A wind park system is disclosed. Briefly described, one embodiment includes at least one wind power installation having a rotor; an electrical generator drivable by the rotor, such that the electrical generator is operable to deliver electrical power to an electrical network; and a control device operable to control at least a voltage and a current of the electrical power delivered into the electrical network, and operable to implement a phase angle change in dependence on a network voltage measured at least one location within the electrical network, wherein the phase angle determines the phase position between the current and the voltage of the electrical power delivered by the electrical generator.

1. Field of the Invention
The invention concerns a method of operating a wind park and also a wind park as such.
2. Description of the Related Art
Wind power installations were initially always erected in the form of individual units and it is only in recent years that, caused also by administrative and building regulations, wind power installations are frequently installed in wind parks. In that respect a wind park in its smallest unit is an arrangement of at least two wind power installations, but frequently markedly more. By way of example mention may be made of the wind park at Holtriem (East Frisia) where more than 50 wind power installations are set up in an array. It is to be expected that the number of units and also the installed power of the wind power installations will also increase greatly in the forthcoming years. In most cases the wind potential is at its greatest in regions of the power supply networks with a low level of short-circuit power and low population density. It is precisely there that the technical connection limits are quickly reached by the wind power installations, with the result that it is then no longer possible for further wind power installations to be set up at such sites.
A conventional wind park which is connected, by way of example, to a 50 MW transformer substation can therefore provide, at a maximum, only 50 MW total power, that is to say for example 50 wind power installations each providing 1 MW nominal power.
Bearing in mind that wind power installations are not constantly operated in the nominal mode and thus the overall wind park also does not constantly reach its maximum power, referred to herein as the nominal power, it can be found that the wind park is not put to optimum use if the nominal power of the wind park corresponds to the maximum possible total power which can be fed into the electric grid connected to the wind park.
Another issue is that in the case of low-power electrical networks, some of which are called island networks, the network frequency rises very quickly and abruptly if a relatively large consumer is separated from the electrical network. The drive machines such as for example diesel engines, water wheels and the like require some time in order then to reduce their mechanical and electrical power. During that period of time those generators produce more energy than is taken from the electrical network. That energy is then consumed for accelerating the generators. That means that the rotary speed and thus also the network frequency rises.
As many electrical devices, for example computers, electric motors and the like, which are connected to the electrical network, are however not designed for fluctuating network frequencies or abrupt changes therein, that can result in damage to electrical machines, even going as far as destruction thereof.


In a liquid crystal display device where each unit pixel p arranged on a liquid crystal panel 101A is constituted by a plurality of pixels p1, p2, and p3, the pixels p1, p2, and p3 are divided into sub-pixels p11 and p12, sub-pixels p21, and p22, and sub-pixels p31 and p32, respectively. The liquid crystal display device is provided with driver ICs 201 and 202 for driving the sub-pixels p11, p21, and p31, and the sub-pixels p12, p22, and p32 constituting the pixels so that different gradation-brightness value characteristics may be given. Due to this, multi-gradation display can be performed.

