Doxycycline-regulated growth hormone gene expression system for swine.

Although growth hormone gene transgenic animals are much larger than normal animals, they manifest differences that have adverse effects on survival due to overexpression of growth hormone. We developed a stable pig embryonic fibroblast cell line expressing pig growth hormone (pGH) using the Tet-On system, with which we can conditionally manipulate expression of pGH in vivo. Inducible expression of pGH was achieved by combining reserve Tet-controlled transcriptional activator and tetracycline-responsive element in a single plasmid. The mRNA expression of pGH was significantly increased compared to the non-induced group by about 10-fold. The controlled secretion of pGH induced by doxycycline was further tested in stably transfected cells. We conclude that inducible GH expression can be achieved in pig embryonic fibroblasts.

Testing the utility of mitochondrial cytochrome oxidase subunit 1 sequences for phylogenetic estimates of relationships between crane (Grus) species.

Morphology and biogeography are widely used in animal taxonomy. Recent study has suggested that a DNA-based identification system, using a 648-bp portion of the mitochondrial gene cytochrome oxidase subunit 1 (CO1), also known as the barcoding gene, can aid in the resolution of inferences concerning phylogenetic relationships and for identification of species. However, the effectiveness of DNA barcoding for identifying crane species is unknown. We amplified and sequenced 894-bp DNA fragments of CO1 from Grus japonensis (Japanese crane), G. grus (Eurasian crane), G. monacha (hooded crane), G. canadensis (sandhill crane), G. leucogeranus (Siberian crane), and Balearica pavonina (crowned crane), along with those of 15 species obtained from GenBank and DNA barcoding, to construct four algorithms using Tringa stagnatilis, Scolopax rusticola, and T. erythropus as outgroups. The four phylum profiles showed good resolution of the major taxonomic groups. We concluded that reconstruction of the molecular phylogenetic tree can be helpful for classification and that CO1 sequences are suitable for studying the molecular evolution of cranes. Although support for several deeper branches was limited, CO1 data gave remarkably good separations, especially considering that our analysis was based on just a fragment of the gene and that CO1 has generally been viewed as useful only for resolving shallow divergences.

Antibody isotype responses to Schistosoma japonicum antigens in subjects from a schistosomiasis area with repeated praziquantel chemotherapy compared with a new endemic zone in Hunan Province, P.R. China.

To demonstrate the dynamics of specific antibody isotypes against schistosome adult worm (AWA) and soluble egg (SEA) antigens, we evaluated (in 1999-2000) 112 subjects infected with Schistosoma japonicum from 2 regions of Hunan Province, China. Fifty-eight subjects were from Area A, a well-known endemic area with repeated chemotherapy. Area B (n = 54) is a new endemic focus in another part of the same province. Serum samples were collected prior to praziquantel (PZQ) chemotherapy, and at 2 and 12 months post-treatment. IgM, IgA, IgG, IgG2, IgG4 and IgE antibodies to AWA and SEA were measured by quantitative enzyme-linked immunosorbent assay (ELISA). Pre-treatment antibody isotype levels from Area A, except IgA against AWA and SEA, were significantly higher than those from Area B. In response to chemotherapy, most antibody isotype levels fell or remained stable. However, in Area A there was a significant increase in the IgA, IgE and IgG4 responses to AWA 2 months after PZQ--which fell to approach pre-treatment levels by 12 months. A similar response was seen in Area B with IgE and IgG4 to AWA. Levels of all AWA-specific IgE and IgG4 were significantly higher in subjects from Area A compared with Area B at all time-points. AWA-IgE levels demonstrated significant linear correlations with age and number of previous PZQ treatments in Area A only. All SEA-specific isotypes in both areas fell significantly in response to treatment--except IgE, which remained stable in both area. All SEA-specific isotype levels (except IgA) were significantly higher from Area A at baseline. This significant difference was maintained through 12-months follow-up for IgE, IgG2 and IgG4 only. This study suggests that multiple episodes of schistosome infection may be required to generate antibody isotype levels that have been associated with resistance to re-infection in other studies. Further, a surrogate marker of successful chemotherapy (AWA-IgG4) performed less effectively in patients with previous treatment courses.

