Molecular Characterization of the North American Lung Fluke Paragonimus kellicotti in Missouri and its Development in Mongolian Gerbils

Abstract

Human paragonimiasis is an emerging disease in Missouri. To characterize local parasites, we examined crayfish from three rivers. Metacercaeriae consistent with Paragonimus kellicotti were detected in 69%, 67%, and 37% of crayfish from the Big Piney, Huzzah, and Black Rivers, respectively. Sequencing of the second internal transcribed spacer and other DNA markers confirmed the species identification and the presence of identical parasite sequences in clinical specimens from two human cases. Mongolian gerbils were infected by intraperitoneal injection with 3–8 metacercariae. Most gerbils died 15–49 days post-infection. Necropsies showed pulmonary hemorrhage with necrosis, and flukes as long as 8 mm were recovered from intrathoracic tissues. Western blot analysis using P. kellicotti antigen showed a strong antibody response in gerbils 39 days post-infection. These results demonstrate that P. kellicotti is common in Missouri crayfish. The gerbil model may be useful for research on the pathogenesis, immunology, and treatment of paragonimiasis.

Serological Diagnosis of North American Paragonimiasis by Western Blot Using Paragonimus kellicotti Adult Worm Antigen

“…Molecular phylogenetic studies using mitochondrial or non-coding DNA markers indicate that P. westermani and P. kellicotti are not closely related within the genus Paragonimus. 5,13,14 However, these studies provide little information about cross-reactivity of diagnostic antigens. In the past, worm extracts or e/s antigens from a number of old-world Paragonimus species such as P. westermani, P. heterotremus, and Paragonimus africanus have been used for serological diagnosis of paragonimiasis.…”

“…Mongolian gerbils were infected with P. kellicotti metacercariae as previously described. 5 Venous blood was collected from infected Mongolian gerbils at various time points after infection (p.i.). Plasma was separated and preserved at −20 C until use.…”

“…Adult P. kellicotti worms were obtained from experimentally infected Mongolian gerbils, and soluble total worm antigen was prepared as described previously. 5 A P. westermani Chafee extract of adult worms was provided by CDC. 6,7 Excretory/secretory (e/s) antigens of P. kellicotti were obtained by culturing two adult flukes (65 days p.i.)…”

“…Western blot was performed with three types of antigen (P. kellicotti total worm antigen, P. kellicotti e/s antigen, and P. westermani total worm antigen) as described previously for P. kellicotti. 5 Briefly, 10 μg of antigen per cm was separated on a 4-12% reducing gel, blotted onto a nitrocellulose membrane, blocked at room temperature for 30 min using 5% non-fat dry milk (BioRad, Hercules, CA) in PBS, and incubated with patient or animal serum or plasma at a dilution of 1:100 or 1:50, respectively. Serum and secondary antibody were diluted in PBS-tween and incubated at 37 C for 1 and 2 h, respectively.…”

A review on the molecular characterization of digenean parasites using molecular markers with special reference to ITS region

“…ITS-2 motifs can be utilized for species identifi cation along with RNA secondary structures. Fischer et al (2011) genetically characterized metacercariae of Paragonimus kellicotti found in cray fi sh from USA using ITS-2 region, ITS-1, 5.8S and 28S rDNA sequences. The ITS-2 sequences from metacercariae of P. kellicotti showed 99 % similarity with GenBank sequences of P. kellicotti along with one bp deletion at site 55 and transversion at site 298.…”