[Capillaria hepatica infection in rodents from Anning Prefecture of Yunnan Province and experimental research on host animals].

OBJECTIVE: To investigate the prevalence of Capillaria hepatica in rodents from Anning Prefecture, Yunnan, and observe the susceptibility of C. hepatica to SD rats and KM mice. METHODS: Rodents were trapped in a cultivated filed of Wenquan Town, Annning from March 2010 to March 2012. The species of rodents were identified. The liver was examined and a microscopic examination of tissue was performed by the tissue press technique for the presence of the typical bipolar eggs, adults or larval stages. The prevalence of C. hepatica in rodents was calculated. C. hepatica eggs were collected and cultured in vitro. Each SD rat or KM mouse was orally infected with approximately 1 000 C. hepatica eggs. The control groups with 4 SD rats or 4 KM mice received only normal saline. The experimental animals were euthanized at the 30th and 80th day post infection. Collected liver samples were processed for gross pathological and histological section examination. RESULTS: A total of 115 rodents were captured and examined. C. hepatica eggs were found in 26 (22.6%) rodents. There was no significant difference in the prevalence between female (22.5%, 18/80) and males (22.9%, 26/115) (P > 0.05). The highest prevalence was found in Rattus norvegicus (10/11). Pathologi cal findings showed numerous white-yellow small nodules ranged from 0.1-0.2 cm in diameter. Under light microscope, C. hepatica eggs were ovoid [(50-65) microm x (25-30) microm]. At the 30th day post-infection, there were several adult worms and their eggs delimited by a fibrous capsule, and septal fibrosis formations occurred in the liver of SD rat. No worm or eggs were found in the mouse liver, but the liver presented inflammatory cell infiltration. At the 80th day post-infection, live worms disappeared from the focal lesions in the liver of SD rat, being replaced by partially calcified worm debris. Mature worms and eggs were seen in the KM mouse liver, however, septal fibrosis was absent. CONCLUSION: This study has documented a high prevalence of C. hepaticum in R. norvegicus from Anning Prefecture. SD rat and KM mouse are the susceptible hosts of C. hepatica.

[Effect of male Trichinella spiralis to female fertility].

OBJECTIVE: To study the life span of Trichinella spiralis male adult and its effect on female fertility. METHODS: 22 Kunming mice were divided into three groups. Group A was orally inoculated by encysted larvae, dissected at day 5, 10, 20, and 30 post infection, adult worms were then collected and counted. Group B was fed with 5-day adults (70 females + 30 males/mouse). Group C was fed with 5-day females (70/mouse) only. Groups B and C were sacrificed 30 days post inoculation, larvae were collected to calculate reproductive capacity index (RCI). Male worms of day 5, 10, 20 and 30 were fixed in glutaraldehyde for scanning electron microscopy (SEM) and transmission electron microscopy (TEM). RESULTS: 20 days after infection, there were more males than females in the intestines of group A mice (female: male = 1:1.3), but much more males than females on the 30th day after infection (female: male =1:8.5). Mice of groups B and C were infected by 5-day adults successfully with a RCI of 154.90 +/- 2.62 and 13.77 +/- 1.67 respectively (t=111.26, P<0.01). SEM revealed the copulatory bell of 5-day male adult, alae erected, and genital pore uncovered; while alae of 20-day male collapsed, genital pore was covered. Under TEM, there were large number of mature sperms within spermaductus of 5-day and 10-day males, but only a few immature sperms in the testis of 20-day and 30-day males. CONCLUSION: Male adults can survive longer than one month in host. 5-day female and male adults and 5-day females can infect mice orally, with a significantly higher fertility in the former.

[Preliminary investigation on Paragonimus in Lvchun county of Yunnan province].

69 crabs were collected from Daxing, Gekui and Niukong townships of Lvchun county, Yunnan Province in 2006 and excysted metacercariae were only obtained from crabs of Niukong. The infection rate was 27.6% (8/29) with an average metacercaria number of 2.25 each crab. No encysted metacercariae were found. The excysted metacercariae were morphologically identified as Paragonimus proliferus.

