Prasarn Tangkawattana

A Portrait of the Transcriptome of the Neglected Trematode, Fasciola gigantica—Biological and Biotechnological Implications

Fasciola gigantica (Digenea) is an important foodborne trematode that causes liver fluke disease (fascioliasis) in mammals, including ungulates and humans, mainly in tropical climatic zones of the world. Despite its socioeconomic impact, almost nothing is known about the molecular biology of this parasite, its interplay with its hosts, and the pathogenesis of fascioliasis. Modern genomic technologies now provide unique opportunities to rapidly tackle these exciting areas. The present study reports the first transcriptome representing the adult stage of F. gigantica (of bovid origin), defined using a massively parallel sequencing-coupled bioinformatic approach. From >20 million raw sequence reads, >30,000 contiguous sequences were assembled, of which most were novel. Relative levels of transcription were determined for individual molecules, which were also characterized (at the inferred amino acid level) based on homology, gene ontology, and/or pathway mapping. Comparisons of the transcriptome of F. gigantica with those of other trematodes, including F. hepatica, revealed similarities in transcription for molecules inferred to have key roles in parasite-host interactions. Overall, the present dataset should provide a solid foundation for future fundamental genomic, proteomic, and metabolomic explorations of F. gigantica, as well as a basis for applied outcomes such as the development of novel methods of intervention against this neglected parasite.

High prevalence of Opisthorchis viverrini infection in reservoir hosts in four districts of Khon Kaen Province, an opisthorchiasis endemic area of Thailand.

Khon Kaen, a northeastern province of Thailand, has been considered as one of the human opisthorchiasis endemic areas with continuing high prevalence. Unsuccessful eradication of the disease is probably from the culture of eating raw and undercooked fish of local residence and the parasitic persistency in animal reservoir hosts, such as cats and dogs. In cooperation with the other human opisthorchiasis control programs in an endemic area of 29 villages in Ban Haet, Ban Phai, Chonnabot and Muncha Khiri Districts, Khon Kaen, this study investigated the prevalence of Opisthorchis viverrini infection using a formalin-ether sedimentation method as the gold standard, and hematology and blood chemistry of the reservoir hosts in this endemic area. The results showed that cats had much higher prevalence (76 of 214, 35.51%) than dogs (3 of 821, 0.37%). Hematology between the infected and uninfected cats was not different. Complete blood count and biochemistry reflected some altered hepatic functions. However, only severely infected cats showed apparent clinical signs, including lethargy, diarrhea, ocular and nasal discharges. Moreover, the ultrasonogram of infected cats with very high egg per gram (>1500 EPG) showed apparent thickening of the gall bladder wall with hyperechoicity of hepatic parenchyma. This study suggests that cat is the most important animal reservoir of human opisthorchiasis, especially in this endemic area. It is also interesting that villages with infection are mostly located in the vicinity of Chi River and two large water reservoirs (Lawa and Nong Kongkaew Lakes), but people without infection were away from Chi River, on the south of Kudkhow Lake. Further investigation on this particular geofactor is essential for effective opisthorchiasis control programs.

Comparative study of the characteristics and properties of tendinocytes derived from three tendons in the equine forelimb.

The aim of this study was to determine the characteristic differences in tendinocytes derived from tendons in the equine forelimb, superficial digital flexor tendon (SDFT), deep digital flexor tendon (DDFT) and common digital extensor tendon (CDET), in morphology, proliferation, collagen production ability and ability for synthesis of matrix metalloproteinases (MMPs). Significant differences were observed in cell number in vivo. The cellular number was largest in the SDFT and smallest in the CDET. The values of in vitro proliferation ratios and ability for synthesis of collagen and MMPs were largest in the SDFT and smallest in the CDET. Addition of TNFalpha to culture of all three types of tendinocytes increased the synthesis of both proMMP-9 (except CDET) and collagen and decreased proMMP-13 synthesis and had no effect on proMMP-2 synthesis. Flexor tendons in forelimbs (SDFT and DDFT) restore energy during locomotion and are more easily injured than are extensor tendons. This structural property would cause active ECM and MMPs synthesis. And CDET have very low turnover potential; in the small number of cells, low cellular proliferation, lower ability for synthesis of collagen and MMPs. The isolated tendinocytes provided much information on the characteristics and properties of tendons for the ECM turnover system and responsiveness of tendinocytes to complex inflammatory responses in tendinopathy.

