Occurrence and distribution of Naegleria species from thermal spring environments in Taiwan.

UNLABELLED: Naegleria spp. is a free-living amoeba that can be found in the natural environment. A number of Naegleria spp. can cause fatal infections in the central nervous system in humans and animals, and the most important source of infection is through direct water contact. In this study, water samples from various thermal springs were taken from four thermal spring areas. Naegleria spp. was detected via culture confirmation and molecular taxonomic identification. Among the 60 samples obtained, Naegleria spp. was identified in 26 (43·3%) samples. The identified species included Naegleria australiensis, Naegleria gruberi, Naegleria lovaniensis and Naegleria mexicana. The presence of living Naegleria spp. was significantly associated with elevated pH value in the water sample. SIGNIFICANCE AND IMPACT OF STUDY: In this study, we examined the presence of living Naegleria spp. in thermal spring waters in south-eastern Taiwan. Naegleria spp. was isolated and culture-confirmed from thermal spring water. Naegleria fowleri was not found in all water samples, and Naegleria australiensis was the most common Naegleria genotype.

Comparison of two molecular techniques for the detection of avian reoviruses in formalin-fixed, paraffin-embedded chicken tissues.

Reverse transcription (RT) in situ polymerase chain reaction (PCR) and in situ hybridization (ISH) techniques were used to detect the sigma c-encoded gene of avian reovirus (ARV) in chicken tissue sections. The advantage of using in situ methods is to make more rapid and accurate diagnosis of ARV infections. The sensitivity of these two techniques were compared. Of the two techniques, the RT in situ PCR test was found to be more sensitive than ISH and provided the rapid, sensitive, and specific detection of ARV infections.

Pathogenicity and drug susceptibility of the Pasteurella anatis isolated in chickens in Taiwan.

A strain of Pasteurella anatis (PA) was isolated from the sinus of an adult leghorn laying chicken with sinusitis, nasal discharge, drop in egg production, and low mortality, symptoms initially thought to indicate infectious coryza. The tiny, smooth, whitish colonies were identified as PA. To compare its pathogenicity with that of commercial broilers, nine groups, 10 birds per group, of 10-day-old broilers were individually inoculated with the strain of PA, Pasteurella multocida (PM), or Escherichia coli (EC) by intravenous, intraperitoneal, intramuscular, or subcutaneous inoculation. The PA was determined to cause the signs, lesions, and septicemic death, which are similar to the symptoms of PM or EC infection. At 1 wk postinfection (PI), the mortality rate was between that of PM and EC infection at 1 wk PI. Twenty antimicrobial-containing discs were evaluated, and the isolate was highly sensitive to cetiofer, amoxicillin, lincopectin, and furazolidone. Furthermore, it was moderately sensitive to tetracycline and enrofloxacin and only slightly sensitive to cephalothin, chloramphenicol, flumequine, nalidixic acid, neomycin, oxolinic acid, streptomycin, and trimethoprim. The PA infection was treated successfully with amoxicillin.

Diagnosis of Penaeus monodon-type baculovirus by PCR and by ELISA of occlusion bodies.

The black tiger prawn Penaeus monodon is a valuable aquaculture product in Taiwan. Two specific diagnostic methods were established for P. monodon-type baculovirus, one using polymerase chain reaction (PCR) technology and the other enzyme-linked immunosorbent assay (ELISA) technology. Monodon-type baculovirus (MBV) was purified by sucrose gradient centrifugation from occlusion bodies of MBV-infected postlarvae of P. monodon. MBV DNA was subsequently purified from the occlusion bodies and its presence was confirmed by PCR using primers of the polyhedrin gene. Based on conserved sequences of the DNA polymerase genes of Autographa californica nuclear polyhedrosis virus (AcMNPV) and Lymantria dispar nuclear polyhedrosis virus (LdMNPV), primers were designed and synthesized to yield a 714 bp PCR fragment from MBV. However, the sequence of this fragment revealed low homology with that of LdMNPV and AcMNPV. From the DNA sequence of this fragment, a second set of primers was designed, and using these primers, a 511 bp DNA fragment was amplified only when MBV DNA was the template. DNA templates from AcMNPV, white spot syndrome diseased shrimp, or PMO cells (a cell line derived from the Oka organ of Penaeus monodon) did not give any amplified DNA fragment. Therefore, this primer pair was specific for the diagnosis of MBV. By using intraspleenic immunization of rabbits with purified MBV occlusion bodies, a polyclonal rabbit antiserum against MBV was obtained. This antiserum could detect nanogram levels of MBV, but did not cross react with white spot syndrome virus (WSSV), homogenates of PMO cells, postlarvae, hepatopancreatic tissue or intestinal tissue of black tiger prawns by competitive ELISA. This sensitive method could detect MBV even in tissue homogenates.

In situ hybridization and RT-PCR detection of Macrobrachium rosenbergii nodavirus in giant freshwater prawn, Macrobrachium rosenbergii (de Man), in Taiwan.