Efficacy of praziquantel against Schistosoma japonicum: field evaluation in an area with repeated chemotherapy compared with a newly identified endemic focus in Hunan, China.

This study conducted in 1999/2000 was designed to evaluate the efficacy of praziquantel against Schistosoma japonicum in an area with repeated chemotherapy (Area A) compared with a newly identified endemic focus (Area B) in Hunan Province, China. The population size was 2015 and 2180 in Areas A and B, respectively, of which 1129 and 1298 subjects received stool examination. A total of 230 subjects were identified by the Kato-Katz technique (4 smears per person) as being infected with S. japonicum, 124 in Area A (prevalence 11%) and 106 in Area B (prevalence 8.2%). They were treated with a single oral dose of praziquantel (40 mg/kg) in the non-transmission season. A follow-up stool examination was made 50 days after treatment. Among the 220 cases followed, 22 were found stool-egg-positive, with an overall cure rate of 90%, and 99% reduction of infection intensity (eggs per gram stool). No significant difference was found in cure rates between the 2 areas (89.7% vs 90.3%). The efficacy of the drug in the area with repeated chemotherapy was not significantly different from that in the newly identified endemic focus. This study, therefore, suggests that the efficacy of praziquantel against S. japonicum has not changed in the Dongting Lake region after more than 14 years of mass chemotherapy, and there is no evidence of tolerance or resistance of S. japonicum against praziquantel.

Susceptibility of Schistosoma japonicum to praziquantel in China.

To look for possible evidence of the development of resistance in Schistosoma japonicum to praziquantel, we conducted a field study in China. During the non-transmission period of schistosomiasis a random sample of 2860 individuals from six villages in three provinces of China were examined using a parasitological stool examination. Of the 372 stool-positive subjects, 363 subjects were treated with a single oral dose of 40 mg/kg of praziquantel. Six to Seven weeks after treatment, of 334 subjects examined using the same stool examination, stool-negative results were found in 319 patients which represents a 95.5% parasitologic cure rate. Fifteen subjects still excreting eggs were treated a second time with the same dose of praziquantel. All stool samples, including those from participants re-treated with praziquantel, were re-examined 12 weeks after the first treatment and no stool-positive subjects were found. The results indicate that there was no evidence for reduced susceptibility of S. japonicum to praziquantel despite its extensive use in the main endemic areas of China for more than 10 years. The in vitro responses to praziquantel of cercariae, miracidia and eggs of S. japonicum compared with S. mansoni demonstrate that the cercariae, miracidia and eggs of S. japonicum are more sensitive to praziquantel than those of S. mansoni. More sensitive worms would be less likely to develop resistance and this could explain why no evidence for resistance was found in S. japonicum in China.

Determinants of hepato- and spleno-megaly in Hunan, China: cross-sectional survey data from areas endemic for schistosomiasis.

In order to understand the determinants of schistosome-related hepato- and spleno-megaly better, 14,002 subjects aged 3-60 years (59% male; mean age = 32 years) were randomly selected from 43 villages, all in Hunan province, China, where schistosomiasis caused by Schistosoma japonicum is endemic. The abdomen of each subject was examined along the mid-sternal (MSL) and mid-clavicular lines, for evidence of current hepato- and/or spleno-megaly, and a questionnaire was used to collect information on the medical history of each individual. Current infections with S. japonicum were detected by stool examination. Almost all (99.8%) of the subjects were ethnically Han by descent and most (77%) were engaged in farming. Although schistosomiasis appeared common (42% of the subjects claiming to have had the disease), only 45% of the subjects said they had received anti-schistosomiasis drugs. Overall, 1982 (14%) of the subjects had S. japonicum infections (as revealed by miracidium-hatching tests and/or Kato-Katz smears) when examined and 22% had palpable hepatomegaly (i.e. enlargement of at least 3 cm along the MSL), although only 2.5% had any form of detectable splenomegaly (i.e. a Hackett's grade of at least 1). Multiple logistic regression revealed that male subjects, fishermen, farmers, subjects aged > or = 25 years, subjects with a history of schistosomiasis, and subjects who had had bloody stools in the previous 2 weeks were all at relatively high risk of hepato- and/or spleno-megaly. In areas moderately endemic for Schistosoma japonicum, occupational exposure and disease history appear to be good predictors of current disease status among older residents. These results reconfirm those reported earlier in the same region.

