Identification of risk factors of Coxiella burnetii (Q fever) infection in veterinary-associated populations in southern Taiwan.

The first case of Q fever in Taiwan was reported in 1993. The disease is considered to be emerging in Taiwan, but the route of transmission has remained unclear. The annual number of confirmed Q fever cases has been increasing up to more than 100 cases since 2005, comparing with less than 30 before 2003. The purpose of this study was to determine the seroprevalence and risk factors of Coxiella burnetii infection in veterinary-associated populations in southern Taiwan. A total of 228 serum samples of high risk individuals engaging in veterinary-related work or animal-farm work, were collected between March and June in 2007. The study individuals were interviewed by a structured questionnaire designed for Q fever investigation. Serum samples from different animal species were also obtained for Q fever analysis in the same study areas. Serological test was conducted by indirect immunofluorescence antibody assay (IFA). The result demonstrated the overall seroprevalence of Q fever was 26.3% in individuals engaging in veterinary and animal-related work in southern Taiwan. After multiple logistic regression analysis, goat exposure was significantly associated with seropositivity of Q fever in the study population in southern Taiwan (adjusted odds ratio: 2.62; 95% CI: 1.06-6.46). In addition, the highest seroprevalence (43.8%) of Q fever was identified in goats (P < 0.05). Finally, this study documented that people with prior knowledge of Q fever were less likely to be seropositive for C. burnetii. It was concluded that goat exposure was the most important risk factor associated with C. burnetii infection and appropriate health education could be useful to prevent high risk individuals from the infection in southern Taiwan.

Purification and characterization of a recombinant beta-galactosidase with transgalactosylation activity from Bifidobacterium infantis HL96.

A beta-galactosidase isoenzyme, beta-Gall, from Bifidobacterium infantis HL96, was expressed in Escherichia coli and purified to homogeneity. The molecular mass of the beta-Gall subunit was estimated to be 115 kDa by SDS-PAGE. The enzyme appeared to be a tetramer, with a molecular weight of about 470 kDa by native PAGE. The optimum temperature and pH for o-nitrophenyl-beta-D-galactopyranoside (ONPG) and lactose were 60 degrees C, pH 7.5, and 50 degrees C, pH 7.5, respectively. The enzyme was stable over a pH range of 5.0-8.5, and remained active for more than 80 min at pH 7.0, 50 degrees C. The enzyme activity was significantly increased by reducing agents. Maximum activity required the presence of both Na+ and K+, at a concentration of 10 mM. The enzyme was strongly inhibited by p-chloromercuribenzoic acid, divalent metal cations, and Cr3+, and to a lesser extent by EDTA and urea. The hydrolytic activity using lactose as a substrate was significantly inhibited by galactose. The Km, and Vmax values for ONPG and lactose were 2.6 mM, 262 U/mg, and 73.8 mM, 1.28 U/mg, respectively. beta-Gall possesses strong transgalactosylation activity. The production rate of galactooligosaccharides from 20% lactose at 30 and 60 degrees C was 120 mg/ml, and this rate increased to 190 mg/ml when 30% lactose was used.

Molecular and biochemical analysis of two beta-galactosidases from Bifidobacterium infantis HL96.

