Development of an immunoassay using recombinant outer membrane protein A and flagellin for diagnosis of goats with melioidosis.

Among domestic animals, melioidosis is one of the most common diseases reported in goat, sheep, and swine. To evaluate the specific antibodies in goats with melioidosis, we developed a serology test using recombinant outer membrane protein A (OmpA) and flagellin (FliC) of Burkholderia pseudomallei as antigens. DNA corresponding to each antigen was cloned into a pET32a vector and expressed in Escherichia coli. Essentially, the recombinant OmpA and FliC were expressed in a soluble form that could be isolated with 95% homogeneity. Both recombinants could be recognized by rabbit antibodies prepared against heat-inactivated B. pseudomallei (1:1,000) on a Western blot. Subsequently, we demonstrated that both recombinants could capture the antibodies present in goat with naturally occurring melioidosis (optimized titer 1:40) while not cross-reacting with the serum samples of goats naturally infected by Corynebacterium pseudotuberculosis or Staphylococcus aureus. Finally, an enzyme-linked immunosorbent assay (ELISA) using 20 goat serum samples without melioidosis and 10 goat serum samples with melioidosis demonstrated that the infected group has significantly higher antibody titer levels than the normal group (P<0.001) when using either OmpA or FliC as an antigen. However, the sensitivity (100%) of the assay using OmpA was superior to that (90%) from using FliC. Serological tests that are commonly used often rely on antigens from crude cell extracts, which pose risks for laboratory-acquired infections and inconsistency in their preparation; however, use of recombinant OmpA is safe; it can potentially be used as a reagent in testing for goat melioidosis.

Apoptosis Induction by Pseudorabies Virus via Oxidative Stress and Subsequent DNA Damage Signaling.

BACKGROUND: Pseudorabies virus (PRV) infection induces apoptosis in swine cells both in vitro and in vivo; however, the mechanism associated with host-cell signaling has not been studied. This study investigated the role of free radicals caused by cellular oxidative stress after viral infection and examined whether the DNA damage response plays an important role in PRV-induced apoptosis. METHODS: Several apoptosis assays and western blotting confirmed PRV-induced apoptosis. PRV-mediated oxidative stress was evaluated by reactive oxygen species (ROS) assay. RESULTS: Our results showed that PRV caused apoptosis in a porcine kidney cell line, PK15, and induced expressions of proapoptotic Bcl family proteins in a dose- and time-dependent manner. Expressions of specific DNA damage sensors and phosphorylation of histone H2AX were also significantly increased, which subsequently activated the expressions of checkpoint kinase 1/2 and proapoptotic p53. Caffeine, a known DNA damage inhibitor, was found to inhibit caspase-3 activation and protect cells from PRV-induced apoptosis. Additionally, the antioxidant N-acetyl-L-cysteine was shown to prevent the production of cellular ROS, protecting DNA from cleavage. CONCLUSIONS: Our results confirmed that oxidative stress and free radicals arising from PRV infection cause DNA damage, which consequently triggers apoptosis.

Neutrophils as one of the major haptoglobin sources in mastitis affected milk.

The antioxidant haptoglobin (Hp) is an acute-phase protein responsive to infectious and inflammatory diseases. Hp and somatic cell counts (SCC) are sharply elevated in bovine milk following intramammary administration of endotoxin or bacteria. However, the sources of milk Hp responsible for such increases are not fully understood. The purpose of this study was to define the source of milk Hp from dairy cows with naturally occurring mastitis. Quarter milk samples selected from 50 dairy cows were separated into four groups according to SCC as group A: < 100 (n = 19); B: 100-200 (n = 10); C: 201-500 (n = 10); and D: > 500 x 10(3) (n = 11) cells/mL. Our results reveal that milk Hp concentrations were correlated with SCC (r = 0.742; P < 0.01), and concentrations in group D were approximately 10-fold higher than in group A. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicates that the milk somatic cells from group D were not only capable of synthesizing Hp but could also markedly increase Hp mRNA expression. Western blot, immunocytochemistry, double confocal immunofluorescence, and Hp releasing experiments demonstrate that neutrophils were associated with the biosynthesis and release of Hp in milk. It further shows that Hp was significantly elevated in the epithelium of mammary gland tissue with mastitis and was also expressed in the cultured mammary epithelial cells. We propose that neutrophils and epithelial cells may play an essential role in elevating milk Hp in addition to previous suggestions that Hp may be derived from mammary tissues and circulation.

Low levels of haptoglobin and putative amino acid sequence in Taiwanese Lanyu miniature pigs.

