C-Terminal Part of Glutamate-Ammonia-Ligase Adenyltransferase Gene Identified by RAPD-HRM with 3H Primer for E. Coli Screening.

A single random oligonucleotide 3H primer has been previously applied in random-amplified- polymorphic-DNA (RAPD)-PCR to distinguish stocked bacteria E. coli within a cocktail mixture also containing Enterococcus faecalis, Bifidobacterium longum and Ruminococcus gnavus. In this study, we demonstrate that a 702 base pair (bp) gene fragment can be amplified as a unique pattern by RAPD-PCR using a 3H primer in human faeces containing E. coli. This unique 702 bp amplicon contained a 687 bp gene fragment identified as the C-terminal region of the glutamate-ammonia-ligase adenyltransferase (glnE) gene of E. coli. By high-resolution melt (HRM) analysis, a mean melt-curve temperature of this 702 bp amplicon was determined to be approximately 88.1 ± 0.22 degrees Celsius (°C). A combination of RAPD with HRM in one single reaction based on this amplicon can achieve semi-quantitative detection of up to 102 CFU/ml of E. coli. To increase the signal intensity of HRM, a primer pair capable of screening E. coli directly from fresh human faeces was re-designed from the 687 bp gene segment, giving a mean peak melt-curve temperature at 88.35 ± 0.11 °C. Finally, single-nucleotide polymorphisms of this 687 bp gene segment were analysed for pathogenic E. coli strains, including UMN026, O83:H1, O104:H4, O157:H7 and O169:H41. We conclude that this 687 bp segment of the glnE gene has a high potential for screening of human faecal E. coli, including pathogenic strains, in contaminated food and water.

Underlying mechanism of regulatory actions of diclofenac, a nonsteroidal anti-inflammatory agent, on neuronal potassium channels and firing: an experimental and theoretical study.

Diclofenac (DIC), a nonsteroidal anti-inflammatory drug, is known to exert anti-nociceptive and anti-convulsant actions; however, its effects on ion currents, in neurons remain debatable. We aimed to investigate (1) potential effects of diclofenac on membrane potential and potassium currents in differentiated NSC-34 neuronal cells and dorsal root ganglion (DRG) neurons with whole-cell patch-clamp technology, and (2) firing of action potentials (APs), using a simulation model from hippocampal CA1 pyramidal neurons based on diclofenac's effects on potassium currents. In the NSC-34 cells, diclofenac exerted an inhibitory effect on delayed-rectifier K⁺ current (I(KDR)) with an IC50 value of 73 µM. Diclofenac not merely inhibited the I(KDR) amplitude in response to membrane depolarization, but also accelerated the process of current inactivation. The inhibition by diclofenac of IK(DR) was not reversed by subsequent application of either naloxone. Importantly, diclofenac (300 µM) increased the amplitude of M-type K⁺ current (I)(KM)), while flupirtine (10 µM) or meclofenamic acid (10 µM) enhanced it effectively. Consistently, diclofenac (100 µM) increased the amplitude of I(KM) and diminished the I(KDR) amplitude, with a shortening of inactivation time constant in DRG neurons. Furthermore, by using the simulation modeling, we demonstrated the potential electrophysiological mechanisms underlying changes in AP firing caused by diclofenac. During the exposure to diclofenac, the actions on both I(KM) and I(KDR) could be potential mechanism through which it influences the excitability of fast-spiking neurons. Caution needs to be made in attributing the effects of diclofenac primarily to those produced by the activation of I(KM).

Prevalence of Chlamydophila abortus infection in domesticated ruminants in Taiwan.

This study is to (1) investigate the prevalence of Chlamydophila abortus infection in cows and goats in Taiwan, and (2) compare the genetic properties of Taiwanese isolates with abortion strains from other sources. Approximately 71% of aborted cows and 58% of aborted does had IgG against C. abortus in their sera. The seroprevalence rate in cows may be overestimated, because a certain degree of cross-reactivity with C. pecorum cannot be ruled out. Only 22.7% (from aborted cows) and 33.3% (from aborted dogs) of vaginal swabs that tested positive by polymerase chain reaction led to successful isolation of C. abortus by inoculation into chicken embryos, equivalent to 7.1% and 7.9% of isolation rates, respectively. The major outer membrane protein gene of 15 Taiwanese abortion isolates was compared with that of various strains by restriction fragment length polymorphism (RFLP) and nucleotide sequencing. Restriction enzyme CfoI was able to distinguish Taiwanese ruminant isolates, which have identical RFLP patterns, from C. felis (feline) and C. psittaci (avian) strains. Taiwanese isolates had 98.8-100% homology with known ruminant abortion strains and were phylogenetically closest to bovine LW508 strain.

Nucleotide sequencing of S-RNA segment and sequence analysis of the nucleocapsid protein gene of the newly isolated Akabane virus PT-17 strain.

The nucleotide sequences of the S-RNA of Akabane viruses JaGAr-39, OBE-1, Iriki and the newly isolated PT-17 strains and the Aino virus were determined and compared. The results reveal that the S-RNAs of the four Akabane strains share 96.9% homology in nucleotide sequences. Only one amino acid difference out of the 233 amino acids of the nucleocapsid protein (N) and three amino acid differences in the 91 amino acids of the nonstructural protein (NSs) were found among the Akabane viruses. Amino acid sequences of N and NSs proteins of the Aino virus have approximately 80% identity as compared with the Akabane viruses. The results also demonstrate that the four Akabane viruses and the Aino virus can be clearly differentiated by RFLP (restriction fragments length polymorphism) analysis using RT-PCR generated nucleocapsid protein genes and digested with HaeIII and HindIII. The phylogenetic tree based on the UPGMA (Unweighted Pair Group Method with Arithmetic Mean) analysis of the sequences of nucleocapsid protein genes and the S-DNAs revealed that the newly isolated PT-17 strain is most closely related to Iriki strain, than the JaGAr-39 or OBE-1 strains.