1. Field of the Invention
The present invention relates to a liquid crystal display device capable of performing multi-gradation display. Particularly, the present invention relates to a liquid crystal display device which can be driven by a driver of an existing type and yet can perform multi-gradation display with higher performance than expected with the existing type of driver.
2. Description of the Related Art
A liquid crystal display device and a plasma display device are known as image display devices using a flat panel. Usually, a digital signal is used at the input interface of these display devices. In a display device using a digital signal at its input interface, the number of displayable gradations depends on the number of bits contained in a signal to be used. As the number of gradations increases, the number of bits increases too. In case of a liquid crystal display device, a source driver achieving the largest number of gradations among source drivers now in practical use is an 8-bit type (256 gradations). Gradations more than this can not be displayed.
Suppose that a 12-bit type source driver is developed by simply increasing the number of bits. When such a 12-bit type source driver is compared with an 8-bit type source driver, the number of resistors comprised in a digital-analog converter (hereinafter referred to as DCA) for generating each gradation and the number of switch circuits for selecting the resistors required in the 12-bit type source driver are 16 (2122/8=4096/256=16) times larger than those required in the 8-bit type source driver. Consequently, the size of the circuit becomes considerably large, and an increase in costs is inevitable because of expansion of the chip size. Hence, there occurs an idea of enabling display of a larger number of gradations than achieved by an existing circuit system yet with the use of an existing circuit system. As one method therefore a method of using each unit pixel by dividing it into a plurality of pixels has been proposed.
Unexamined Japanese Patent Application KOKAI Publication No. 2001-34232 proposes one such method. FIG. 16 is a block diagram showing an example of the structure of a liquid crystal display device according to a prior art, to which the present invention will be applied. As shown in FIG. 16, the liquid crystal display device 100 comprises a color liquid crystal panel 101, a backlight 102, a cell driver 103, a data processing unit 104, and an input/output (I/F) unit 105.
The color liquid crystal panel 101 displays a color image by liquid crystal cells arranged on a plane. The backlight 102 is a light source which emits white-color light from the back of the liquid crystal panel, so that the liquid crystal panel may perform color image display by transmissive light. The cell driver 103 generates a drive signal for driving each liquid crystal cell of the liquid crystal panel based on input data. The data processing unit 104 performs data processing for supplying input data to the cell driver 103 in response to an input digital signal. The I/F unit 105 constitutes an interface for external inputting and outputting. The cell driver 103 is built up by a source driver (not shown) and a gate driver (not shown). The source driver controls the source of each transistor for driving each liquid crystal cell along the arrangement in the vertical direction (column direction). The gate driver controls the gate of each transistor along the arrangement in the horizontal direction (row direction).
FIG. 17 are diagrams for explaining an example of a display screen of a conventional liquid crystal display device, which is disclosed in the aforementioned Unexamined Japanese Patent Application KOKAI Publication No. 2001-34232. FIG. 17(A) is a view of the color liquid crystal panel in partial enlargement. FIG. 17(B) is a diagram showing an example of how to divide each unit pixel. As shown in FIG. 17(A), in case of using color filters, the display screen of the color liquid crystal panel 101 of the conventional liquid crystal display device is structured in a way that an R (red) pixel, a G (green) pixel, and a B (blue) pixel are sequentially and repeatedly arranged horizontally in each row. With the use of the color filters, color display is performed through these R pixels, G pixels, and B pixels based on red image data, green image data, and blue image data respectively. However, a monochrome image is displayed in each liquid crystal cell constituting each pixel of the color liquid crystal panel 101.
Specifically, in the color liquid crystal panel 101, one set of an R pixel, a G pixel, and a B pixel is used as a unit pixel and monochrome display is performed in each unit pixel. Since a unit pixel of a color image is constituted by an R pixel, a G pixel, and a B pixel in case of using color filters, the number of brightness levels displayable by one unit pixel is three times as large as the number of brightness levels displayable by each of the R pixel, the G pixel, and the B pixel.
Therefore, it is possible to break the gradation levels of a display image into more minutely-stepped levels by dividing the brightness level range into, for example, three and scale-marking each divided range. Let it be assumed that one unit pixel p is divided into three pixels p1, p2, and p3 as shown in FIG. 17(B), and each of the pixels p1, p2, and p3 performs 8-bit display. Since the brightness level range displayable by each pixel is from 0 to 255, the brightness level range displayable by the unit pixel p is from 0 to 765 (255×3). A display image including a high gradation level can be achieved by arranging that the smallest value 0 in the brightness level range correspond to the smallest value in image data and the largest value 765 in the brightness level range correspond to the largest value in the image data.
When the data processing unit 104 supplies a brightness value converted from image data to the unit pixel p, it divides the value almost equally among the three pixels p1, p2, and p3. Specifically, let a case be considered where 8-bit image data is input to a color display for performing 8-bit display. The 8-bit image data is composed of values of 0 to 255. In this case, it is arranged that the smallest value in the image data correspond to the smallest brightness value 0 of the color display and the largest value in the image data correspond to the largest brightness value 765 of the color display.
FIG. 18 shows a relationship between brightness values of a unit pixel and brightness values of each pixel in a conventional liquid crystal display device. The data processing unit 104 divides a brightness value acquired from image data among the pixels p1, p2, and p3 as shown in FIG. 18. For example, in case of a brightness value 0 for the unit pixel p, the pixels p1, p2, and p3 are given shares of 0, 0, and 0 respectively. In case of a brightness value 1 for the unit pixel p, the pixels p1, p2, and p3 are given shares of 0, 0, and 1 respectively. In case of a brightness value 2 for the unit pixel p, the pixels p1, p2, and p3 are given shares of 0, 1, and 1 respectively. Likewise, until the brightness value 765 for the unit pixel p, the brightness value for each pixel is determined in the same way. To sum up, according to the conventional liquid crystal display device 100 shown in FIG. 17, a brightness value is equal to the gradation level input to the liquid crystal display device 100. According to the prior art, in the liquid crystal display device 100, the unit pixel p is divided into three homogeneous pixels p1, p2, and p3, thereby achieving an almost three-times-larger number of gradations by adding the gradations (input data to the driver) of all the three pixels. FIG. 19 shows a relationship between input gradation levels and brightness values in the conventional liquid crystal display device 100. FIG. 19 reveals that the relationship between gradation levels input to the liquid crystal display device 100 (or data for each pixel input to the driver) and brightness values (standardized brightness values in FIG. 18) is linear. Therefore, the sum of brightness values of all the pixels p1, p2, and p3 is equal to the brightness value of the unit pixel p.
Further, the Publication of Japanese Patent No. 2700903 discloses a technique for regarding a plurality of neighboring pixels as one display unit, controlling the gradation level of each display unit by changing combinations of the lighting and non-lighting states of each pixel in the display unit or the gradation level of each pixel in the display unit, and arranging that the center of the display unit correspond to the center of the densities of the middle tones.
The invention disclosed in the Publication of Japanese Patent No. 2700903 is directed to a liquid crystal display device of a so-called simple matrix type, and for performing gradational display by varying the width of a data electrode.

Liquid crystal display device and driving method for liquid crystal display device


A process for gas entrainment of wastewater in an anaerobic wastewater treatment system includes the steps of withdrawing wastewater from an anaerobic digester and piping it into a flotation container. Thereafter the wastewater is pumped into a free standing tank having pressure in the tank maintained at a predetermined level through the injection into the tank of compressed gas generated by anaerobic digestion occurring in the treatment system. The wastewater is removed from the tank and pumped through a multiple jet configuration wherein gas from the tank is entrained in the wastewater as the water is injected back into the tank. The gas entrained water is then discharged from the tank back into the flotation container where the gas bubbles particles to the surface of the water to form a sludge layer. The sludge layer formed on the surface of the water in the flotation container is recycled to the digester for further increased digestion, thereby providing an efficient flotation process with increased wastewater clarification, reduced disturbance of the anaerobic bacterial sludge layer, and efficient utilization of the gas generated by anaerobic digestion in the system.

The treatment of wastewater has historically been accomplished through anaerobic and aerobic processes. In the aerobic treatment of wastewater, a system exposes the wastewater to an oxygen atmosphere. A dissolved air flotation ("DAF") system is an example of a system using an aerobic process.
In a typical DAF system, recycled water or the raw wastewater is pressurized in a pressurization tank. Compressed air is then added to the water in the pressurization tank. This water, along with flocculating chemicals is introduced into the DAF vessel. Bubbles are formed as the air entrained in the water is released from pressure. The particles attach themselves to or become trapped by the bubbles. As the bubbles rise to the surface of the water, the particles are carried along. When the bubbles reach the surface the air escapes, forming a foam or sludge layer on the surface. The sludge layer contains the particles deposited by the escaping air bubbles. The sludge layer is kept intact and skimmed from the vessel to be processed by various means. Thus, through aeration, the wastewater is fed needed air and chemicals and the water clarified.
In an anaerobic process, digestion of the organic matter by gas forming bacteria occurs in a digester. Historically, anaerobic treatment of wastewater has not included aeration of the wastewater for further clarification where the use of oxygen or air to aerate the wastewater will cause the resultant sludge to become inactive since anaerobic treatment requires an oxygen-free atmosphere. In presently available anaerobic processes the wastewater is not well flocculated, making it difficult to separate solids from the liquor, resulting in less clarity than achieved in wastewater treated aerobically.