Two genes encoding beta-galactosidase isoenzymes, beta-galI and beta-galIII, from Bifidobacterium infantis HL96 were revealed on 3.6- and 2.4-kb DNA fragments, respectively, by nucleotide sequence analysis of the two fragments. beta-galI (3,069 bp) encodes a 1,022-amino-acid (aa) polypeptide with a predicted molecular mass of 113 kDa. A putative ribosome binding site and a promoter sequence were recognized at the 5' flanking region of beta-galI. Further upstream a partial sequence of an open reading frame revealed a putative lactose permease gene transcribing divergently from beta-galI. The beta-galIII gene (2,076 bp) encodes a 691-aa polypeptide with a calculated molecular mass of 76 kDa. A rho-independent transcription terminator-like sequence was found 25 bp downstream of the termination codon. The amino acid sequences of beta-GalI and beta-GalIII are homologous to those found in the LacZ and the LacG families, respectively. The acid-base, nucleophilic, and substrate recognition sites conserved in the LacZ family were found in beta-GalI, and a possible acid-base site proposed for the LacG family was located in beta-GalIII, which featured a glutamate at residue 160. The coding regions of the beta-galI and beta-galIII genes were each cloned downstream of a T7 promoter for overexpression in Escherichia coli. The molecular masses of the overexpressed proteins, as estimated by polyacrylamide gel electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, agree with their predicted molecular weights. beta-GalI and beta-GalIII were specific for beta-D-anomer-linked galactoside substrates. Both are more active in response to ONPG (o-nitrophenyl-beta-D-galactopyranoside) than in response to lactose, particularly beta-GalIII. The galacto-oligosaccharide yield in the reaction catalyzed by beta-GalI at 37 degrees C in 20% (wt/vol) lactose solution was 130 mg/ml, which is more than six times higher than the maximum yield obtained with beta-GalIII. The structure of the major trisaccharide produced by beta-GalI catalysis was characterized as O-beta-D-galactopyranosyl-(1-3)-O-beta-D-galactopyranosyl-(1-4)-D-glucopyranose (3'-galactosyl-lactose).

Molecular cloning, characterization and nucleotide sequence of the gene for secreted alpha-amylase from Xanthomonas campestris pv. campestris.

alpha-Amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) of apparent molecular mass 45 kDa was secreted by Xanthomonas campestris pv. campestris grown in medium containing starch or maltose. We isolated its structural gene from a recombinant lambda library and located it on a 2.7 kb DNA fragment. Nucleotide sequencing of the fragment revealed a potential ORF encoding a protein of 475 amino acid residues, including a potential signal sequence of 35 amino acids. The signal processing site was confirmed by N-terminal amino acid sequence analysis of the exported alpha-amylase. The deduced amino acid sequence of the mature protein is very similar to that of the alpha-amylase of Aeromonas hydrophila. It also contains all four amino acid sequences highly conserved in the alpha-amylases from a wide range of organisms. Expression of the amy gene in Escherichia coli was poor from its own promoter, but was enhanced by the upstream promoter on the vector. The alpha-amylase synthesized in E. coli was located in the periplasm.

Cloning and characterization of a gene required for the secretion of extracellular enzymes across the outer membrane by Xanthomonas campestris pv. campestris.

Nonpathogenic mutants of Xanthomonas campestris pv. campestris, generated from transposon mutagenesis, accumulated extracellular polygalacturonate lyase, alpha-amylase, and endoglucanase in the periplasm. The transposon Tn5 was introduced by a mobilizable, suicidal plasmid, pSUP2021 or pEYDG1. Genomic banks of wild-type X. campestris pv. campestris, constructed on the broad-host-range, mobilizable cosmid pLAFR1 or pLAFR3, were conjugated with one of the mutants, designated XC1708. Recombinant plasmids isolated by their ability to complement XC1708 can be classified into two categories. One, represented by pLASC3, can complement some mutants, whereas the other, represented by a single plasmid, pLAHH2, can complement all of the other mutants. Restriction mapping showed that the two recombinant plasmids shared an EcoRI fragment of 8.9 kb. Results from subcloning, deletion mapping, and mini-Mu insertional mutation of the 8.9-kb EcoRI fragment suggested that a 4.2-kb fragment was sufficient to complement the mutant XC1708. Sequence analysis of this 4.2-kb fragment revealed three consecutive open reading frames (ORFs), ORF1, ORF2, and ORF3. Hybridization experiments showed that Tn5 in the genome of XC1708 and other mutants complemented by pLASC3 was located in ORF3, which could code for a protein of 83.5 kDa. A signal peptidase II processing site was identified at the N terminus of the predicted amino acid sequence. Sequence homology of 51% was observed between the amino acid sequences predicted from ORF3 and the pulD gene of Klebsiella species.