Damin Luo

Multiple primer PCR for the identification of anisakid nematodes from Taiwan Strait

There were six major larval anisakid species found in commercial marine fishes caught in the Minnan fishing ground in the Taiwan Strait: Anisakis physeteris, Anisakis pegreffii, Raphidascaris trichiuri, Contracaecum aduncum, Contracaecum muraenesoxi, Contracaecum sp. For rapid identification of the parasite species above, a single and a multiple primer PCR (multiplex PCR) method, using specific primers based on aligned sequences of the internal transcribed spacer ITS-1, 5.8S, and ITS-2 of nuclear ribosomal DNA, were jointly used for the rapid identification of these anisakid larvae. The primers yielded distinct PCR products for each of the anisakid nematodes, providing rapid and accurate tools for identifying anisakid nematodes with distinct geographical distribution.

Anisakis pegreffii: A quantitative fluorescence PCR assay for detection in situ

To facilitate improved diagnosis and detection of the third stage larva (L3) of Anisakis pegreffii from the Minnan-Taiwan bank fishing ground in Taiwan Strait, a real-time PCR method for the detection in situ and differentiation was developed to amplify a region of the second internal transcribed spacer (ITS-2) of this parasite. The real-time PCR assay was capable of detecting 1/3 of a single L3 in 30 mg of marine fish tissue, and also exhibited a high level of specificity for A. pegreffii, no fluorescence signals were observed in other five major larval anisakid species found in commercial marine fishes caught in this fishing ground.

Cathepsin B-like and hemoglobin-type cysteine proteases: Stage-specific gene expression in Angiostrongy cantonensis

Three cysteine protease genes, cathepsin B-like enzyme gene 1, 2 (AC-cathB-1, AC-cathB-2) and hemoglobin-type cysteine protease gene (AC-hem) were isolated and described from Angiostrongylus cantonensis adult. The deduced amino acid sequence of Ac-cathB-1 and AC-cathB-2 contain all of the conserved regions of cathepsin B. AC-cathB-2 is similar to a host intrusion-related cysteine protease B from Parelaphostrongylus tenuis, and the AC-hem shares high similarity to legumain from Haemonchus contortus. AC-cathB-1 was expressed significantly higher in L1 as compared with AC-hem, the AC-cathB-2 followed; AC-cathB-2 transcripts in L3 were found increased rapidly and obviously abundant, suggesting that AC-cathB-1 and AC-cathB-2 may play an important role in intermediate and final host invasion, separately. The cysteine protease genes were more or less expressed in adult stage excepted for AC-cathB-2. As the AC-cathB-1 and AC-hem highly expressed in adult worms, suggesting AC-hem may activate AC-cathB-1 which involved in the host invasion and feeding process.

Immunolocalization and developmental expression patterns of two cathepsin B proteases (AC-cathB-1, -2) of Angiostrongylus cantonensis

In this study we have investigated the anatomic sites of expression and developmental expression patterns of two cathepsin B-like cysteine proteases (AC-cathB-1, -2) of Angiostrongylus cantonensis. The immunolocalization results revealed that native AC-cathBs were found present in the L1 and L3 larvae, female and male adults, and the AC-cathBs were localized mainly on the digestive tract of A. cantonensis and expressed at varied levels and in different patterns in the internal tissues according to their developmental stage. Consistent with the infective stage of L3 is a much more intense staining of AC-cathBs in the esophagus compared with the intestine. In contrast to L3, more abundant signals were located to the intestine of adults, suggesting that nutrition digestion likely to be the main function of the protease at this point. AC-cathBs fluorescent signals were present in excretory pore, excretory tube in lateral cords, and muscular esophagus of larvae, further supported the AC-cathB-1, -2 likely to be released by A. cantonensis as excretory/secretory products. Additionally, only the protein AC-cathB-2 was detected in the reproductive system, especially in the wall of vas deferens, uterus, and oviduct of the parasites, whether the AC-cathB-2 has some function in germ cells development and maturation need to be further characterized. Although the anatomic sites and expression patterns were different in larvae and adults and the corresponding function might not the same, AC-cathB-1 and -2 involved in the host-parasite interaction in addition to digestive function.

Angiostrongylus cantonensis cathepsin B-like protease (Ac-cathB-1) is involved in host gut penetration

Although the global spread of the emerging zoonosis, human angiostrongyliasis, has attracted increasing attention, understanding of specific gene function has been impeded by the inaccessibility of genetic manipulation of the pathogen nematode causing this disease, Angiostrongylus cantonensis. Many parasitic proteases play key roles in host-parasite interactions, but those of A. cantonensis are always expressed as the inactive form in prokaryotic expression systems, thereby impeding functional studies. Hence, a lentiviral system that drives secreted expression of target genes fused to a Myc-His tag was used to obtain recombinant Ac-cathB-1 with biological activity. Although this class of proteases was always reported to function in nutrition and immune evasion in parasitic nematodes, recombinant Ac-cathB-1 was capable of hydrolysis of fibronectin and laminin as well as the extracellular matrix of IEC-6 monolayer, so that the intercellular space of the IEC-6 monolayer increased 5.15 times as compared to the control, while the shape of the adherent cells partly rounded up. This suggests a probable role for this protease in intestinal epithelial penetration. The inhibition of Ac-cathB-1 enzymatic activity with antiserum partly suppressed larval penetration ability in the isolated intestine. Thus, an effective system for heterologous expression of parasite proteases is presented for studying gene function in A. cantonensis; and Ac-cathB-1 was related to larval penetration ability in the host small intestine.