Process for dissolved gas flotation in anaerobic wastewater treatment


Provided herein are detection systems and related methods for detecting moving objects in an airspace surrounding the detection system. In an aspect, the moving object is a flying animal and the detection system comprises a first imager and a second imager that determines position of the moving object and for moving objects within a user selected distance from the system the system determines whether the moving object is a flying animal, such as a bird or bat. The systems and methods are compatible with wind turbines to identify avian(s) of interest in airspace around wind turbines and, if necessary, take action to minimize avian strike by a wind turbine blade.

There is an interest and need in the art for reliable and robust detection of flying avians. Avian detection systems have many applications, ranging from avian counts, classification and/or identification in a specific geographical location, to deterrence and counter-measure systems for aviation and wind production systems. A common objective of such systems is the replacement of subjective and inaccurate human-based counts with an automated and reliable detection system. This is a reflection that human-based detection of flying avians requires intensive training to be able to properly identify avians and species thereof, is highly labor intensive and is inherently inaccurate.
One specific application of bird detection systems is for wind energy generation. There is concern as to the risk to avians arising from avian-wind turbine collision. One challenge for accurately assessing the risk of wind turbine collision by a flying avian is the difficulty in reliably determining the number of birds and the species of such birds in an area of a turbine or a to-be-located turbine. It is difficult to continuously monitor airspace, and so conventional bird strike fatality searches are conducted using systematic schedules with an attendant estimate of fatalities based on a uniform distribution over time, as explained in "Impacts of Wind Energy Facilities on Wildlife and Wildlife Habitat" Technical Review 07-2. September 2007 (available at: wildlife.org/documents/technical-reviews/docs/Wind07-2.pdf). This has numerous disadvantages, including not accounting for cluster fatalities, injured avians that leave the immediate area or are removed by scavengers, and the challenge associated with reliably and consistently locating carcasses. Regardless of such inaccuracies, there has been documentation of raptor fatalities at wind turbine fatalities. See, e.g., Id. at p. 15 and references cited therein, including for California-based wind-farm facilities such as the Altamont Pass Wind Resource Areas (APWRA), San Goronio and Tehachapi. Estimates for raptor kills at APWRA per year range from between 881-1300 or about 1.5-2.2 raptor fatalities/MW/year, including about 75 to 116 Golden Eagles. With these statistics in mind, there is interest in bird detection systems including for use with wind-farm planning, development, expansion and operation.
One example of a bird detection and dissuasion system is Dtbird® by Liquen (description available at dtbird.com/index.php/en/technology/detection). A fundamental limitation of that system is the reported detection efficiency of 86-96% for a distance of only 150 m from the wind turbine, with an efficiency that falls off with increasing distances.
Other implementations of avian detection systems are based on radar including, for example, Merlin Avian Radar Systems by DeTect (www.detect-inc.com/avian.html). Those systems, however, require bulky and expensive radar equipment and are not suited to distinguishing between avian species of interest. For example, a fundamental drawback is the inability to distinguish between an endangered or valued raptor species and another bird species that is neither endangered or of commercial importance. For example, it would be beneficial to distinguish between a golden eagle and a turkey vulture, for example with action implementation for wind blade speed tailored to species of interest only. Radar systems are not suited for such applications, as they do not obtain visual details that would otherwise distinguish between different bird species that are similarly sized and/or have similar flight characteristics. Furthermore, radar-based systems produce many false-positives, including arising from moving objects such as a turbine blade.
U.S. Pat. Pub. 2013/0050400 (Stiesdal) describes an arrangement to prevent collision of a flying animal with a wind turbine. Stiesdal, however, is limited in that there is not full spatial coverage, but instead focuses on imaging horizontal directions. U.S. Pat. No. 8,598,998 describes an animal collision avoidance system. Other systems are described, for example, in U.S. Pat. Pub. Nos. 2009/0185900 (Hirakata) and 2008/0298692 (Silwa). Each of those systems have inherent limitations, such as not providing full coverage of all directions of the surrounding airspace, do not provide sufficient detection efficiency and/or cannot reliably distinguish between avian species and confine detection to a specific avian species.
Because of the risk to migratory birds, raptors and other avians of interest including bats, it is desirable to have a reliable, cost-effective and robust system for identifying certain avian species, including before siting of wind turbine(s) as well as during wind turbine operation. Provided herein are various methods and systems for avian detection, including highly reliable and sensitive detection systems over sufficiently large detection ranges that provide sufficient time to take action to minimize or avoid unwanted contact between a specific avian species and the wind turbine, while minimizing unnecessary wind turbine shutdown for avian species or other moving objects that are not of interest, while avoiding the need for large groups of human observers.

This patent is subject to a terminal disclaimer.


A handle for a confectionery product that envelopes a housing detachably engaged to a support. The housing receives a novelty item. After the confectionery product is eaten the novelty item is released by breaking the support from the housing.

Confectionery products for eating take many varied forms. Candies often are picked up by hand for consumption. Other confectionery products such as frozen ice cream, fruit-flavored frozen liquids, and hard candy suckers are typically attached to a handle. The confectionery product envelopes an upper portion of the handle. A person eating the confectionery product holds a lower portion of the handle to orient the product during consumption. The handle thereafter is discarded. Often these handles are made of wood or food-grade plastic.
As a further inducement to purchase and consume confectionery items, handles have been constructed with ornamental features and characters. The ornamental character typically is molded integral with the handle. The confectionery product then envelopes the character. The ornamental features and characters are revealed by consuming the confectionery product. After the confectionery product was completely consumed, the ornamental character could be used for play. Other handles have been made of a hollow tube having slotted openings. Following consumption of the confectionery product, the handle could be used as a wind instrument, such as a whistle and the like, for play.
While providing an additional incentive to purchase a confectionery product, the ornamental characters attached to the handles had drawbacks. The handle restricted the play use of the ornamental character. The character would be carried by the handle for play. Separating the ornamental character from the handle however, would leave a rough and potentially sharp projection which may prevent the ornamental character from sitting evenly, or may cause injury to a person using the separated character for play.
To further encourage purchase of confectionery products, the novelty item also takes the form of contest entry blank or mail-in redemption coupons. Typically these blanks and coupons are printed as part of the wrapper. Such wrappers however, may become sticky from the confectionery product or torn. This makes it difficult to use the entry form or the coupon.
Accordingly, there is a need in the art for an improved holder for confectionery products which contains a novelty item as a premium.

