Simple modification to improve reliability of copro-DNA examinations for diagnosing Echinococcus multilocularis infections in red foxes.

Epidemiological studies of Echinococcus multilocularis infections in definitive hosts require a reliable and economic diagnostic method. In this study, the current copro-DNA examination technique was modified by increasing the faecal amounts tested and adding a step to neutralize the faeces before DNA extraction. Reliability of the modified method was evaluated using rectal faecal samples from red foxes and comparing them with intestinal worms detected using the sedimentation and counting technique (SCT) following necropsy. The modified copro-DNA examination method demonstrated 93.9% sensitivity (138/147) on the SCT. Its detectability increased depending on the worm burden, and the sensitivity was 100% in cases harbouring over 1000 worms. From 111 SCT-negative cases, six (5.4%) were copro-DNA-positive, and all were confirmed as E. multilocularis via sequencing analysis. Five of the remaining 105 SCT-negative cases (4.8%) retained polymerase chain reaction (PCR) inhibitors in the extracted solution, suggesting that approximately 5% of the red fox faeces retained these inhibitors after treatment with the present copro-DNA extraction method. Although further evaluation is needed for faeces deposited in the wild, the present copro-DNA examination technique will help monitor the E. multilocularis prevalence in definitive hosts. When used for detailed evaluations of endemicity (e.g. changes in infection pressure or spread in non-endemic areas), the absence of PCR inhibitors should be confirmed, and multiple trials on faecal subsamples are recommended.

The timing of worm exclusion in dogs repeatedly infected with the cestode Echinococcus multilocularis.

Experimental Echinococcus multilocularis infection and deworming was repeated three or five times in nine dogs at various re-infection schedules. The mean number of worms decreased more than 91% in dogs with repeated infection, compared to first infection controls (n= 6). The copro-antigen assay and the egg count in the faeces suggested that the worm burden gradually decreased each time the dogs were re-infected. To examine whether such worm exclusion was a non-specific response, five dogs were sequentially infected with the parasite four times and subsequently fed freely for 6 months. Even after the 6-month interval, the five dogs that were infected five times with the parasite were still able largely to exclude the adult worms. The results suggested that the ability of worm exclusion in dogs that developed a resistance did not become rapidly extinct. Observation of the condition of faeces and the excretion of hooks in the faeces of repeatedly infected dogs revealed that the exclusion of worms started at the first week after the re-infection, and it continued during the patent period. Serum antibodies specific to the parasite antigen increased gradually until the third infection and significantly decreased during the 6-month interval. There was little enhancement of serum antibodies after the fifth infection in most dogs, although no clear correlation was observed between the antibody response and the worm burden. These findings suggested the possibility of developing a vaccine.

Role of C-terminal region of HA-33 component of botulinum toxin in hemagglutination.

Using SDS-PAGE, we found that one subcomponent, hemagglutinin (HA-33), from the Clostridium botulinum progenitor toxin of type D strain 1873 and type C strain Yoichi had slightly smaller molecular sizes than those of type C and D reference strains, but other components did not. Based on N- and C-terminal sequence analyses of HA-33, a deletion of 31 amino acid residues from the C-terminus at a specific site was observed in the HA-33 proteins of both strains. The progenitor toxins from both strains showed poor hemagglutination activities, titers of 2(1) or less, which were much lower than titers from the reference strains (2(6)), and did not bind to erythrocytes. These results suggest strongly that the short C-terminal region of the HA-33 plays an essential role in the hemagglutination activity of the botulinum progenitor toxin. Additionally, a sequence motif search predicted that the C-terminal region of HA-33 has a carbohydrate-recognition subdomain.

Characterization and reconstitution of functional hemagglutinin of the Clostridium botulinum type C progenitor toxin.

The purified progenitor toxin of Clostridium botulinum type C strain 6814 (C-6814) forms a large complex composed of 150-kDa neurotoxin (NT), 130-kDa nontoxic-nonhemagglutinin (NTNHA), and hemagglutinin (HA) components. The HA component consisted of a mixture of several subcomponents with molecular masses of 70, 55, 33, 26-21 and 17 kDa. We isolated the HA subcomponents from the progenitor toxin by chromatography in the presence of denaturants. The isolated HA subcomponents, designated as i-HA-33, i-HA-55, i-HA-70 and i-HA-33/17, were nearly homogeneous on SDS/PAGE, but the HA-17 and HA-26-21 components were not purified. Some HA subcomponents, designated as f-HA-33 and f-HA-33/17 complex, existed free of the progenitor toxin in the culture medium and they were separately purified. Every HA subcomponent so far isolated shows binding activity to erythrocytes. The hemagglutination activities of each HA subcomponent had a titer of 25 for the f-HA-33/17 complex, and below 23 for the other f- and i-HA subcomponents, while the parent progenitor L toxin was 28. The reconstitution of various combinations of f- and i-HA subcomponents was attempted via mixing and tested for hemagglutination activity. When the i-HA-33/17 complex and i-HA-55 were mixed, the hemagglutination activity was recovered to a titer of 29, which was slightly higher than that of the parent toxin. These data imply that a combination of at least HA-33, -17 and -55 subcomponents is required for full hemagglutination activity of the botulinum progenitor toxin, but each single HA subcomponent shows weak or no aggregation of erythrocytes.

