Fumarate respiration of Fasciola flukes as a potential drug target.

Fascioliasis is a neglected tropical zoonotic disease caused by liver flukes belonging to the genus Fasciola. The emergence of resistance to triclabendazole, the only World Health Organization-recommended drug for this disease, highlights the need for the development of new drugs. Helminths possess an anaerobic mitochondrial respiratory chain (fumarate respiration) which is considered a potential drug target. This study aimed to evaluate the occurrence of fumarate respiration in Fasciola flukes. We analyzed the properties of the respiratory chain of Fasciola flukes in both adults and newly excysted juveniles (NEJs). Fasciola flukes travel and mature through the stomach, bowel, and abdominal cavity to the liver, where oxygen levels gradually decline. High fumarate reductase activity was observed in the mitochondrial fraction of adult Fasciola flukes. Furthermore, rhodoquinone-10 (RQ10 Em'= -63 mV), a low-potential electron mediator used in fumarate respiration was found to be predominant in adults. In contrast, the activity of oxygen respiration was low in adults. Rotenone, atpenin A5, and ascochlorin, typical inhibitors of mitochondrial enzymes in complexes I, II, and III, respectively, inhibit the activity of each enzyme in the adult mitochondrial fraction. These inhibitors were then used for in vitro viability tests of NEJs. Under aerobic conditions, NEJs were killed by rotenone or ascochlorin, which inhibit aerobic respiration (complex I-III), whereas atpenin A5, which inhibits complex II involved in fumarate respiration, did not affect NEJs. Moreover, ubiquinone-10 (UQ10 Em'= +110 mV), which is used in oxidative respiration, was detected in NEJs, in addition to RQ10. In contrast, under anaerobic conditions, rotenone and atpenin A5, which inhibit fumarate respiration (complex I-II), were crucial for NEJs. These findings demonstrate that NEJs have active hybrid respiration, in which they can properly use both oxygen and fumarate respiration, depending on oxygen availability. Thus, fumarate respiration is a promising drug target for Fasciola flukes, because it plays an essential role in both adults and NEJs.

Molecular characterization and phylogenetic analyses of Fasciola gigantica of buffaloes and goats in Punjab, Pakistan.

Fasciola gigantica is considered to be a major pathogen causing fasciolosis in the Indian subcontinent, resulting in production losses of millions of dollars in the livestock industry. Understading the dispersal origin and the patterns of spread of F. gigantica is important. A total of 53 Fasciola flukes collected from buffaloes and goats in Punjab, Pakistan between 2017 and 2018 were identified as F. gigantica based on the multiplex PCR for the phosphoenolpyruvate carboxykinase (pepck) and the PCR-restriction fragment length polymorphism (RFLP) for DNA polymerase delta (pold). A significant genetic difference between F. gigantica from buffaloes and goats was indicated by the genetic analyses of mitochondrial markers, NADH dehydrogenase subunit 1 (nad1) and cytochrome C oxidase subunit 1 (cox1). Phylogenetic analysis of the seventeen nad1 haplotypes of F. gigantica from Pakistan with those in neighbouring countries of the Indian subcontinent revealed that all the haplotypes identified in Pakistan were clustered in haplogroup A. fasciola gigantica with the eight haplotypes might be expanded in Pakistan from Indian origin, along with the migration of the domestic animals, since they were related to Indian haplotypes. In contrast, the remaining nine haplotypes were not shared with any neighbouring countries, suggesting independent origin, probably from neighbouring Middle East countries. However, cautious interpretation is required due to the very limited samples size of this study. Our study provides a proof of concept for a method that could be used to investigate the epidemiology of F. gigantica.

Specific increase of Fusobacterium in the faecal microbiota of neonatal calves infected with Cryptosporidium parvum.

The faecal microbiota plays a critical role in host health, with alterations in the human faecal microbial composition associated with various conditions, particularly diarrhoeal diseases. However, little is known about microbial changes during cryptosporidiosis, one of the most important diarrhoeal diseases caused by protozoa in cattle. In this study, alterations in the faecal microbiota of neonatal calves as a result of Cryptosporidium parvum infection were investigated on a C. parvum-positive farm. Comparisons were made among groups of C. parvum-infected, rotavirus-infected, and the pathogen-negative calves. A specific increase in the abundance of Fusobacterium was observed in the faecal microbiota of C. parvum-infected animals. Diarrhoea severity increased in accordance with the abundance of C. parvum and Fusobacterium. Moreover, the specific increase of Fusobacterium appeared to be a universal feature of C. parvum infection, since neonatal calves from geographically separated areas showed the same result. These observations indicated that the growth of Fusobacterium may be an important aggravating factor of cryptosporidiosis.