Novelty-containing handle for confectionery product and method


A Multiple Protocol Signal Detector, MPSD (320, 500) has a mixer (406) that receives signal energy from a multi-band antenna system (324) via a low noise amplifier (402) and from a variable frequency synthesizer (408) via a first variable band pass filter (410). The output of the mixer (406) passes through a second variable band pass filter (416) to an envelope detector (418) a power detector (419) and a low resolution A/D (420) that inputs digitized samples to a binary modulation detector, BMD (422). A controller (424) configures the aforementioned devices of the MPSD (320) to detect communications using different protocols based on a scan Look Up Table (426).

The last fifteen years has seen widespread adaptation of cellular telephony systems for wireless wide area networks (WWANs). At present there are believed to be over two billion cellular telephone users globally. Countries, or in some cases supra-national groupings, regulate electromagnetic spectrum usage independently of each other. This means that cellular networks in different parts of the world do not necessarily use the same frequencies. Additionally, competition between different corporations, and in some cases industry groups, have produced different cellular telephony communication protocols such as GSM, CDMA, WCDMA, and iDEN. In some cases, a particular protocol may be used within different carrier frequency bands in different countries. Some cellular telephone handsets (cell phones) are cable of operating in multiple frequency bands for use in different countries.
Furthermore, it is anticipated that more wireless devices that support multiple competing cellular telephony protocols will be developed. To make these wireless communication devices even more useful, manufacturers have added the ability to communicate using one or more shorter-range wireless communication protocols such as WiFi for wireless local area networks (WLANs), WiMax for wireless metropolitan area networks (WMANs), or Bluetooth for wireless personal area networks (WPANs).
A wireless communication device is, of course, meant to be mobile. By design, a wireless communication device's receiver frequently scans for base station signals in its vicinity so that, when a user moves from one cell's coverage to another, the communications link can be handed off seamlessly. Additionally, a user may move out of a zone of service using one RAT into a zone covered using a second RAT, for example an area still using a legacy protocol, and need to scan a large amount of spectrum to find a compatible network. In a similar case, a cell phone may be activated after a user has deplaned in a foreign country, in which case the cell phone methodically scans all frequencies for any RAT it is capable of using. Wireless mobile communication devices may also automatically scan for shorter range communication signals as well as WWAN signals (or instead of WWAN signals). All of the scanning activities occur even if the cell phone is in idle mode (e.g., not in a call), keeping the cell phone's receiver busy, and draining precious battery power. Given the inconvenience of reduced battery life and the increased awareness of the need to conserve energy globally, it would be desirable to be able to reduce the power consumed by scanning activities.
Skilled artisans will appreciate that elements in the figures are illustrated for simplicity and clarity and have not necessarily been drawn to scale. For example, the dimensions of some of the elements in the figures may be exaggerated relative to other elements to help to improve understanding of embodiments of the present invention.


A method and apparatus to effect tanning in humans. The method utilizes very short discrete pulses, with a dark period of low to zero energization between discrete pulses, of ultraviolet or visible light irradiation of selected wave length to effect the tanning. Different apparati are disclosed to carry out the invention. One type of apparatus continuously generates irradiation of the selected wave length and projects that irradiation through mechanical pathblockers to create the discrete pulses. Another type of apparatus intermittently generates irradiation in the desired discrete pulse and of the desired wave length to create the desired effect.

This invention relates to a new technique to induce "tanning" in humans, by utilizing very short, discrete pulses of ultraviolet ("UV") and/or visible light energy of selected wave length, for example about 250-400 nanometers ("nm"), preferably about 280-300 nm with an optimum of about 290 nm.
The prior art discloses several apparati to induce artificial tanning of human skin. See, U.S. Pat. No. 4,862,886 (Clarke); U.S. Pat. No. 4,794,925 (Kei Mori); U.S. Pat. No. 4.711,448 (Tsvi Goldenberg); U.S. Pat. No. 4,611,327 (Clarke;) and U.S. Pat. No. 4,469,102 (Fish). The prior art devices generally utilize a source of continuous fluorescent ultraviolet radiant energy, usually within the Ultraviolet A ("UVA") spectrum (wave length 320 to 390 nm), with very limited amounts of Ultraviolet B ("UVB") spectrum (wave length, 286 to 320 mm); the Ultraviolet C spectrum ("UVC", wave length 40-286 nm) is seldom utilized in tanning machines.
Tanning occurs largely through a process known as melanogenesis, a process which turns skin darker by stimulating the melanin-containing cells known as melanocytes to generate small packets of pigment. When an individual increases the pigment in his or her skin the emotional result is generally a sense of well-being and perceived improved appearance. UVB light is a known initiator for melanogenesis with a peak "tanning efficiency" at a wave length of about 290 nm. However, UVB has heretofore been saddled with safety concerns. This is because, even though it is known that peak tanning efficiency occurs at about 290 nm, it is also true that exposure to UVB energy has a significant risk of the toxic result of erythema, or "sun burn". Because of this risk, Federal Regulations (e.g., 21 CFR .sctn.1040.20(ii)(c)(1)), place strict limitations on the amount of UVB radiation a recipient may be exposed to (usually, the total energy source must emit less than 0.3% UVB). Consequently, prior art tanning devices are ordinarily required to utilize UVA energy sources (e.g., a continuously emitting fluorescent light tube). As a result, currently available tanning booths and the like are required to use the least efficient source of radiant energy to promote tanning.
Light above but near 320 nm is within the UVA spectrum. UVA will induce the so called "immediate tanning" related to the oxidation of pre-existing melanin, the dark pigment within skin. It is thought that this stimulus of tanning is most likely dependent upon the minimal but still present UVB within commercially available tanning light sources. These currently available suntanning light sources use continuous light sources that produce a series of potentially toxic results , for example the rapid destruction of genetic (thymine dimer formation) and protein structure through the build up of cellular toxins, presumably superoxides and resulting products. Such toxic effects are often divided into both short and long term events. The short term events include sun burn, corneal clouding, and retinal damage. The long term effects include premature aging of the skin and accelerated cancer incidence (e.g., melanoma).