Decreased risk of cholangiocarcinogenesis following repeated cycles of Opisthorchis viverrini infection-praziquantel treatment: Magnetic Resonance Imaging (MRI) and histopathological study in a hamster model

It has been suggested that repeated infection of Opisthorchis viverrini followed by repeated treatment with praziquantel (PZQ) increases risk of development of cholangiocarcinoma (CCA). Evidence for the prediction has accumulated based on findings of indirect approaches involving molecular changes and epidemiological trends. By contrast, here we directly monitored the impact of repeated liver fluke infection and treatment with PZQ on cholangiocarcinogenesis in a rodent model of human opisthorchiasis, using magnetic resonance imaging (MRI) and histopathology. Twenty five Syrian golden hamsters were assigned to five treatment groups: 1) infection with O. viverrini (OV group), 2) treatment with the carcinogen N-nitrosodimethylamine (NDMA) at 12.5ppm (DMN), 3) O. viverrini infection in tandem with NDMA (OD), 4) O. viverrini infection, NDMA, and treatment with PZQ (ODP), and 5) uninfected, untreated control. The repeated infections were established by intragastric inoculation of 50 metacercariae of O. viverrini to the OV, OD and ODP hamsters at weeks 0, 5 and 10. PZQ at 300mg/kg body weight was given to each hamster of the ODP group on weeks 4, 9 and 13 (four weeks after each infection). Imaging by MRI was undertaken on weeks 5, 10 and 14 (i.e. one week after each PZQ treatment). MRI revealed that the ODP hamsters did not develop CCA, whereas necropsy at week 40 revealed CCA in hamsters of the OD and DMN groups. Findings for histopathology and for proliferating cell nuclear antigen index conformed to the MRI findings. In overview, and notwithstanding that the immune response of individual hosts may play roles in cholangiocarcinogenesis, three cycles of the infection with O. viverrini followed treatment of the infection with PZQ did not increase the risk of bile duct cancer in this hamster model of liver fluke infection-induced CCA.

Efficacious and safe dose of praziquantel for the successful treatment of feline reservoir hosts with opisthorchiasis

Opisthorchiasis caused by Opisthorchis viverrini is a major food-borne zoonosis in Greater Mekong sub-region. Even though campaigns discouraging the consumption of raw fish have been launched to public, the disease still remains highly endemic. The unsuccessful eradication of the disease is probably because of the persistence of the parasite in animal reservoir hosts, particularly felids. Praziquantel (PZQ) is the drug of choice for morbidity control of opisthorchiasis in humans and animals. However, there is no specific study on its dosage regimen for feline opisthorchiasis. Thus, the effective treatment dose of PZQ, as well as its adverse effects, was evaluated in O. viverrini infected cats. Twenty-eight infected male and female cats from the endemic area of Khon Kaen and Maha Sarakham Provinces, Thailand were enrolled in this study. Physical, hematological, blood chemical and urine examinations were analyzed, as indicators of health status, on the day before and 30days after treatment. Intensity of the infections was determined by the formalin-ethyl acetate sedimentation technique. Cats were equally allotted into the low infection group of 14 cats with egg count per gram of feces (EPG) <300 and the high infection group of 14 cats with EPG higher than 300. Cats in each group were equally divided into two subgroups of 7 cats; thus, there were two low infection subgroups (L1 and L2 subgroups) and two high infection subgroups (H1 and H2 subgroups). A single dose of 25mg/kg PZQ was orally administered to each cat in the L1 and H1 subgroups and a single oral dose of 40mg/kg PZQ was administered to the L2 and H2 subgroups. Complete clearance of O. viverrini eggs was found in all cats in the L1, L2 and H2 subgroups; thus, the cure rate (CR) and egg reduction rate (ERR) were 100%. However, partial clearance was observed in two cats with high EPG (1502 and 1518) in the H1 subgroup, which received 25mg/kg PZQ. Regards, CR and ERR for these two animals was 71.4 and 99.5%. No significant difference among the 4 subgroups was seen. Almost all hematological, blood chemical and urinalysis data were within normal ranges, except for the eosinophilia and an increase of alanine aminotransferase (ALT). Hookworm infection seen in all cats would cause eosinophilia. As for drug safety, there was no side effect observed in any cats. In conclusion, this study suggested that 40mg/kg PZQ is a highly effective and safe dosage for the treatment of feline reservoir hosts of human opisthorchiasis.

Reservoir Animals and Their Roles in Transmission of Opisthorchis viverrini

Although any fish-eating mammals could be potential definitive hosts of Opisthorchis viverrini, only a few, especially cats and dogs, are actually known reservoir hosts for this parasite. Both animals usually get infected via consuming raw or undercooked contaminated fish, fish dishes or food remains from households. The infected animals sustain parasite egg spread via open environment defecation. Cats are the most important reservoir with higher prevalence rates of O. viverrini infection than dogs in endemic areas. Usually Opisthorchis-infected animals do not exhibit apparent clinical symptoms or specific abnormalities in laboratory examinations. Pathological findings in these animal reservoirs are basically similar to those seen in humans and experimental animals, namely periductal inflammation, biliary hyperplasia and periductal fibrosis. However, O. viverrini-associated cholangiocarcinoma has not yet been reported in the reservoir animals at present. Praziquantel is a treatment of choice not only for humans but also for animal reservoirs. Integrated control of opisthorchiasis in animal reservoirs is based on holistic approaches such as EcoHealth/One Health concepts. In fact integrated control of opisthorchiasis in humans in ecosystem has also proved successful, for example, the Lawa model for opisthorchiasis control in the endemic area of Khon Kaen, Thailand. Other feral and wild animals in endemic areas might also be potential reservoirs, and this requires more investigation. In addition, genetic diversity and evolution of the flukes might also influence zoonotic capability.

