Yuanan Lu

Establishment and characterization of 13 cell lines from a green turtle (Chelonia mydas) with fibropapillomas.

SummaryThirteen cell lines were established and characterized from brain, kidney, lung, spleen, heart, liver, gall bladder, urinary bladder, pancreas, testis, skin, and periorbital and tumor tissues of an immature male green turtle (Chelonia mydas) with fibropapillomas. Cell lines were optimally maintained at 30° C in RPMI 1640 medium supplemented with 10% fetal bovine serum. Propagation of the turtle cell lines was serum dependent, and plating efficiencies ranged from 13 to 37%. The cell lines, which have been subcultivated more than 20 times, had a doubling time of approximately 30 to 36 h. When tested for their sensitivity to several fish viruses, most of the cell lines were susceptible to a rhabdovirus, spring viremia carp virus, but refractory to channel catfish virus (a herpesvirus), infectious pancreatic necrosis virus (a birnavirus), and two other fish rhabdoviruses, infectious hematopoietic necrosis virus and viral hemorrhagic septicemia virus. During in vitro subcultivation, tumor-like cell aggregates appeared in cell lines derived from lungs, testis, and periorbital and tumor tissues, and small, naked intranuclear virus particles were detected by thin-section electron microscopy. These cell lines are currently being used in attempts to isolate the putative etiologic virus of green turtle fibropapilloma.

Detection of Green Turtle Herpesviral Sequence in Saddleback Wrasse Thalassoma duperrey: A Possible Mode of Transmission of Green Turtle Fibropapilloma

Samples of DNA were prepared from various tissues and organs (including snout, gill, eye, brain, heart, liver, gut content, intestine, swim bladder, spleen, gallbladder, spinal cord, gonad, and muscle) of six healthy appearing reef cleaner fish, saddleback wrasses Thalassoma duperrey, captured from a cleaning station in North Kaneohe Bay, Oahu, Hawaii. The DNA samples were tested for evidence of green turtle herpesvirus infection by nested polymerase chain reaction (PCR). Green turtle herpesviral sequences were detected in snout (3/6), gill (2/6), and liver (1/6). All other tissues were negative. Except for a single nucleotide substitution (from A to G at position 48, resulting in a single amino acid change from isoleucine to methionine at position 16), the DNA sequences detected in the fish were identical to that of a newly reported green turtle herpesvirus. Although preliminary, these data represent the first evidence for an association of a herpesvirus with saddleback wrasse, suggesting that cleaner fish may serve as vectors or carriers for the transmission of the agent causing green turtle fibropapilloma.

Clinical isolates of Streptococcus pneumoniae resistant to levofloxacin contain mutations in both gyrA and parC genes

Thirty Streptococcus pneumoniae clinical isolates resistant to levofloxacin were analyzed for the quinolone resistance-determining DNA sequences to identify point mutations and were tested for in vitro susceptibility to multiple drug classes. Of these isolates, 29 had mutations in both gyrA and parC genes of DNA gyrase and topoisomerase IV, respectively. In GyrA, an amino acid change from Ser-81-->Phe was detected in 27 isolates and a Glu-85-->Lys change was found in the remaining three. Of the 29 isolates for which ParC data were available, Ser-79-->Tyr or Phe were the predominant mutations observed. MICs for levofloxacin were 4-16 mg/l, whereas those for moxifloxacin were 1-2 mg/l. Twenty-four (80%) isolates were susceptible to erythromycin, 25 (83%) to azithromycin, 26 (87%) to clarithromycin, 27 (90%) to clindamycin, 20 (67%) to penicillin, 21 (70%) to ceftriaxone and 30 (100%) to amoxycillin/clavulanate. These results confirm the presence of double mutations among clinical isolates of S. pneumoniae from diverse geographical regions of North America and also suggest that quinolone resistance may develop independently of resistance to other drug classes.