Method of inducing tanning by pulsed light and apparatus to effect same


The disclosed invention relates to assays for detecting and quantifying A.beta. peptide, using solid supports that are coated with heavy metal cations, such as zinc (II) or copper (II) form of a nitriloacetic acid. Further, diagnostic kits are described which are used to carry out the assays of the present invention. An improvement in an assay for detection of A.beta. peptide is suggested which comprises forming a heavy metal cation/solid support complex. The preferred heavy metal cations for this improvement are zinc (II) or copper (II) form of a nitriloacetic acid. Finally, methods and kits for bulk purification of A.beta. peptides from biological fluids are taught.

1. Field of the Invention
The purpose of this invention is to assay the quantity and quality of A.beta. peptide in Alzheimer's disease (AD) and A.beta. amyloidotic disorders related to Alzheimer's disease. Specifically, the invention proposes to achieve this end by enriching the peptide by capturing it from biological fluids such as plasma, serum, cerebrospinal fluid or urine with a zinc- or copper-chelated microwell plate, and then measuring the amounts of captured A.beta. with specific anti-A.beta. antibodies in an ELISA.
2. Related Art
Alzheimer's disease is characterized pathologically by the accumulation in the brain of A.beta. protein. The A.beta. protein is a small peptide that is also found in cerebrospinal fluid and plasma. Much evidence implicates the accumulation of A.beta. in the pathogenesis of the disease, either as the neurotoxic agent itself or as a hallmark which accompanies neurotoxicity in the disorder. A.beta. accumulates as a highly insoluble deposit within neuronal tissues. It is desirable to discover a treatment which would reverse the deposition and relieve or arrest clinical deterioration.
Aggregation of A.beta. in the brain is believed to contribute to the progressive dementia, characteristic of Alzheimer's disease (AD) and to the premature AD observed among Down's syndrome patients. A.beta., a 4.3-kDa peptide, is the principal constituent of the cerebral amyloid deposits, a pathological hallmark of Alzheimer's disease (AD) (Masters et al., Proc. Natl. Acad. Sci. USA 82:4245-4249 (1985); Glenner & Wong, Biochem. Biophys. Rev. Commun. 120:885-890 (1984)). A.beta. is derived from the much larger amyloid protein precursor (APP) (Kang et al., Nature 325:733-736 (1987); Tanzi et al., Science 235:880-884 (1987); Robakis et al., Proc. Natl. Acad. Sci. USA 84:4190-4194 (1987); Goldgaber et al., Science 235:877-880 (1987)), whose physiological function remains unclear. The cause of Alzheimer's disease remains elusive; however, the discovery of mutations of APP close to or within the A.beta. domain (Goate et al., Nature 349:704-706 (1991); Levy et al., Science 248:1124-1126 (1990); Murrell et al., Science 254:97-99 (1991); Hendricks et al., Nature Genet. 1:218-221 (1992), linked to familial AD (FAD) (E. Levy et al., Science 248:1124 (1990); A.beta. Goate et al., Nature 349:704 (1991); M. Chartier-Harlin et al., Nature 353:844 (1991); J. Murrell, M. Farlow, B. Ghetti, M. D. Benson, Science 254:97 (1991); L. Hendricks et al., Nature Genet. 1:218 (1992); M. Mullan et al., Nature Genet. 1:345 (1992)), indicates that the metabolism of A.beta. and APP is likely to be intimately involved with the pathophysiology of this disorder.
Alzheimer's disease affects 10% of individuals over the age of 60, however, the existence of A.beta. deposits in 40% of the brains of normal individuals in their forties suggests an even larger subclinical prevalence. Hence, the disease process is likely to be very common, with individual thresholds of neuronal and functional reserve being responsible for the varying onset of clinical symptoms. The disease is debilitating, chronic, incurable and very expensive to treat and an effective prevention or therapy would have an enormous commercial market. However, there are no reliable biochemical markers for AD.
FAD patients with the "Swedish" APP mutation overproduce the soluble, secreted form of A.beta. and suffer from early onset (<60 years) AD (Citron et al., Nature 360:672-674 (1992)). A potential neuropathogenic mechanism has been reported by Younkin and colleagues (Society for Neuroscience, Mol. Genet. Med. 3:95-137 (1993)) for the APP 717 mutations which account for 90% of the APP mutations causing FAD. These mutations apparently lead to an increase in the ratio of "long" A.beta. (1-42) to Ad (1-40). A.beta. (1-40) is the predominant species in the cerebrospinal fluid (CSF), and is a relatively soluble peptide. A.beta. (1-42) is significantly more amyloidogenic, and its overproduction relative to the 1-40 species appears to lead to early-onset AD in these patients. Therefore, levels of A.beta. (1-40) and A.beta. (1-42) in the cerebrospinal fluid (CSF), plasma, serum or urine may be expected to correlate with cerebral pathology in sporadic AD cases, the predominant clinical form of the disorder.
Two protocols currently exist for the estimation of A.beta. levels in biological fluids. The first involves the immunoprecipitation of A.beta. with specific anti-A.beta. antibodies (e.g. Haass et al., Nature 359:322-325 (1992); Citron et al., Nature 360:672-674 (1992)), a technique which is, at best, semiquantitative. This technique was used in combination with western blotting to measure A.beta. levels in CSF (Shoji et al., Science 258:126-129 (1992)) but found no gross differences between AD and control specimens. A double antibody capture ELISA using monoclonal antibodies raised against A.beta. appears to give specific A.beta. quantification with a sensitivity limit at about 0.6 nM (Seubert et al., Nature 359:325-327 (1992)).
The double antibody ELISA is more widely used and is the only described means of accurately quantifying A.beta.. It has two important limitations. It requires an abundance of expensive antibody to coat the wells of microwell plates in order to capture the A.beta. from the biological fluid. A second anti-A.beta. antibody, at a higher dilution, is used to detect captured A.beta.. The second limitation of the double-antibody capture ELISA technique for A.beta. assay is that it requires a fluorescence-generating enzyme-conjugated detection antibody and a fluorescence microwell plate reader for the final step of the assay.
Fluorescence plate readers are highly specialized and expensive (about $30,000, Millipore Cytofluor), which limits the accessibility of the technique. Fluorescence has been preferred over more versatile, and cheaper, chromogenic assays (e.g., horse radish peroxidase-conjugated detection antibody acting on a chromogenic substrate), because it lowers the limit of sensitivity allowing the measurement of A.beta. at the levels found in biological fluids. No A.beta. assay has been described where the development of a chromogenic substrate was measured by a visible-light microwell plate reader, a far less expensive instrument (e.g., $8,000).
The A.beta. species assay of the present invention will provide a rational basis to monitor response to putative treatments for AD, as well as providing early diagnostic information if clinical outcome studies validate the correlation of the A.beta. levels in the blood or CSF with disease progression.