Neutralizing formaldehyde in chicken cadaver with urea and urea fertilizer solution

This study demonstrated the potential of using urea and urea fertilizer to neutralize formaldehyde (Fd) in chicken cadavers. Initially, in vitro Fd neutralization with various concentrations of urea solution (US) and urea fertilizer solution (UFS) was conducted; subsequently, 18% US and 27% UFS were selected for infusing into the formalinized chickens. The measurement at 48 hr after infusion showed that both solutions could effectively lower Fd in chicken cadavers to below a permissible exposure limit without affecting cadaveric and histological quality. In addition, neutralizing power of 18% US was approximately 1.3 times that of 27% UFS. This is the first demonstration of neutralizing potential of US and UFS against Fd both in vitro and in vivo.

Fibrillar architecture at three different sites of the bovine superficial digital flexor tendon

Superficial digital flexor tendon (SDFT) of the bovine hindlimb originates from the caudolateral aspect of the distal femur and finally inserts onto the plantar aspect of the middle phalanges. In the present study, morphology and morphometry of the bovine SDFT at the muscle-tendon junction (MTJ), middle metatarsus (mM) and tendon-bone interface (TBI) were investigated. Cross-sectional morphology at the three regions of SDFT were oval, semioval and ring-formed, respectively. Significant difference in cross-sectional area was found only between MTJ-mM and mM-TBI (P<0.05). Functional compression and friction from the adjacent structures could be the most potential interactions affecting such appearances. Morphometric data of tenocyte number, water content, and glycosaminoglycan (GAG) length and angle were found increasing in the proximodistal direction, except the fibril diameter and collagen fibril index (CFI). Statistical analyzes could reveal significant differences in average number of tenocytes (P<0.0001), CFI (between MTJ-mM and MTJ-TBI, P<0.05), water content (between MTJ-TBI, P<0.05), length of GAG chains (between MTJ-TBI, P<0.05), and angle of GAG chains (P<0.0001), respectively. The fibrillar characteristics at the three different areas, including fibril diameter distribution and interfibrillar distance, existed in conforming to the tensional axes in situ. In addition, length and angle of GAG chains were relevant to moving directions of the collagen fibrils.

Morphometric analysis of cornea in the Slc39a13/Zip13-knockout mice

Ehlers-Danlos syndrome (EDS) is a group of hereditary diseases caused by mutation of extracellular matrix-related genes. Recently, spondylodysplastic EDS-Zip13 (spEDS-Zip13: OMIM 612350) was recognized as a new EDS type. This current study could reveal various morphometric differences of collagenous population in the proper substance of cornea between the wild type and spEDS-Zip13-knockout (Zip13-KO) mice. Blockade of Smad-signaling pathway might initiate these alterations. Predilected dissimilarity in level of transcriptional activity probably dictated morphology of keratocyte and shape and electron density of its nucleus. In addition, the imbalance of proteoglycans and glycosaminoglycans would also affect the diameter and arrangement of collagen fibrils. These findings would be considered as vulnerable characteristics of corneal stroma of the Zip13-KO mice.

Morphometric analysis of growing tenocytes in the superficial digital flexor tendon of piglets

The fine structures of different tendons in various animals at different ages have been studied extensively to reveal their arrangement and growth patterns. However, knowledge of the microstructures of the growing tenocytes in the tendons of piglets is still lacking. Thus, we performed the first morphometric analysis to describe the characteristics of tenocytes in the metacarpal superficial digital flexor tendon of 0-, 10- and 20-day-old piglets. In the present study, hydrochloric acid/collagenase digestion was applied to remove the interstitial connective tissue to obtain clear visualization of intact tenocytes and their cytoplasmic processes (Cp). Then, the morphometry of the tenocytes was investigated by optical and electron microscopy. The mean ± SE values of the fascicle area, number of tenocytes/fascicle, cell density, number of Cp/tenocyte, length of Cp, and thickness of Cp were compared among the three age groups. Significant differences (judged at P<0.05) were found in almost all morphometric aspects among the age groups, except for the number of Cp/cell (P=0.545) and thickness of the Cp (P=0.105). A decrease of cell density corresponded with an increase in the length of the Cp, which were extended to connect either with the Cp of the other tenocytes or the surrounding endotendineum. Moreover, an increase of the fascicle area reflected the increase in tendon diameter. The revealed morphometric characteristics are thus the outcome of tendon growth.