Octaarginine Improves the Efficacy of Nitazoxanide against Cryptosporidium parvum.

Cryptosporidiosis is an intestinal disease that affects a variety of hosts including animals and humans. Since no vaccines exist against the disease till date, drug treatment is the mainstay of disease control. Nitazoxanide (NTZ) is the only FDA-approved drug for the treatment of human cryptosporidiosis. However, its efficacy in immunocompromised people such as those with AIDS, in malnourished children, or those with concomitant cryptosporidiosis is limited. In the absence of effective drugs against cryptosporidiosis, improving the efficacy of existing drugs may offer an attractive alternative. In the present work, we have assessed the potential of the cell-penetrating peptide (CPP) octaarginine (R8) to increase the uptake of NTZ. Octaarginine (R8) was synthetically attached to NTZ in an enzymatically releasable manner and used to inhibit growth of Cryptosporidium parvum in an in vitro culture system using human ileocecal adenocarcinoma (HCT-8) cell line. We observed a significant concentration-dependent increase in drug efficacy. We conclude that coupling of octaarginine to NTZ is beneficial for drug activity and it represents an attractive strategy to widen the repertoire of anti-cryptosporidial therapeutics. Further investigations such as in vivo studies with the conjugate drug will help to further characterize this strategy for the treatment of cryptosporidiosis.

Linguatula serrata in an imported dog in Germany: Single-case or emerging disease?

Linguatula serrata is a worm-like parasite with zoonotic potential that inhabits the nasal cavities of canids. Although most cases of linguatulosis are associated with unspecific and rather mild respiratory symptoms, cases of unusual infestations and severe courses in both animals and humans have been reported. In central and northern Europe, the pathogen used to appear only sporadically, however, within the last few years the number of detections has increased noticeably. In July 2020 an approximately nine-month-old dog, imported from Romania, was presented in a veterinary practice in Gotha, central Germany, due to persistent worsening cough. Despite antibiotic treatment the tussis became more severe until the dog expectorated multiple worm-like structures. Three of these specimens were sent to the Institute of Parasitology (Faculty of Veterinary Medicine, University of Leipzig, Leipzig) for morphological and genetic species identification. The latter was based on a 1000-bp fragment of the mitochondrial cytochrome c oxidase subunit I gene (cox1) and the complete nuclear 18S rRNA gene. The dog presented in this study suffered from a severe respiratory impairment caused by worm-like parasites inhabiting its upper respiratory tract. The detected parasites were morphologically identified as female specimens of the so-called tongue-worm L. serrata, which was confirmed by pairwise alignment and phylogenetic analysis of the produced sequences. We report an unusually severe case of L. serrata infection in an imported dog and discuss the spread of this potentially dangerous parasite in central and northern Europe.

A simple and efficient transfection protocol for Cryptosporidium parvum using Polyethylenimine (PEI) and Octaarginine.

The transfection of Cryptosporidium represents a major challenge, and current protocols are based on electroporation of freshly excysted sporozoites using a rather large amount of plasmid DNA which typically has a very poor yield. In this study, we report a fast and simple protocol for transfection of Cryptosporidium parvum that takes advantage of the DNA condensing power of the poly cationic polymer polyethylenimine (PEI) and the gene delivery property of the short cell-penetrating peptide octaarginine. Our novel protocol requires a very low amount of plasmid DNA and does not necessitate special laboratory equipment to be performed. Transfection appears to be more efficient in oocysts just triggered for excystation than the excysted sporozoites. Altogether, the application of octaarginine with PEI allows efficient transfection. To the best of our knowledge, this is the first report on an electroporation-free protocol for transfection of sporozoites of a Cryptosporidium species.