Diagnostic assay for Alzheimer's disease: assessment of A.beta. abnormalities


A flick knife with a lever frame has a hilt, a blade, a lever frame and a spring. The blade has a proximal end pivotally mounted in the hilt. The lever frame is mounted through the hilt and has a front connector that abuts the proximal end of the blade to rotate the blade, and a rear connector. The spring is mounted inside the hilt and connects the hilt to the rear connector of the lever frame and eases opening of the blade and prevents the blade from opening. Because the spring is mounted in the hilt, the knife is thinner than prior art and therefore more convenient.

1. Field of the Invention
The present invention relates to a flick knife, especially to a flick knife that has a lever frame mounted on the hilt that abuts a proximal end of the blade to help people unfold the blade and reduces a size of the knife.
2. Description of the Prior Arts
A foldable knife where the blade is folded into the hilt is popular since it can be safely stored and conveniently taken out for outdoor activities. A flick knife is one type of foldable knives and has a spring mounted between the hilt and the blade and pushing the blade out of the covers to unfold the blade.
A conventional flick knife has two covers, a blade and a torsion spring. The two covers are separately connected to each other to form a hilt. The blade is mounted between the two covers, and has a proximal end pivotally connected to the hilt and a locking structure to selectively hold the blade unfolded. The torsion spring is mounted in one of the covers, and connects to the blade and the cover.
Therefore, the cover must be thick enough to hold a torsion spring and this is inconvenient to carry. Since the torsion spring is mounted on one side of the blade, the torsion spring imposes a greater force to one side over the other which may prevent smooth rotation.
To overcome the shortcomings, the present invention provides a flick knife with a lever frame to mitigate or obviate the aforementioned problems.


A method for a fast color saturation control of digital color images using one color saturation factor has been achieved. Said method can be performed in any color space having three primaries as e.g. the R-G-B or in the CMY color space without the requirement to convert all the pixels of said color image into another color space as e.g. HSI to perform the color saturation control. Thus a significant computational effort for the conversion of the pixels from one color space into another and back can be avoided. Saving computational effort saves time and reduces the electrical power consumption. Right after the color saturation control process the corrected images can be viewed, if the colors are not satisfactory the process can be easily repeated using a modified color saturation factor. Said method can be used for new pictures taken by a digital camera as well as for downloaded pictures from any source. Additionally said method of color saturation control can be combined with a color correction process.