Between January and March 2006, suspected outbreaks of white tail disease were observed in post-larvae and juveniles of Macrobrachium rosenbergii in hatcheries and nursery ponds at Kaohsiung and Pingtung Counties in southern Taiwan. Pathognomonic lesions showed the presence of large oval and/or irregular basophilic, cytoplasmic inclusion bodies in the infected muscles and hepatopancreas. Reverse transcriptase polymerase chain reaction (RT-PCR) assay and in situ hybridization (ISH) revealed evidence of M. rosenbergii nodavirus (MrNV) infection but did not detect extra small virus. Phylogenetic analysis indicated that there were very high identities between nucleotide sequences among six strains obtained in this study (99.5-100%), moderate identities with Caribbean and Indian strains (98.2-98.6%), but slightly lower identity with a Chinese strain (95.2%). This is the first confirmation of MrNV in giant freshwater prawns, using an RT-PCR and ISH, in Taiwan.

Properties of a fructose-1,6-diphosphate-activated lactate dehydrogenase from Acholeplasma laidlawii type A.

Acholeplasma laidlawii A possesses a nicotinamide adenine dinucleotide (NAD)-dependent l(+)-lactate dehydrogenase (LDH) which is activated specifically by low concentrations of fructose-1, 6-diphosphate (FDP). Studies with partially purified enzyme show that the kinetic response to FDP is hyperbolic. The enzyme is inhibited by inorganic phosphate, adenosine triphosphate, and high concentrations of reduced NAD (NADH). Low activity is demonstrable in the absence of FDP at pH 6.0 to 7.2, but FDP is absolutely required in the region of pH 8. FDP causes an upward shift in the optimum pH of the enzyme, which is near 7.2 in tris (hydroxymethyl)aminomethane buffer. Activation of the enzyme by FDP is markedly affected by substrate concentration; FDP lowers the apparent K(m) for pyruvate and NADH. The affinity of the enzyme for pyruvate is also influenced by H(+) concentration. The pyruvate analogue alpha-ketobutyrate serves as an effective substrate for the enzyme; when it is utilized, the enzyme is still activated by FDP. Reversal of the pyruvate reduction reaction catalyzed by the enzyme can be demonstrated with the 3-acetylpyridine analogue of NAD. The catalytic properties of the A. laidlawii enzyme and the known FDP-activated LDHs which occur among lactic acid bacteria are discussed.

Comparison of antigen binding capabilities of various membrane filters and filter papers in dot immunoassay.

Antigen binding activities of 25 kinds of filter papers, including nitrocellulose (NC), nylon or polyvinylidine difluoride (PVDF), in the binding of 5 viruses, 3 bacteria, 2 mycoplasmas and 1 chicken serum protein antigens in dot immunoassay were compared. Immobilon affinity membrane type D (IAM-D) was found best in binding viral antigens, followed by Ultrabind SV-450 (SV-450). SV-450 was found best in binding bacterial antigen, followed by Ultrabind US-450 (US-450), IAM-D and NC 0.45 micron. IAM-D was the best in binding mycoplasma antigen, followed by Ultrabind HP, US-450, NC 0.2 micron. Overall, IAM-D had the best capability in the binding of the antigens.

The optimal dose of hyperbaric tetracaine spinal anesthesia for cesarean section.

Seventy-five term parturients undergoing cesarean section were anesthetized with 9 mg, 12 mg, 15 mg and 18 mg of tetracaine in 1.5 cc of 10% using a standardized spinal anesthetic technique. The four groups were compared for their maximal spread of anesthesia, quality of analgesia, incidence of hypotension, mgs of ephedrine used to correct hypotension, and the Apgar scores of the infant. It was found that maximal segmental spread of analgesia for 9 mg. 12 mg and 15 mg groups were not different significant regardless of height or weight. But with the 18 mg-group there was significant excessive spread of analgesia. While there was no difference in maximal spread of analgesia, there was a progressive increase in good quality of analgesia with increasing dosages. There were 67%, 81%, 96%, 100% of the patients had good analgesia for their operation in the four respective group. The percentages of hypotension were significanly more in the 15 mg and 18 mg groups. However, we did not find the hypotension in the 15 mg and 18 mg group need more ephedrine to correct than the lower dosages. In addition, all babies had good Apgar scores and there was no statistical difference among the four groups.

[The effect of epidural anesthesia on tourniquet pain: a comparison of 2% lidocaine and 0.5% bupivacaine].

The incidence of tourniquet pain was evaluated in two groups of patients with 20 each undergoing orthopedic surgery of the lower extremities during epidural anesthesia using plain solution of either 2% lidocaine or 0.5% bupivacaine. The drugs were administered in a randomized fashion. Measurement of the levels of sensory loss to pinprick and incidence of tourniquet pain were made by blind-trust. The maximum analgesia level, time between 1st injection and onset of pain, time between tourniquet inflation and onset of pain were recorded similarly in both groups of patients. The incidence of tourniquet pain was significantly greater in patients given 2% lidocaine (40%) than in patients given 0.5% bupivacaine (10%). The incidence of pain was not related to the time of tourniquet inflation, because patients in the bupivacaine group had a significant longer duration of tourniquet inflation than did patients in the lidocaine group. The incidence of pain was also not related to tachyphylaxis, because 7 of 8 patients who complained tourniquet pain in lidocaine group received less than 3 injections for maintenance of analgesia when tourniquet pain started. In summary, it is apparent that tourniquet pain occurs less frequently when bupivacaine is employed for epidural anesthesia as compared to lidocaine.