Natural antibodies in Microtus fortis react with antigens derived from four stages in the life-cycle of Schistosoma japonicum.

The levels of antibodies which react with the cercarial antigens (CA), schistosomulum stage antigens (SSA), adult-worm antigens (AWA) and soluble egg antigens (SEA) of Schistosoma japonicum were investigated in Microtus fortis and albino mice, using an indirect ELISA. The M. fortis studied fell into three groups: animals caught in the wild; laboratory-bred animals left unchallenged; and laboratory-bred animals that had been challenged with S. japonicum (30 cercariae/animal) 15 days previously. There were also three groups of albino mice: those without infection; those studied 15 days after challenge infection; and those investigated 42 days after infection. The antibodies detected at the highest levels in the laboratory-bred, uninfected voles and in the wild-caught animals were those reacting with SSA, followed, in descending order, by those reacting with AWA, CA and SEA. The levels of natural antibodies to SSA and AWA in these voles were significantly higher than the corresponding levels observed in the uninfected mice and even in the mice infected 15 days previously. The levels of antibodies reacting with CA, SSA, SEA and AWA in the experimentally infected M. fortis were 1.9-, 2.2-, 1.5- and 2.1-fold higher, respectively, than those in the laboratory-bred but uninfected voles. The observations indicate that even uninfected M. fortis produce antibodies which react with S. japonicum, and this presumably results in the natural resistance to infection which has been reported in these rodents.

An evaluation of Schistosoma japonicum infections in three villages in the Dongting lake region of China. I. Prevalence, intensity and morbidity before the implementation of adequate control strategies.

We examined three Chinese villages (one farming village and two fishing villages) in an area highly endemic for schistosomiasis japonica in order to study the prevalence, intensity of infection and the associated morbidities before the implementation of adequate control strategies. Socio-economic status, medical histories including the frequency and type of water contact, physical examinations, parasitological examinations and questionnaires relevant to the knowledge of schistosomiasis were performed on a random sample of 1542 individuals (45% female; 55% male). The prevalence of Schistosoma japonicum was 9.4% in the farming village and 16.5 and 26.2% in the fishing villages. Eighty-three percent of the infected population had light infections (8-100 eggs per gram stool (epg)) and only 6% had heavy infections (> 400 epg). Both the prevalence and intensity of infection varied significantly (P < 0.01) with the frequency of water contact. All the morbidity indicators (weakness, inability to work, diarrhoea, hepatomegaly and splenomegaly) were significantly higher (P < 0.01) among those infected with S. japonicum. Knowledge of schistosomiasis, in general, was unsatisfactory in all three villages; 12.4% of the population was infected when their knowledge of schistosomiasis was good, whereas 26.6% of the population was infected when their knowledge was poor. Further, it appears that schistosomiasis control based on selective chemotherapy (praziquantel) of randomly selected stool-positive individuals was ineffective in significantly reducing the prevalence of S. japonicum and its associated clinical manifestations in the villages under study.

Schistosoma japonicum infection among migrant fishermen in the Dongting Lake region of China.

An epidemiological survey of Schistosoma japonicum infection was made among 316 persons, fishermen and their family members, in 5 sites of the Dongting Lake region. Their medical history was taken and physical and stool examinations were carried out. The infected persons were divided into 3 groups; 2 were treated with one or 2 doses of praziquantel and one, the control group, was given no treatment. The results showed a high overall prevalence of 41% with an intensity of infection of 34 eggs per gram (epg) in a single stool specimen. The individuals with a history of treatment had a lower prevalence (45/167, 26.9%) than those with no history of treatment (85/149, 57%) (P less than 0.01). The mean intensity of infection of the treated group, 27.8 epg, was also lower than that of the untreated group, 39.1 epg (P less than 0.01). Symptoms within the past 2 weeks were reported by 31% of the individuals surveyed. Individuals excreting eggs had a higher rate of symptoms (56.2%) than those not excreting eggs (13.4%) (P less than 0.01). The proportions of those not excreting eggs in the treated groups were 77.8% and 84.1%, compared with 14.6% in the control group. The survey revealed relatively high endemicity among fishermen and their family members. Treatment and health education of fishermen should be encouraged to control transmission.