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.

Figure 1.

A, Carapace of the cephalothorax removed to show a Paragonimus kellicotti–type metacercaria (arrow) in the heart of a Orconectes punctimanus crayfish from the Huzzah River near Steelville, Missouri. B, Isolated metacercaria of P. kellicotti showing the outer and inner cyst walls. C, P. kellicotti larvae hatching from a metacercarial cyst. D, Small unidentified metacercaria commonly found in the heart and tail muscle of Missouri crayfish. Cy = cyst; icy = inner cyst; ocy = outer cyst. Scale bars: A = 1 cm, B–D = 100 μm.

Figure 2.

A, Agarose electrophoresis showing detection of Paragonimus kellicotti DNA in clinical specimens by polymerase chain reaction. Lane 1, no template control; lane 2, template was genomic DNA from a non-infected human; lane 3, template DNA isolated from a lung biopsy specimen from a patient with paragonimiasis; lane 4, 100-basepair DNA marker; lane 5, template DNA isolated from the sputum of a patient with paragonimasis that contained P. kellicotti eggs; DNA was isolated from metacercariae isolated from Missouri crayfish. B, Western blot using P. kellicotti native antigen probed with serum samples obtained from experimentally infected gerbils. Lanes 1–3, serum samples from three gerbils that died 41, 39, and 45 days after intraperitoneal injection with four or five metacercariae, respectively. All gerbils had adult worms in lungs and eggs in the pleural cavity at necropsy; lane 4, serum sample from 25 days post-infection from the same gerbil that was tested in lane 3; lanes 5–7, serum samples from uninfected control gerbils.

Figure 3.

A, Opened cyst with four adult Paragonimus kellicotti (arrows) in the lung of an experimentally infected Mongolian gerbil 42 days post-infection, B, P. kellicotti egg recovered from the pleural cavity an infected gerbil. C, Subadult P. kellicotti from the pleural cavity of an infected gerbil 35 days post-infection. Ventral suckers were larger than oral suckers in all specimens. D, Midbody region of an adult P. kellicotti showing the branched ovary and numerous eggs in the uterus at 42 days post-infection. Ca = cecum; op = operculum; os = oral sucker; ut = uterus; vs = ventral sucker; te = testis; vi = vitelline glands; ov = ovary. Scale bars: A = 1 cm, B = 10 μm, C–D = 1 mm.