Infection of sheep by Echinococcus multilocularis in Gansu, China: evidence from mitochondrial and nuclear DNA analysis.

BACKGROUND: In the normal life cycle of the parasite (Echinococcus multilocularis) that causes alveolar echinococcosis, domestic and wild carnivores act as definitive hosts, and rodents act as intermediate hosts. The presented study contributes to the research on the distribution and transmission pattern of E. multilocularis in China having identified sheep as an unusual intermediate host taking part in the domestic transmission of alveolar echinococcosis in Gansu Province, China. METHODS: From 2020 to 2021, nine whitish different cyst-like were collected from the liver of sheep in Gansu Province for examination. A near complete mitochondrial (mt) genome and selected nuclear genes were amplified from the cyst-like lesion for identification. To confirm the status of the specimen, comparative analysis with reference sequences, phylogenetic analysis, and network analysis were performed. RESULTS: The isolates displayed ≥ 98.87% similarity to E. multilocularis NADH dehydrogenase sub-unit 1 (nad1) (894 bp) reference sequences deposited in GenBank. Furthermore, amplification of the nad4 and nad2 genes also confirmed all nine samples as E. multilocularis with > 99.30% similarity. Additionally, three nuclear genes, pepck (1545 bp), elp-exons VII and VIII (566 bp), and elp-exon IX (256 bp), were successfully amplified and sequenced for one of the isolates with 98.42% similarity, confirming the isolates were correctly identified as E. multilocularis. Network analysis also correctly placed the isolates with other E. multilocularis. CONCLUSIONS: As a result of the discovery of E. multilocularis in an unusual intermediate host, which is considered to have the highest zoonotic potential, the result clearly demonstrated the necessity for expanded surveillance in the area.

Update on the genetic diversity and population structure of Echinococcus granulosus in Gansu Province, Tibet Autonomous Region, and Xinjiang Uygur Autonomous Region, Western China, inferred from mitochondrial cox1, nad1, and nad5 sequences.

The identification of additional Echinococcus granulosus sensu lato (s.l.) complex species/genotypes in recent years raises the possibility that there might be more variation among this species in China than is currently understood. The aim of this study was to explore intra- and inter-species variation and population structure of Echinococcus species isolated from sheep in three areas of Western China. Of the isolates, 317, 322, and 326 were successfully amplified and sequenced for cox1, nad1, and nad5 genes, respectively. BLAST analysis revealed that the majority of the isolates were E. granulosus s.s., and using the cox1, nad1, and nad5 genes, respectively, 17, 14, and 11 isolates corresponded to Elodea canadensis (genotype G6/G7). In the three study areas, G1 genotypes were the most prevalent. There were 233 mutation sites along with 129 parsimony informative sites. A transition/transversion ratio of 7.5, 8, and 3.25, respectively, for cox1, nad1, and nad5 genes was obtained. Every mitochondrial gene had intraspecific variations, which were represented in a star-like network with a major haplotype with observable mutations from other distant and minor haplotypes. The Tajima's D value was significantly negative in all populations, indicating a substantial divergence from neutrality and supporting the demographic expansion of E. granulosus s.s. in the study areas. The phylogeny inferred by the maximum likelihood (ML) method using nucleotide sequences of cox1-nad1-nad5 further confirmed their identity. The nodes assigned to the G1, G3, and G6 clades as well as the reference sequences utilized had maximal posterior probability values (1.00). In conclusion, our study confirms the existence of a significant major haplotype of E. granulosus s.s. where G1 is the predominant genotype causing of CE in both livestock and humans in China.

PCR-RFLP assay confirms the existence of different mitochondrial lineages of Taenia hydatigena including a possible geographically restricted group.

Taenia hydatigena is a widespread tapeworm of canids (primarily dogs) that causes cysticercosis in ruminants (domestic and wild) and manifests as depression and weakness secondary to various hepatic damages and sometimes mortality in young animals, although, commonly encountered cases are asymptomatic. In most taeniids, genetic polymorphism has been found to impact host preferences, distribution, disease epidemiology and management. Recently, we identified two main mitochondrial lineages of T. hydatigena in China, and here, we examined the mitochondrial nad4-nad5 genes of T. hydatigena from China, Nigeria, Pakistan and Sudan to assess the intraspecies variation of isolates from these countries and also the distribution of the distinct mitochondrial groups. In addition to China, haplogroup B variant was found in Pakistan, while haplogroup A demonstrated a widespread distribution. We then designed a PCR-restriction fragment length polymorphism (PCR-RFLP) assay using XmiI (AccI) and RsaI (AfaI) restriction enzymes to differentiate members of both haplogroups. This result provides more molecular evidence supporting the existence of distinct mitochondrial variants of T. hydatigena. The epidemiological significance of these different mitochondrial groups remains to be explored further. The current PCR-RFLP assay offers a useful molecular approach for investigating the genetic population structure of T. hydatigena in enzootic regions and in identifying/discriminating the different mitochondrial groups (haplogroups A and B).

A new molecular nomenclature for Taenia hydatigena: mitochondrial DNA sequences reveal sufficient diversity suggesting the assignment of major haplotype divisions.