Epidemiology of bovine ephemeral fever virus infection in Taiwan.

Sick animals with excessive nasal discharges and protruding tongue as a result of dyspnea were observed in the August of 1996. Eight strains of BEF virus were isolated from heparinized blood samples of the affected cattle. Most of the affected cattle were difficult to be treated and had a poor prognosis. A total of 516 farms in the 9 districts of Taiwan were affected in 1996. Among a population of 110,247 dairy cattle, 14,993 (13.6%) cattle were found to be clinically ill. During the epidemic, 1,685 (11.3%) affected cattle were culled or dead after the onset of the disease. Furthermore, a strain of Ibaraki virus was isolated from the blood sample of a sick cattle that showed pyrexia, labored respiration and solitary behavior in the affected farm. The cattle with Ibaraki virus infection had typical symptoms of BEF at the early stage of the disease, but neither stomatitis nor pharyngoesophageal paralysis was observed at the onset of the disease. The outbreak was presumably brought about by the low level or non-immune status of a large cattle population due to the negligence of BEF vaccination. Therefore, the disease easily recurred in Taiwan after a typhoon episode in the August of 1996, which resulted in the proliferation of biting midges in the field. No difference in the antigenicty was found between the new and the previous isolates of BEF virus. As analyzed by cross neutralization test, the isolated BEF viruses showed no relationship to the Kimberley and Berrimah viruses that were isolated from the blood of cattle and related to BEF virus in Australia. We have tried to advise farmers that they must vaccinate their cattle annually to prevent BEF outbreak in the future.

Epizootic of Chlamydia psittaci infection in goats in Taiwan.

Epizootic abortion in goats has been frequently reported in Taiwan since 1993. The outbroken flocks were found in most of districts in Taiwan. No apparent clinical signs were found in aborted doe. The typical abortion occurred in the last two months of pregnancy. The incidence of abortion was from 10% to 87% in outbroken farms in 1993, and a total of 976 out of 2130 pregnancies (46%) were found abortion in our investigation. Gross lesions in aborted fetuses included generalized haemorrhage and swollen liver. Chlamydia psittaci was isolated from tissues of aborted fetuses and from vaginal swabs of aborted does. Chlamydial antibodies were detected among 67% to 100% of aborted does from epizootic flocks. The C. psittaci was diagnosed as the causal agent in enzootic abortion. This is the first report on chlamydial isolation and antibody surveys in epizootic abortion in goats in Taiwan.

The isolation of Akabane virus (Iriki strain) from calves in Taiwan.

Nonsuppurative encephalitis in calves aged 4-12 months, cow abortion and fetal deformities were endemic in dairy farms in Taiwan in recent years. A virological investigation emphasizing on Arthpodborn virus (Arbovirus) was conducted. Total of 11 strains of Akabane virus were isolated from endemic districts between June and July of 1992. Among them, seven viruses were isolated from blood samples of 15 calves showing nervous signs. Another 4 Akabane viruses were isolated from clinically healthy calves from three of six dairy farms investigated. All the six investigated farms had a recent history of abortion and fetal deformities. The isolates caused prominent cytopathic effects in HmLu-1 cells and could reach a high virus titers (5 x 10(6) TCID50/ml). As demonstrated by a cross neutralization test, the isolates had identical antigenicity to Iriki strain of Akabane virus, but were antigenically more distant to JaGar-39 and OBE-1 strain of Akabane virus. This is the first report on the isolation of Akabane virus in Taiwan, and also the second report on the isolation of Iriki virus in the world.

Infectious bill atrophy syndrome caused by parvovirus in a co-outbreak with duck viral hepatitis in ducklings in Taiwan.

In October 1989, an epizootic duckling disease with high mortality occurred in Taiwan. The disease was characterized by droopiness, inappetence, ataxia, ruffled feathers, and watery diarrhea. Affected ducklings were lame, were unable to stand, showed opisthotonos, and often died 3 or 4 days after the onset of the disease. Tolerant maturing ducklings displayed atrophic upper bills with a protruding tongue and became stunted as they reached maturity. No diagnostic histopathologic lesions were found in these ducklings. Fourteen parvovirus isolates, 33 duck viral hepatitis virus (DVHV) isolates, two adenovirus isolates, and two reovirus isolates were obtained and identified from more than 500 sick ducklings in the epizootic. The epizootic was diagnosed as a co-outbreak of duck parvovirus infections and duck viral hepatitis. The high mortality in ducklings and the bill atrophy syndrome were reproduced in ducklings by inoculating the parvovirus isolates alone. The epizootic was controlled by an emergency immunization program of ducklings with sera collected from recovered ducks or a bivalent inactivated vaccine composed of local DVHV and parvovirus isolates.

[A comparative identification of four Gynostemma spp. herbs and their various species from Guangxi].

In this paper, the authors have described the morphological and histological character of the four herbs of Gynostemma spp. and their confused species from Guangxi viz Gynostemma pentaphyllum, G. guangxiense, G. laxum and G. longipes, and their confused species of four Hemsleya daxienensis, H. chinensis, Cayratia japonica and C. japonica var. pubifolia. The description is illustrated with line-drawings for comparative identification.