[Scanning electron microscopy of Paragonimus proliferus].

OBJECTIVE: To identify the species of Paragonimus proliferus with scanning electron microscopy (SEM) based on the surface structure of excysted metacercariae, adult worms and eggs. METHODS: Crabs were collected from the endemic area of P. proliferus and excysted metacercariae were separated. Adult worms at different ages and eggs were obtained from the experimentally infected rats. After being fixed by 2.5% glutardialdehyde and 1% osmic acid, alcohol dehydration, gilded by ion spatter, the specimens were observed under SEM by STEREOSCAN-100. RESULTS: The cuticular spines of excysted metacercariae distributed in single pattern, bayonet-shaped or scale-shaped. There were 6 dome-shape papillae around the rim of the ventral sucker symmetrically arranged. The cuticular spines of different age adult worms distributed in group pattern, relatively denser and more regularly arranged in the anterior part than the posterior part of the worm body. The shape and arrangement of the cuticular spines on adult worms at different ages were basically uniform. The surface of eggshell including the operculum was generally smooth. The shell rim joining the operculum was thick and prominent. A knot-like prominence was observed at the aboperculum end. CONCLUSION: The cuticular spines of both excysted metacercariae and adult worms of P. proliferus show its own characteristics, but the size and shape of the cuticular spines among individuals or different parts of the same specimen show certain differences.

Sequence analyses of ITS2 and CO1 genes of Paragonimus proliferus obtained in Yunnan province, China and their similarities with those of P. hokuoensis.

Among about 50 Paragonimus species, Paragonimus proliferus is a rare species characterized by extremely large metacercariae, most of which are present excysted in the crab hosts. Recently, this species was discovered by us in northern Vietnam as the first record outside of China. DNA sequences of both second internal transcribed spacer region (ITS2) and cytochrome oxidase subunit 1 gene (CO1) genes of the metacercariae and adult worms of P. proliferus of the Vietnamese isolates were identical with those of Paragonimus hokuoensis in the DNA database of the GenBank. To confirm those observations and to clarify the molecular phylogenetic status of P. proliferus, we determined the ITS2 and CO1 sequences of the metacercariae of P. proliferus obtained in Yunnan province, China where the original specimen was discovered. The results show that both ITS2 and CO1 sequences of P. proliferus of the Chinese isolates are identical with those of P. proliferus of the Vietnamese isolates and are also identical with those of P. hokuoensis that appeared in the DNA database (obtained in Yunnan province), suggesting the synonymy of P. hokuoensis with P. proliferus. By phylogenetic tree analyses, all samples of P. proliferus from China and Vietnam together with P. hokuoensis constructed a distinct group within, or very close to, Paragonimus skrjabini complex in both trees.

Effects of frozen storage on the structure of sarcocysts in pig muscle and implications in taxonomic studies.

UNLABELLED: The morphology of the cyst wall of Sarcocystis has unique characteristics that can be used in species identification. To find a suitable way to preserve Sarcocystis cyst samples for species identification, by light microscopy and electron microscopy, we recorded the morphological changes in the cysts of Sarcocystis suihominis and Sarcocystis miescheriana from pig muscle, induced by storage at -20 degrees C. Comparisons were made between fresh cysts and those subjected to frozen storage for periods of 3 days, 20 days and 30 days. RESULTS: cyst wall of the two Sarcocystis species appeared unaffected by storage. There was no obvious change in the length, nor in the width of the protrusions after storage (P>0.05), but the structure of the bradyzoite in the sarcocyst was in many cases disintegrated at -20 degrees C in 20 days for S. miescheriana and 30 days for S. suihominis. To our knowledge this is the first report that Sarcocystis cyst in muscle can be stored at -20 degrees C before and remain suitable for ultrastructural morphological study. Consequently, this paper proposes freezing as a convenient storage method for samples used in taxonomic studies of Sarcocystis.