(1) Field of the Invention
This invention relates generally to digital color image processing and relates more particularly to a method for a fast digital color saturation control with low computational effort.
(2) Description of the Prior Art
Color is the perceptual result of light in the visible region of the spectrum, having wavelengths in the region of 400 nm to 700 nm, incident upon the retina. The spectral distribution of light relevant to the human eye is often expressed in 31 components each representing a 10 nm band.
The human retina has three types of color photoreceptors cone cells, which respond to incident radiation with somewhat different spectral response curves. Because there are exactly three types of color photoreceptor, three numerical components are necessary and sufficient to describe a color, providing that appropriate spectral weighting functions are used.
Pixel values in accurate gray-scale images are based upon broadband brightness values. Pixel values in accurate color images are based upon tristimulus values. Color images are sensed and reproduced based upon tristimulus values, whose spectral composition is carefully chosen according to the principles of color science. As their name implies, tristimulus values come in sets of three. In most imaging systems, tristimulus values are subjected to a non-linear transfer function that mimics the lightness response of vision. Most imaging systems use RGB values whose spectral characteristics do not exactly match the tristimulus values of the human eyes.
A combination of real world physical characteristics determines what the human vision system perceives as color. A color space is a mathematical representation of these characteristics. Color spaces are often three-dimensional. There are many possible color space definitions.
Digital imagery often uses red/green/blue color space, known simply as RGB. Said RGB space is illustrated in FIG. 1 prior art. The red/green/blue values start at zero at the origin and increase along the three axes. Because each color can only have values between zero and some maximum (255 for 8-bit depth), the resulting structure is a cube. We can define a color simply by giving its red, green, and blue values, or coordinates, within the color cube. These coordinates are usually represented as an ordered triplet. Several colors are shown in FIG. 1 prior art mapped into their locations in the 8-bit RGB cube, or color space. Black has zero intensity in red, green, or blue, so it has the coordinates (0,0,0). At the opposite corner of the color cube, white has maximum intensities of each color, or (255, 255, 255). Cyan, magenta and yellow, which are combinations of green and blue, red and blue, and red and green, respectively, are at (0, 255, 255), (255, 0, 255) and (255, 255,0). Finally, note that a middle gray is at the exact center of the cube at location (128, 128,128). Other colors can be described by specifying their coordinates within this cube.
The Cyan, Magenta, Yellow, known as CMY, is often used in printing. The CMY color space is related to the RGB space by being inverse of it. The origin of this color space is not black, but is white, and the primary axes of the coordinate system are not red, green and blue but are cyan, yellow and magenta. The color red in this space is a combination of yellow and magenta, while green is composed of yellow and cyan. In the printing industry, where images start with a white piece of paper (the origin) and ink is applied to generate colors, the CMY color space is commonly used.
Another color space, often used by artists, is Hue, Saturation and Intensity (or HSI). In this color space scenes are not described in terms of red, green, and blue, but as hue, saturation, and intensity (HSI). We see things as colors, or hues that either have a washed-out look or have deep, rich tones. This means having low or high saturation, respectively. Hue is the attribute of a visual sensation according to which an area appears to be similar to one of the perceived colors, red, green and blue, or a combination of them. Saturation is the colorfulness of an area judged in proportion to its brightness.
Images that digital cameras deal with are often obtained through tri-color filter sets, like RGB or CMY, and normally the processing of the images are done in either RGB or CMY color space. Some image operations are, however, complicated to perform in these color spaces, but become trivial in a different color space. This is the case with saturation control, which is best done in HSI space. This is obvious since saturation is one of the original coordinates of HSI. The conversion between color spaces, however, requires significant computer power that takes time and consumes significant electrical power in a battery-powered device as e.g. a digital camera.
By color saturation control is meant the process to increase or decrease the amount of color in an image without changing the image contrast. When saturation is lowered the amount of white in the colors is increased (washed out). By adjusting the color saturation the same image can be everything from a black and white image to a fully saturated image having strong colors.
The color saturation control is best explained using the Hue, Saturation, and Intensity (HSI) color space as shown in FIG. 2 prior art. HSI is a very different three-dimensional color space from RGB or CMY. FIG. 2 prior art illustrates a common representation of HSI color space. The cone shape has one central axis representing intensity. Along this axis are all gray values, with black at the pointed end of the cone and white at its base. The greater the distance along this line from the pointed end, or origin, the brighter or higher is the intensity. If the cone is viewed from above, it becomes a circle. Different colors, or hues, are arranged around this circle—the familiar color wheel used by artists. Hues are determined by their angular location on this wheel. Saturation, or richness of color, is defined as the distance perpendicular to the intensity axis. Colors near the central axis have low saturation and look pastel. Colors near the surface of the cone have high saturation.
In prior art said HSI color space is used to change the color saturation of an image. This is relatively simple to do in HSI color space. First, the original image would have to be converted to HSI. Second, the saturation would be modified. Said modification of the saturation is simple because saturation is one of three coordinates of the HSI color space and thus only one coordinate of the pixels has to be changed. The other two coordinates remain unchanged. Finally, the image would have to be converted back to RGB. The same process applies to the CMY color space, which is very close in structure to the RGB color space. The image would have to be converted from CMY to HSI, then the color saturation would have to be modified and finally it would have to be converted back to CMY. It is obvious that a significant computational effort is required for all these conversions.
U.S. Pat No. (5,555,031 to Van Rooij) describes how in a video signal processing circuit, an adaptive signal compression is realized by correcting the color saturation by multiplication of color difference signals (R-Y, G-Y) by a same correction factor in such a way that color signal values (R, G, B) remain below their respective maximally allowed values without the luminance (Y) being limited as well. Preferably, the correction factor is obtained in dependence upon a non-linearly compressed luminance signal (Y′).
U.S. Pat. No. (5,561,474 to Kojima et al.) discloses a video camera having a processing circuit, which converts electrical signals obtained from an imager into a luminance signal and color-difference signals. The color video camera further includes a memory, which stores a table of values designating color saturation levels and corresponding to a specific hue of a background color. The memory outputs a color saturation level based on the color difference signals obtained by imaging an object on a background of the specific hue. This color saturation level is compared with the luminance signal, and based on the comparison results the luminance signal and color difference signals are separated into components corresponding to the background area and components corresponding to the object area.
U.S. Pat. No. (5,852,502 to Beckett) shows an apparatus and a method for producing series of high-resolution color composite images. The digital camera has an optical assembly that directs visual images to a high-resolution monochrome sensor and a lower resolution color sensor. During the processing of the composite image, the monochrome grayscale value becomes the composite frame grayscale value, the color frame hue value becomes the composite frame hue value, and the color frame saturation value becomes the composite frame saturation value. Processing of the monochrome and color images is achieved on the pixel level. A processor calculates the grayscale value for each pixel in each successive monochrome and color image frame. The processor also calculates the hue value (color) and the saturation value (amount of color) for each pixel in each successive color image frame.

Method for fast color saturation control


A gondola car has a body for lading carried on an underframe. The underframe includes a center sill and cross-bearers. The car has deep side beams having top chords, side sills, and side sheets. The lower portion of the car includes tubs that seat between the cross-bearers. The car may have an internal volume of more than 8000 cu. ft. The car may have rotary dump claw sockets. The car has opposed internal and external stiffeners aligned at the longitudinal stations of the cross-bearers. The internal stiffeners may be triangular cantilevers extending upwardly inside the side sheets. The side sheet lies intermediate the stiffeners and their flanges. The top chords may be wider in cross-section than the side sills. The side sills may define torque tubes that co-operate with the sidewall stiffeners and the top chords to resist lateral deflection. The car may include a false deck, or dog-house at one end to accommodate the brake reservoir and brake valve, such that the car is longitudinally asymmetric.

It is often desirable for rail road cars to weigh out at the same time as they bulk out. For example, the maximum gross weight on rail of a "110 Ton" railroad freight car in North America is 286,000 lbs. If the car carries less than the maximum allowed lading by weight, then an unnecessarily high proportion of the weight being hauled is the weight of the car itself—which is also backhauled empty. Therefore, it follows that most often when relatively low density lading is to be carried it is desirable to have a high volume. This reflects conventional understanding in the railroad industry. Municipal waste tends to provide an example of relatively low density lading. Wood chips may provide another example.
It may be desirable to increase the size of the car by making the car taller. However, a fully laden car must not have a center of gravity more than 98 inches above top of rail (TOR). Therefore, is may also be desirable to extend the lading carrying envelope downward, below the upper flange (or top cover plate) of the center sill, below the height of the center of the couplers, and even below the bottom flange (or bottom cover plate) of the center sill. For this purpose a designer might consider the "bathtub" feature, of which the classic example is the bathtub gondola car shown and described in U.S. Pat. No. 4,361,097 of Jones et al., issued Nov. 30, 1982. As the walls of the gondola increase in height, there may not only be a center of gravity concern, but also a concern that the sidewalls of the car may begin to lack adequate side-ways stiffness. This may be particularly so where the car is to be emptied in a rotary dumping apparatus and where the length of the car has also been increased, with the truck centers being spaced more than, and possibly quite substantially more than, 46′-3″ apart. Finally, it may be desired partially to compress the lading in the car. That is, in one example, it may be desired partially to compact municipal waste in the car as it is loaded.