Characterization of nicking of the nontoxic-nonhemagglutinin components of Clostridium botulinum types C and D progenitor toxin.

Clostridium botulinum C and D strains produce two types of progenitor toxins, M and L. Previously we reported that a 130-kDa nontoxic-nonhemagglutinin (NTNHA) component of the M toxin produced by type D strain CB16 was nicked at a unique site, leading to a 15-kDa N-terminal fragment and a 115-kDa C-terminal fragment. In this study, we identified the amino acid sequences around the nicking sites in the NTNHAs of the M toxins produced by C. botulinum type C and D strains by analysis of their C-terminal and N-terminal sequences and mass spectrometry. The C-terminus of the 15-kDa fragments was identified as Lys127 from these strains, indicating that a bacterial trypsin-like protease is responsible for the nicking. The 115-kDa fragment had mixtures of three different N-terminal amino acid sequences beginning with Leu135, Val139, and Ser141, indicating that 7-13 amino acid residues were deleted from the nicking site. The sequence beginning with Leu135 would also suggest cleavage by a trypsin-like protease, while the other two N-terminal amino acid sequences beginning with Val139 and Ser141 would imply proteolysis by an unknown protease. The nicked NTNHA forms a binary complex of two fragments that could not be separated without sodium dodecyl sulfate.

Dichain structure of botulinum neurotoxin: identification of cleavage sites in types C, D, and F neurotoxin molecules.

Botulinum neurotoxin (NT) is synthesized by Clostridium botulinum as about a 150-kDa single-chain polypeptide. Posttranslational modification by bacterial or exogenous proteases yielded dichain structure which formed a disulfide loop connecting a 50-kDa light chain (Lc) and 100-kDa heavy chain (Hc). We determined amino acid sequences around cleavage sites in the loop region of botulinum NTs produced by type C strain Stockholm, type D strain CB16, and type F strain Oslo by analysis of the C-terminal sequence of Lc and the N-terminal sequence of Hc. Cleavage was found at one or two sites at Arg444/Ser445 and Lys449/Thr450 for type C, and Lys442/Asn443 and Arg445/Asp446 for type D, respectively. In culture fluid of mildly proteolytic strains of type C and D, therefore, NT exists as a mixture of at least three forms of nicked dichain molecules. The NT of type F proteolytic strain Oslo showed the Arg435 as a C-terminal residue of Lc and Ala440 as an N-terminal residue of Hc, indicating that the bacterial protease cuts twice (Arg435/Lys436 and Lys439/Ala440), with excision of four amino acid residues. The location of cleavage and number of amino acid residue excisions in the loop region could be explained by the degree of exposure of amino acid residues on the surface of the molecule, which was predicted as surface probability from the amino acid sequence. In addition, the observed correlation may also be adapted to the cleavage sites of the other botulinum toxin types, A, B, E, and G.

Molecular composition of progenitor toxin produced by Clostridium botulinum type C strain 6813.

The molecular composition of the purified progenitor toxin produced by a Clostridium botulinum type C strain 6813 (C-6813) was analyzed. The strain produced two types of progenitor toxins (M and L). Purified L toxin is formed by conjugation of the M toxin (composed of a neurotoxin and a non-toxic nonhemagglutinin) with additional hemagglutinin (HA) components. The dual cleavage sites at loop region of the dichain structure neurotoxin were identified between Arg444-Ser445 and Lys449-Thr450 by the analyses of C-terminal of the light chain and N-terminal of the heavy chain. Analysis of partial amino acid sequences of fragments generated by limited proteolysis of the neurotoxin has shown to that the neurotoxin protein produced by C-6813 was a hybrid molecule composed of type C and D neurotoxins as previously reported. HA components consist of a mixture of several subcomponents with molecular weights of 70-, 55-, 33-, 26 through 21- and 17-kDa. The N-terminal amino acid sequences of 70-, 55-, and 26 through 21-kDa proteins indicated that the 70-kDa protein was intact HA-70 gene product, and other 55- and 26 through 21-kDa proteins were derived from the 70-kDa protein by modification with proteolysis after translation of HA-70 gene. Furthermore, several amino acid differences were exhibited in the amino acid sequence as compared with the deduced sequence from the nucleotide sequence of the HA-70 gene which was common among type C (strains C-St and C-468) and D progenitor toxins (strains D-CB16 and D-1873).