SHORT COMMUNICATION; The pathogenicity of a baculo-like virus isolated from diseased penaeid shrimp obtained from China for cultured penaeid species in Hawaii

In 1993, the production of cultivated Penaeus chinensis (also known as P. orientalis) in China dropped dramatically owing to mass mortality due to unidentified causes. The disease was acute and lethal, taking only 2–3 days from onset to 100% mortality of the affected P. chinensis population. Also dramatically affected by the disease was P. japonicus (pink shrimp) cultured in China. The causative agent(s) of the acute mass mortality of the two species of penaeid shrimp was not identified. The present study was directed at identifying the causative agent involved in these mass mortalities and to determine the susceptibility of two other penaeid species, P. stylirostris (blue shrimp) and P. vannamei (white shrimp), commercially cultured in Hawaii and the Western Hemisphere, to the yet-unidentified agent. Moribund pink shrimp (3–8 g) affected by the disease were harvested from Maruha shrimp farm in Dalian, China in July 1994. A 30% cephalothorax tissue suspension was prepared in an antibiotic-incubation mixture (AIM) (10 ml of penicillin/streptomycin, 10 000 U / 10 000 mg ml–1; gentamicin, 0.5 ml of 50 mg ml–1; amphotericin B, 1 ml of 250 mg ml–1; and 88.5 ml MEM-0) with a homogenizer. After removal of cellular debris by low-speed centrifugation, the supernatant was filtered (0.2 mm filter) and the filtrate was stored at 2 70 °C. To test the susceptibility of P. stylirostris and P. vannamei to the unknown agent, animals weighing 3–10 g were experimentally injected with the filtrate, 0.01–0.02 ml per animal by intramuscular (IM) inoculation. Uninfected control animals were injected with the same amount of TNE buffer (pH 7.4). The infectivity experiments were carried out under conditions described previously (Lu et al., 1994). Moribund and dead animals were removed and recorded daily during the course of the test and mortality rates were calculated at the end of the experiment. The results indicated that inoculation of a 30% cephalothorax suspension filtrate prepared from moribund shrimp resulted in 100% mortality of both species of

Examining the Role of Transmission of Chelonid Alphaherpesvirus 5

Marine turtle fibropapillomatosis (FP) is a devastating neoplastic disease characterized by single or multiple cutaneous and visceral fibrovascular tumors. Chelonid alphaherpesvirus 5 (ChHV5) has been identified as the most likely etiologic agent. From 2010 to 2013, the presence of ChHV5 DNA was determined in apparently normal skin, tumors and swab samples (ocular, nasal and cloacal) collected from 114 olive ridley (Lepidochelys olivacea) and 101 green (Chelonia mydas) turtles, with and without FP tumors, on the Pacific coasts of Costa Rica and Nicaragua. For nesting olive ridley turtles from Costa Rica without FP, 13.5% were found to be positive for ChHV5 DNA in at least one sample, while in Nicaragua, all olive ridley turtles had FP tumors, and 77.5% tested positive for ChHV5 DNA. For green turtles without FP, 19.8% were found to be positive for ChHV5 DNA in at least one of the samples. In turtles without FP tumors, ChHV5 DNA was detected more readily in skin biopsies than swabs. Juvenile green turtles caught at the foraging site had a higher prevalence of ChHV5 DNA than adults. The presence of ChHV5 DNA in swabs suggests a possible route of viral transmission through viral secretion and excretion via corporal fluids.

A SDS/Page/Western Blot/EIA Protocol for the Specific Detection of Shrimp Viral Pathogens

Shrimp viral diseases have seriously impacted the sustainability and economic success of the shrimp aquaculture industry worldwide. Among several of the recent viral pathogens which caused massive mortalities in cultured shrimp were the yellow-head virus (YHV) and the white spot baculovirus (WSBV) (also called the Chinese baculovirus [CBV] or the systemic ectodermal and mesodermal baculovirus [SEMBV]). Both YHV and CBV have been reported by our laboratory to be highly pathogenic for Penaeus stylirostris (blue shrimp) and P. vannamei (white shrimp), the two principal penaeid species commercially cultured in Hawaii and the Western Hemisphere (Lu et al. 1997). They are thus potentially a serious problem particularly to the brood stock industry.