Molecular analyses of geographically dispersed Fasciola spp. isolates based on ribosomal, mitochondrial and nuclear molecular markers have revealed high levels of genetic diversity within liver fluke populations. To investigate the Fasciola population substructure across Pakistan 4 molecular markers were compared (fatty acid binding protein, fabp; phosphoenolpyruvate carboxykinase, pepck; random amplified polymorphic DNA, RAPD; mitochondrial NADH dehydrogenase, mt-nd1). Adult parasites (n = 595) were collected from buffalo and cattle across 4 provinces in Pakistan (Baluchistan, Gilgit Baltistan, Khyber Pakhtunkhwa, Punjab). Species classification of all 595 parasites was confirmed by the 3 gel-based markers (pepck, fabp and RAPD) as F. gigantica, except for the fabp marker which unexpectedly could not be amplified in 274 parasites (46%). Analysis of a subset of samples indicates the potential for mis-priming due to multiple genomic loci that match the fabp primer sequences resulting in negative PCR products in some cases. Sequence analysis of the mt-nd1 PCR products identified 29 haplotypes within the samples from Pakistan, the majority of which are unique to this study. None of the 29 haplotype sequences were identified in samples from Africa, highlighting the genetic diversity between geographically disparate liver fluke populations. Inconsistencies between Fasciola spp. molecular markers in this study highlights the need for multiple markers, validated on large numbers of geographically disparate parasites, to generate robust analyses of liver fluke genetic diversity. This study echoes other Fasciola spp. population studies and highlights the genetic diversity of F. gigantica populations in Pakistan that is comparable to observations of diversity throughout Asia.

Phosphoenolpyruvate carboxykinase (PEPCK) is involved in glycolysis in the cestode parasite, Raillietina echinobothrida; whereas, it executes a gluconeogenic role in its host, Gallus domesticus. Because of its differing primary function in the cestode parasite and its host, this enzyme is regarded as a plausible anthelmintic target. Hence, the biological significance of PEPCK in the parasite was analysed using siRNA against PEPCK from R. echinobothrida (RePEPCK). In order to find out the functional differences between RePEPCK and GdPEPCK (PEPCK from its host, G. domesticus), PEPCK genes from both sources were cloned, over-expressed, characterized, and some properties of the purified enzymes were compared. RePEPCK and GdPEPCK showed a standard Michaelis-Menten kinetics with Kmapp of 46.9 and 22.9 µm, respectively, for phosphoenolpyruvate and Kmapp of 15.4 µm for oxaloacetate in GdPEPCK decarboxylation reaction. Here, we report antagonist behaviours of recombinant PEPCKs derived from the parasite and its host. In search of possible modulators for PEPCK, few phytoestrogens were examined on the purified enzymes and their inhibitory constants were determined and discussed. This study stresses the potential of these findings to validate PEPCK as the anthelmintic drug target for parasitism management.

The well-known pathogens of fasciolosis, Fasciola hepatica (Fh) and Fasciola Gigantica (Fg), possess abundant mature sperms in their seminal vesicles, and thus, they reproduce bisexually. On the other hand, aspermic Fasciola flukes reported from Asian countries, which have no sperm in their seminal vesicles, probably reproduce parthenogenetically. The aim of this study was to reveal the origin of aspermic Fasciola flukes. The nuclear single copy markers, phosphoenolpyruvate carboxykinase and DNA polymerase delta, were employed for analysis of Fasciola species from China. The hybrid origin of aspermic Fasciola flukes was strongly suggested by the presence of the Fh/Fg type, which includes DNA fragments of both F. hepatica and F. gigantica. China can be regarded as the cradle of the interspecific hybridization because F. hepatica and F. gigantica were detected in the northern and southern parts of China, respectively, and hybrids flukes were distributed between the habitats of the two species. The Chinese origin was supported by the fact that a larger number of mitochondrial NADH dehydrogenase subunit 1 (nad1) haplotypes was detected in Chinese aspermic Fasciola populations than in aspermic populations from the neighbouring countries. Hereafter, ‘aspermic’ Fasciola flukes should be termed as ‘hybrid’ Fasciola flukes.

Phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.32) is an essential regulatory enzyme of glycolysis in helminths in contrast to its role in gluconeogenesis in their host. Previously we have reported that phytochemicals from Flemingia vestita (Family: Fabaceae), genistein in particular, have vermifugal action and are known to affect carbohydrate metabolism in the cestode, Raillietina echinobothrida. In order to determine the functional differences of PEPCK from the parasite and its avian host (Gallus domesticus), we purified the parasite enzyme apparently to homogeneity, and characterized it. The native PEPCK is a monomer with a subunit molecular weight of 65 kDa. The purified enzyme displayed standard Michaelis-Menten kinetics with Km value of 42·52 μM for its substrate PEP. The Ki for the competitive inhibitors GTP, GMP, ITP and IMP for the carboxylation reaction were determined and discussed. In order to identify putative modulators from plant sources, phytochemicals from F. vestita and Stephania glabra were tested on the purified PEPCK, which resulted in alteration of its activity. From our results, we hypothesize that PEPCK may be a potential target site for anthelmintic action.