Characterization of infectious laryngotracheitis virus isolates: demonstration of viral subpopulations within vaccine preparations

Infectious laryngotracheitis (ILT) is a severe acute respiratory disease of chickens caused by ILT virus. To better understand the epidemiology of the disease, a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assay of the glycoprotein E gene has been developed and utilized to characterize vaccine strains and outbreak-related isolates. Enzymes EaeI and DdeI were used to differentiate the tissue culture origin (TCO) vaccine from chicken embryo origin (CEO) vaccines. Two RFLP patterns were observed with enzyme EaeI, one characteristic of the TCO vaccine and a second characteristic of all CEO vaccines. Three RFLP patterns were observed with enzyme DdeI. Patterns A and B were characterized as single patterns, whereas the type C pattern was a combination of patterns A and B. Analysis of vaccine strains showed the presence of patterns A and C. Pattern A was observed for the TCO vaccine and one CEO vaccine, whereas pattern C was observed for five of the six CEO vaccines analyzed. PCR-RFLP analysis of plaque-purified virus from pattern C CEO vaccine preparations demonstrated the presence of two populations (patterns A and B). Identification of molecularly different populations of viruses within currently used ILT vaccine is the first step to develop better molecular epidemiologic tools to track vaccine isolates in the field.     

Replication and transmission of live attenuated infectious laryngotracheitis virus (ILTV) vaccines

The aim of this study was to evaluate the replication of live attenuated infectious laryngotracheitis virus vaccines in selected tissues and their ability to transmit to contact-exposed birds. Four-week-old specific-pathogen-free chickens were eye drop-inoculated with tissue culture origin (TCO) and chicken embryo origin (CEO) vaccines. Contact-exposed chickens were housed in direct contact with eye drop-inoculated chickens from the first day postinoculation. Virus isolation and real-time polymerase chain reaction were used to detect the presence of live virus and viral DNA, respectively, in the trachea, trigeminal ganglia, eye conjunctiva, cecal tonsils, and cloaca from eye drop-inoculated and contact-exposed birds at days 2, 4, 5 to 10, 14, 18, 21, 24, and 28 postinoculation. No differences were observed in the ability of the TCO and CEO vaccines to replicate in the examined tissues. Both vaccines presented a localized replication in the eye conjunctiva and the trachea. Both vaccines were capable of transmitting to contact-exposed birds, attaining peaks of viral DNA as elevated as those observed in inoculated birds. The CEO vaccine replicated faster and reached higher viral genome copy number than the TCO vaccine in the conjunctiva and trachea of eye drop-inoculated and contact-exposed birds. The viral DNA from both vaccines migrated to the trigeminal ganglia during early stages of infection. Although the CEO and TCO vaccines were not recovered from the cecal tonsils and the cloaca, low levels of viral DNA were detected at these sites during the peak of viral replication in the upper respiratory tract.     

Development and validation of a real-time Taqman polymerase chain reaction assay for the detection of Mycoplasma gallisepticum in naturally infected birds

In this study, we report the development and validation of a real-time polymerase chain reaction (PCR) assay using a Taqman-labeled probe for the detection of Mycoplasma gallisepticum (MGLP assay). The MGLP assay was highly specific with a detection limit of 25 template copies per reaction and a quantification limit of 100 template copies per reaction. Validation of the assay was completed with 1247 samples (palatine cleft and tracheal swabs) from M. gallisepticum-positive and -negative chicken flocks. The MGLP assay was compared to an enzyme-linked immunosorbent assay (ELISA), a conventional polymerase chain reaction assay (mgc2 PCR), and isolation of M. gallisepticum from naturally infected flocks. A total of 805 samples collected from negative flocks, as verified by ELISA and/or mgc2 PCR, were negative by the MGLP assay. A total of 442 samples were collected from positive flocks, of which a total of 228 samples were positive by the MGLP assay. These results agreed for 98.87% of the samples when tested by mgc2 PCR. When comparing the MGLP assay with M gallisepticum isolation, the MGLP assay was more sensitive than isolation for detecting positive birds from a positive flock, 172/265 and 50/265, respectively. Overall, the MGLP assay and M. gallisepticum isolation agreed for 52.8% of the samples tested. In conclusion, the MGLP assay was highly specific, sensitive, and reproducible, and allowed the quantification of template copies directly from clinical samples.     

Characterization of infectious laryngotracheitis virus (ILTV) isolates from commercial poultry by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP)

Infectious laryngotracheitis (ILT) is a highly contagious, acute respiratory disease of chickens, of worldwide distribution, that affects growth and egg production and leads to significant economic losses during periodic outbreaks of the disease. Live attenuated vaccines (chicken embryo origin [CEO] and tissue-culture origin [TCO]) have been widely used to control the disease in the United States. It is believed that most of the outbreaks in the United States are caused by vaccine-related isolates that persist in the field and spill over into na?ve poultry populations. The objective of this study was to utilize the previously developed polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis to genotype recent ILT virus (ILTV) isolates from commercial poultry. Forty-six samples were collected during January 2006 to April 2007 from five poultry production regions of the United States and were characterized within PCR-RFLP groups III-VI. Sixty-three percent of the samples analyzed were categorized as closely related to the vaccine strains (groups III-V), whereas 33% were categorized as group VI viruses that differed in six and nine PCR-RFLP patterns from the CEO and TCO vaccines; a mixture of group IV and V viruses was detected in two samples (4%). In general, groups V and VI were the most prevalent viruses, found in 52% and 33% of the samples tested respectively. Both types of viruses were detected in vaccinated and nonvaccinated flocks. Although genetically different, both viruses produced severe disease in the field.     

Mouse lysozyme production by a monocytoma: isolation and comparison with other lysozymes

A transplantable mouse tumor, GPC-11, produces large amounts of lysozyme. The tumor is a reticulum cell sarcoma, type A, and is a neoplasm of monocytes. The lysozyme was purified from mouse urine in quantities sufficient for structural analysis. Comparison of mouse lysozyme with lysozymes from; chicken egg white and patients with monocytic leukemia reveals similarities in size and electrophoretic mobility and, with human lysozyme, in functional properties; but considerable differences are found in antigenic characteristics and amino acid composition.     

Molecular fossil record of elevated methane levels in late Pleistocene coastal waters

Accumulating evidence suggests that methane has been released episodically from hydrates trapped in sea floor sediments during many intervals of rapid climate warming. Here we show that sediments from the Santa Barbara Basin deposited during warm intervals in the last glacial period contain molecular fossils that are diagnostic of aerobic and anaerobic methanotrophs. Sediment intervals with high abundances of these compounds indicate episodes of vigorous methanotrophic activity in methane-laden water masses. Signals for anaerobic methanotrophy in 44,100-year-old sediment are evidence for particularly intense methane emissions and suggest that the basin's methane cycle can profoundly affect oxygen budgets in the water column.     

A serpentinite-hosted ecosystem: the Lost City hydrothermal field

The serpentinite-hosted Lost City hydrothermal field is a remarkable submarine ecosystem in which geological, chemical, and biological processes are intimately interlinked. Reactions between seawater and upper mantle peridotite produce methane- and hydrogen-rich fluids, with temperatures ranging from <40 degrees to 90 degrees C at pH 9 to 11, and carbonate chimneys 30 to 60 meters tall. A low diversity of microorganisms related to methane-cycling Archaea thrive in the warm porous interiors of the edifices. Macrofaunal communities show a degree of species diversity at least as high as that of black smoker vent sites along the Mid-Atlantic Ridge, but they lack the high biomasses of chemosynthetic organisms that are typical of volcanically driven systems.