Cysticercosis caused by the metacestode larval stage of Taenia hydatigena formerly referred to as Cysticercus tenuicollis is a disease of veterinary importance that constitutes a significant threat to livestock production worldwide, especially in endemic regions due to condemnation of visceral organs and mortality rate of infected young animals. While the genetic diversity among parasites is found to be potentially useful in many areas of research including molecular diagnostics, epidemiology and control, that of T. hydatigena across the globe remains poorly understood. In this study, analysis of the mitochondrial DNA (mtDNA) of adult worms and larval stages of T. hydatigena isolated from dogs, sheep and a wild boar in China showed that the population structure consists of two major haplogroups with very high nucleotide substitutions involving synonymous and non-synonymous changes. Compared with other cestodes such as Echinococcus spp., the genetic variation observed between the haplogroups is sufficient for the assignment of major haplotype or genotype division as both groups showed a total of 166 point-mutation differences between the 12 mitochondrial protein-coding gene sequences. Preliminary analysis of a nuclear protein-coding gene (pepck) did not reveal any peculiar changes between both groups which suggests that these variants may only differ in their mitochondrial makeup.

Preliminary information on the prevalence and molecular description of Taenia hydatigena isolates in Pakistan based on mitochondrial cox1 gene.

Taenia hydatigena is a cestode of veterinary importance. Infection with the metacestode larval stage results in cysticercosis, which poses a serious challenge to the livestock industry worldwide. Globally, there are numerous reports on cysticercosis caused by T. hydatigena in sheep and goat but a lack of data on the prevalence and genetic diversity exists for Pakistan. We designed this study to provide an insight into the disease status as well as investigate the genetic variation among the recovered isolates based on the mitochondrial cox1 gene. In this study, we examined small ruminants (sheep and goats) slaughtered in Faisalabad in eastern Punjab province of Pakistan for T. hydatigena metacestodes and described the population structure and genetic variation using the cytochrome c oxidase subunit 1 (cox1) mitochondrial gene. Overall, a prevalence of 4.40% (goat =4.67% sheep = 4.07%) from a total of 2225 small ruminant carcasses (sheep = 983, goats = 1242) was observed. Based on the NCBI BLAST search and Bayesian phylogeny, the identity of all isolates was confirmed via their nucleotide sequences. The diversity indices indicated a high haplotype and a low nucleotide diversity with 43 haplotypes from 98 isolates. The results also show the existence of unique haplotypes of T. hydatigena in Pakistan as demonstrated by the significant negative values of Tajima's D and Fu's Fs neutrality test suggesting a recent population expansion. The median-joining network of the partial cox1 sequence dataset showed the existence of two main haplotypes detected in both sheep and goat populations. This study shows that the prevalence of cycticercosis due to T. hydatigena is below 5% in sheep and goats in Faisalabad, Punjab, Pakistan. The molecular analysis of the partial cox1 gene also indicates a high degree of genetic variation with the existence of rare haplotypes. These findings represent a preliminary report on the prevalence and genetic variation of T. hydatigena in Pakistan and serve as baseline information for future studies on the prevalence and population structure of T. hydatigena in the country.

A steroid fraction of chloroform extract from bee pollen of Brassica campestris induces apoptosis in human prostate cancer PC-3 cells.

Bee pollen of Brassica campestris L. is widely used in China as a natural food supplement and an herbal medicine in strengthening the body's resistance against diseases including cancer. The present study was carried out to investigate the effect of a steroid fraction of chloroform extract from bee pollen of Brassica campestris L. on human cancer cell viability. Our studies show that among nine cancer cell lines of different origin (PC-3, LNCaP, MCF-7, Hela, BEL-7402, BCG-823, KB, A549 and HO8910), this steroid fraction displayed the strongest cytotoxicity in human prostate cancer PC-3 cells. The mode of cell death appeared to be apoptosis in PC-3 cells, as shown by flow-cytometric analysis and fluorescence microscopes. Caspase-3 activity was obviously enhanced after the cells were treated with the fraction. A time-dependent decrease in the expression of anti-apoptotic protein Bcl-2 was also observed by Western blot analysis. It is suggested that the steroid fraction could induce cytotoxicity in prostate cancer PC-3 cells by triggering apoptosis. The studies indicate that the steroid fraction of chloroform extract from bee pollen of Brassica campestris L. may be a promising candidate for the treatment of advanced prostate cancer.

Brassinolide, a plant sterol from pollen of Brassica napus L., induces apoptosis in human prostate cancer PC-3 cells.

Brassinolide is a plant sterol first isolated from pollen of rape (Brassica napus L.). The present study was carried out to investigate the effect of brassinolide on androgen-independent human prostate cancer PC-3 cell viability. Results showed that brassinolide could induce a time and concentration-dependent cytotoxicity in PC-3 cells. The mode of cell death appeared to be predominately apoptosis, as shown by flow-cytometric analysis, fluorescence and transmission electron microscopes. Caspase-3 activity was obviously increased after brassinolide treatment. Western blot studies indicated that treatment with brassinolide triggered a time-dependent decrease in the expression of anti-apoptotic protein Bcl-2. We suggest that brassinolide could induce cytotoxicity in PC-3 cells by triggering apoptosis. Brassinolide might therefore be a promising candidate for the treatment of prostate cancer.