dopaminergic receptors in rat brain membranes

2-Fluoronorepinephrine (IC₅₀ ≈0.7 μM) is a relatively selective ligand for displacement of radioactive dihydroalprenolol from β₁-adrenergic receptors in membrane preparations from rat cerebral cortex. It is less potent (IC₅₀ ≈10 μM) in displacing dihydroalprenolol from β₂-adrenergic receptors in rat cerebellar membranes and in displacing clonidine from α₂-adrenergic receptors in rat cerebral cortical membranes. It is much less potent (IC₅₀ > 100 μM) in displacing WB-4101 from α₁-adrenergic receptors in rat cerebral cortical membranes. In contrast, 6-fluoronorepinephrine is relatively selective for α-adrenergic receptors, being at least 50–200 times more potent at such receptors than at β-adrenergic receptors. 5-Fluoronorepinephrine like norepinephrine does not exhibit remarkable selectivity towards α- and β-adrenergic receptors. The 2-, 5- and 6-fluorodopamines are more potent ligands at α₁-adrenergic receptors than at α₂- and β-adrenergic receptors but the specificity is not markedly affected by the position of the fluorine substituent. The results suggest that the specificity exhibited by the 2- and 6-fluoronorepinephrine at adrenergic receptors is not primarily due to fluorine-induced changes in the physicochemical properties of the aromatic ring, but instead to stereoselective interactions of the ethanolamine side chain of norepinephrine with fluorine at either the 2- or 6-ring positron. The fluorodopamines like dopamine itself are more potent at dopaminergic than at α- or β-adrenergic receptors. The 2-, 5- and 6-fluorodopamines are all nearly equipotent with dopamine in the displacement of radioactive spiroperidol from dopaminergic receptors in membrane preparations from rat striatum, while the 2- and 6-fluorodopamine are somewhat less potent than dopamine or 5-fluorodopamine in displacement of radioactive apomorphine in striatal membranes.     

Detrimental Effect of Water Submersion of Stools on Development of Strongyloides stercoralis

Strongyloidiasis is prevalent in Thailand, yet its prevalence in the south is lower than in other parts of the country. This might be due to the long rainy season in the south resulting in stool submersion in water inhibiting worm development. In this study, the effect of water submersion of fecal samples on development of Strongyloides stercoralis was investigated. Ten ml of a 1∶5 fecal suspension were placed in 15-ml tubes, 35-mm dishes, and 90-mm dishes producing the depths of 80 mm, 11 mm and 2 mm-suspensions, respectively. The worm development was followed at 1/6, 4, 6, 8, 10, 12, 14, 16, 24, and 36 h, by determining the number of filariform larva (FL) generated from agar-plate cultures (APC). Fecal suspensions kept in tubes and 35-mm dishes showed a decline in FL yield relative to incubation time and reached zero production 14 h after incubation. In contrast, the number of FL generated from the suspension kept in 90-mm dishes remained stable up to 36 h. Cumulatively, all tubes and 35-mm dishes became negative in APC after 14 h while 90-mm dishes remained APC-positive up to 36 h. Adding more water or stool suspension to dishes resulted in a decreased number of FL. Mechanical aeration of the suspensions in tubes restored an almost normal FL yield. It appears that the atmospheric air plays a significant role in growth and development of S. stercoralis in the environment and may be one of factors which contribute to a lower prevalence of human strongyloidiasis in the south of Thailand.     

Molecular Detection of Ancylostoma duodenale,Ancylostoma ceylanicum,and Necator americanus in Humans in Northeastern and Southern Thailand

The 2 principal species of hookworms infecting humans are Necator americanus and Ancylostoma duodenale. Case studies on zoonotic hookworm infections with Ancylostoma ceylanicum and/or Ancylostoma caninum are known mainly from Asian countries. Of these 2 zoonotic species, only A. ceylanicum can develop to adulthood in humans. In the present study, we report a molecular-based survey of human hookworm infections present in southern and northeastern Thailand. Thirty larval hookworm samples were obtained from fecal agar plate cultures of 10 patients in northeastren Thailand and 20 in southern Thailand. Partial ITS1, 5.8S, and ITS2 regions of the ribosomal DNA genes were amplified using PCR. The amplicons were sequenced, aligned, and compared with other hookworm sequences in GenBank database. The results showed that, in Thailand, N. americanus is more prevalent than Ancylostoma spp. and is found in both study areas. Sporadic cases of A. ceylanicum and A. duodenale infection were seen in northeastern Thailand.     

High Prevalence of Haplorchiasis in Nan and Lampang Provinces,Thailand, Proven by Adult Worm Recovery from Suspected Opisthorchiasis Cases

Opisthorchiasis, a risk factor for cholangiocarcinoma in humans, is of public health importance in Thailand. The Annual Surveillance Reports from Nan and Lampang Provinces, Thailand, for the year 2011 showed an opisthorchiasis prevalence of over 70% by recovery of eggs in the feces. This study investigated whether most cases are actually due to minute intestinal flukes (MIF) rather than Opisthorchis viverrini, as the eggs of both can hardly be differentiated by morphology. Fifty and 100 cases from residents in Nan and Lampang, respectively, had stools positive for eggs initially assumed to be those of O. viverrini. Each patient was given praziquantel at 40 mg/kg in a single dose. After 2 hr, 30-45 ml of the purgative magnesium sulfate was given, and stools were collected up to 4 times sequentially. The stools were examined for adult worms by simple sedimentation. It was found that 39 of 50 cases (78.0%) from Nan Province had Haplorchis taichui, with intensities ranging from 5 to 1,250 with an average of 62 worms/case. Taenia saginata (7 cases) and Enterobius vermicularis (1 case) were other helminths recovered as the co-infectants. In Lampang Province, H. taichui was recovered from 69 cases (69.0%). The number of flukes recovered ranged from 1 to 4,277, with an average of 326 worms/case. Four cases had Phaneropsolus bonnei, and 10 T. saginata as the co-infectants. Adult specimens of O. viverrini were not recovered from any stool. Clearly, MIF infection, especially haplorchiasis, is more common in northern Thailand. These findings should encourage the Public Health Office to employ more specific tools than Kato''s method for surveillance of opisthorchiasis in Thailand.