Cathepsin B Homologue at the Interface between a Parasitic Nematode and Its Intermediate Host

Abstract

Parelaphostrongylus tenuis is a parasitic nematode that causes a debilitating neurologic disease in many North American cervids and domestic livestock species. We produced a PCR-based cDNA library from infective larvae (L3) in order to identify molecules that mediate parasitism. A dominant 1,250-bp amplicon encoded a homologue of cathepsin B cysteine proteases. The sequence incorporated a C29G substitution in the putative active site. Antibodies generated against a recombinant form detected the native protein (PtCPR-1) in Western blot assays of L3, but not adult worm, extracts. Immunohistochemical methods revealed that PtCPR-1 synthesis was restricted to larval stages within the snail intermediate host (Triodopsis sp.), beginning as early as 2 days postinfection (dpi) of snails. The protein was present in the intestine and luminal contents and was lost from larvae over time. Concurrent studies showed that larvae induced an immune response in snails beginning at 1 dpi. Layers of hemocytes encapsulated larvae immediately after infection, and granuloma-like structures formed around parasites in chronic infections. Loss of PtCPR-1 from L3 and its accumulation in host tissues coincided with degeneration of granuloma architecture 90 to 105 dpi. Fully developed L3 emerged from the snail at this time. Our data implicate PtCPR-1 in larval development and possibly in the emergence of P. tenuis from the intermediate host. Emerged L3 survived desiccation and cold stress, suggesting that they could remain infectious in the environment. Molecules promoting emergence would facilitate dispersal of L3 and increase the likelihood of transmission to definitive hosts.

Integrative Approach for the Reliable Detection and Specific Identification of the Microsporidium Loma morhua in Atlantic Cod (Gadus morhua)

“…PCR was conducted using 20–50 ng of genomic DNA and amplicons were separated by gel electrophoresis, as described previously (Duffy et al. , ).…”

“…Various primer sets (Table 1) were utilized to amplify DNA from L. morhua (rDNA) and from Atlantic cod (mitochondrial cytochrome b). PCR was conducted using 20-50 ng of genomic DNA and amplicons were separated by gel electrophoresis, as described previously (Duffy et al 2002(Duffy et al , 2006.…”

Cloning and analysis of a Trichinella pseudospiralis muscle larva secreted serine protease gene

“…Sections were deparaffinized and processed for staining with normal rat immunoglobulins (2 μg ml −1 of immunoglobulins preciptated from serum with 40% ammonium sulfate) or with rat mAbs 18H, 9H8 or 1H7 (culture supernatants diluted 1:2) (Beiting et al, 2004; Duffy et al, 2006). Antibody binding was detected with peroxidase-conjugated goat anti-rat IgG diluted in 10% normal mouse serum, using the substrate 3-amino-9-ethyl-carbazole (AEC) (Sigma).…”

Analysis of Heavy-Chain Antibody Responses and Resistance to Parelaphostrongylus tenuis in Experimentally Infected Alpacas

“…The definitive host is the white-tailed deer, Odocoileus virginianus, and terrestrial gastropods serve as intermediate hosts. A variety of grazing animals that are sympatric with white-tailed deer can be exposed to P. tenuis by consuming infected gastropods or vegetation contaminated with the third-stage larvae (L3) that emerge from them (13). Infections are asymptomatic in white-tailed deer; however, in other susceptible species, the parasite migrates aberrantly and can cause severe neurologic disease (reviewed in reference 26).…”

“…Conventional antibodies are known to be protective in nematode infections (5,6,20). The structure of the variable domains of HC IgGs is such that they are effective inhibitors of enzymes (8,12,15,42), and nematode proteases are numerous and prominent at the host-parasite interface (13,35,(38)(39)(40). Thus, HC IgGs have unique potential to contribute to protective immunity against nematodes.…”