Porcine haptoglobin (Hp) is an acute phase protein. Its plasma level increases significantly during inflammation and infection. One of the main functions of Hp is to bind free hemoglobin (Hb) and inhibit its oxidative activity. In the present report, we studied the Hp phenotype of Taiwanese Lanyu miniature pigs (TLY minipigs; n=43) and found their Hp structure to be a homodimer (beta-alpha-alpha-beta) similar to human Hp 1-1. Interestingly, Western blot and high performance liquid chromatographic (HPLC) analysis showed that 25% of the TLY minipigs possessed low or no plasma Hp level (<0.05 mg/ml). The Hp cDNA of these TLY minipigs was then cloned, and the translated amino acid sequence was analyzed. No sequences were found to be deficient; they showed a 99.7% identity with domestic pigs (NP_999165). The mean overall Hp level of the TLY minipigs (0.21 +/- 0.25 mg/ml; n=43) determined by enzyme-linked immunosorbent assay (ELISA) was markedly lower than that of domestic pigs (0.78 +/- 0.45 mg/ml; p<0.001), while 25% of the TLY minipigs had an Hp level that was extremely low (<0.05 mg/ml). In addition, the initial recovery rate (first 40 min) in the circulation of infused fluorescein isothiocyanate (FITC)-Hb was significantly higher in the TLY minipigs with extremely low Hp levels than those with high levels. This data suggests that the low concentration of Hp-Hb complex is responsible for the higher recovery rate of Hb in the circulation. TLY minipigs have been used as an experimental model for cardiovascular diseases; whether they can be used as a model for inflammatory diseases, with Hp as a marker, remains a topic of interest. However, since the Hp level varies significantly among individual TLY minipigs, it is necessary to prescreen the Hp levels of the animals to minimize variation in the experimental baseline. The present study may provide a reference value for future use of the TLY minipig as an animal model for inflammation-associated diseases.

Evidence of tandem repeat and extra thiol-groups resulted in the polymeric formation of bovine haptoglobin: a unique structure of Hp 2-2 phenotype.

Human plasma Hp is classified as 1-1, 2-1, and 2-2. They are inherited from two alleles Hp 1 and Hp 2, but there is only Hp 1 in almost all the animal species. Hp 2-2 molecule is extremely large and heterogeneous associated with the development of inflammatory-related diseases. In this study, we expressed entire bovine Hp in E. coli as a alphabeta linear form. Interestingly, the antibodies prepared against this form could recognize the subunit of native Hp. In stead of a complicated column method, the antibody was able to isolate bovine Hp via immunoaffinity and gel-filtration columns. The isolated Hp is polymeric containing two major molecular forms (660 and 730 kDa). Their size and hemoglobin binding complex are significantly larger than that of human Hp 2-2. The amino-acid sequence deducted from the nucleotide sequence is similar to human Hp 2 containing a tandem repeat over the alpha chain. Thus, the Hp 2 allele is not unique in human. We also found that there is one additional -SH group (Cys-97) in bovine alpha chain with a total of 8 -SH groups, which may be responsible for the overall polymeric structure that is markedly different from human Hp 2-2. The significance of the finding and its relationship to structural evolution are also discussed.

Cloning and expression of human haptoglobin subunits in Escherichia coli: delineation of a major antioxidant domain.

Human plasma haptoglobin (Hp) comprises alpha and beta subunits. The alpha subunit is heterogeneous in size, therefore isolation of Hp and its subunits is particularly difficult. Using Escherichia coli, we show that alpha1, alpha2, beta, and alpha2beta chain was abundantly expressed and primarily present in the inclusion bodies consisting of about 30% of the cell-lysate proteins. Each cloned subunit retained its immunoreactivity as confirmed using antibodies specific to alpha or beta chain. By circular dichroism, the structure of each expressed subunit was disordered as compared to the native Hp. The antioxidant activity was found to be associated with both alpha and beta chains when assessed by Cu(2+)-induced oxidation of low density lipoprotein (LDL). Of remarkable interest, the antioxidant activity of beta chain was extremely potent and markedly greater than that of native Hp (3.5x), alpha chain (10x) and probucol (15x). The latter is a clinically proved potent compound used for antioxidant therapy. The "unrestricted" structure of beta subunit may therefore render its availability for free-radical scavenge, which provides a utility for the future design of a "mini-Hp" in antioxidant therapy. It may also provide a new insight in understanding the mechanism involved in the antioxidant nature of Hp.