A copper substrate 30 is immersed in a liquid 34 (e.g. 0.1 molar concentration hydrochloric acid) and illuminated with collimated radiation 24 (e.g. collimated visible/ultraviolet radiation) produced by a radiation source 20 (e.g. a 200 Watt mercury xenon arc lamp). A window 26 which is substantially transparent to the collimated radiation 24 allows the radiated energy to reach the copper substrate 30. An etch mask 32 may be positioned between the radiation source 20 and the substrate 30 (preferably the mask is also in the liquid). The copper substrate 30 and liquid 34 may be maintained at a nominal temperature (e.g. 25.degree. C.). Without illumination, the copper is not appreciably etched by the liquid. Upon illumination the etch rate is substantially increased. A further aspect is the addition of a passivant (e.g. iodine) to the liquid which forms a substantially insoluble passivation layer 36 on the substrate which is removed or partially removed by the radiation 24. Sidewalls 40 are protected from the etchant by vertical passivation layers 42, further increasing the difference between the illuminated and unilluminated etch rates.

Without limiting the scope of the invention, its background is described in connection with current methods of etching copper.
It has long been known that copper is an excellent metal for use in integrated circuit metallization. Copper exhibits low corrosion, low electromigration and high conductivity. However, copper is not currently easily integrated into integrated circuit fabrication process flows.
Heretofore, in this field, copper has been patterned in a variety of ways. Copper may be plasma etched using a chlorine based etch chemistry at highly elevated temperatures. Ion milling or magnetron etching with argon or chlorine reagents have been somewhat successfully used to pattern copper. Lift-off techniques have been used to pattern copper metal features. Wet chemical etching of copper is also known, as well as dry etching with activated gaseous reagents. Etching can be avoided by selectively eletroplating a copper film onto predisposed regions of a substrate.

Anisotropic liquid phase photochemical copper etch


In forming a donut-like headrest under a foaming process, a sealing garnish element is provided, which includes a garnish portion and an edge abutment portion. Prior to a foaming step, the sealing garnish element is brought to a donut-like trim cover assembly and inserted fit in an annular opening formed in the annular inward wall region of the trim cover assembly, such that the edge portions of the annular opening are abutted against the edge abutment portion of sealing garnish element, while the garnish portion is attached on and along the annular inward wall region of trim cover assembly. Hence, during the foaming step, those edge abutment and garnish portions of sealing garnish element effectively prevents a liquid foaming base material injected in the trim cover assembly, against leakage from the annular opening.

1. Field of the Invention
The present invention relates to a method for forming an headrest for use on an automotive seat by means of a foaming process wherein a liquid base foaming material is injected into within a trim cover assembly of donut-like or tubular ringed shape and cured therein to form a corresponding shape of headrest, and also to the resulting headrest formed by that method.
2. Description of Prior Art
Among various sorts of headrests, there has been known a donut-like or generally tubular ringed shape of headrest having a through-hole formed centrally of and through its body. Typically, forming this headrest is based on a foaming process, using a foaming die device. At an initial step, several separate covering material sections are sewn together to provide a three-dimensional donut-like or generally tubular ringed shape of covering member having an through-hole centrally thereof. Along this covering member, an annular opening portion is defined, which allows entry of a headrest frame with headrest stay portions into within the covering member. Next, the headrest frame is placed in the thus-formed covering member through such annular opening portion, with the headrest stay portions projecting outwardly through another corresponding holes formed in the covering member. Then, as for example disclosed from the Japanese Laid-Open Publication No. 1-153183, a sealing member is secured via plural snapping points to the annular opening portion to close the same, after which, such sealed covering material is placed in a foaming die and subjected to a foaming process. During the foaming operation, the sealing member serves to prevent leakage of the liquid base foaming material from the annular opening portion of covering member.
However, in this sort of conventional method, the material of covering member used is rather soft and unsteady, with the result that a portion of the covering material secured to the foregoing sealing material creates creases or wrinkles therein, which makes incomplete the sealing of the covering member. As a consequence thereof, it has been found defective that, during the foaming process, the liquid base foaming material filled in the sealed covering member is easily leaked from the creased or wrinkled portion of covering material around the sealing member, and an objectionable staining points of cured foaming material appears on the surface of resultant headrest, impairing its outer aesthetic appearance.


Two fluid-operated supplementary speed change mechanisms (9, 10), one of which is formed into a direction-reversing mechanism and the other of which is formed into a high/low speed-selecting mechanism, are provided in a front housing 1 at locations before and behind a bearing support wall (4) which divides the inside of the front housing into front and rear chambers. Two transmission shafts (8, 12), on which fluid-operated clutches (38F, 38R, 45, 46) of the supplementary speed change mechanisms are mounted, are disposed non-coaxially and are supported by the bearing support wall, and rotary joints (54F, 54R, 54L, 55H, 55L) for connecting fluid passages (51F, 51R, 51L, 52H, 52L) in the transmission shafts, which passages are in fluid communication with the fluid-operated clutches, to stationary fluid passages are formed in outer circumferences of the transmission shafts within an inside of the single bearing support wall.

This invention relates to a transmission assembly for a working vehicle comprising a front housing and a transmission case which are arranged in series in a longitudinal direction of the vehicle and are fastened together. The front housing includes at its front end portion an engine flywheel and the transmission case includes at least one speed change mechanism. More particularly, the present invention relates to a transmission assembly in which two fluid-operated supplementary speed change mechanisms are provided within the front housing in such a manner that does not lengthen the front housing.
In a fluid-operated speed change mechanism having plural fluid-operated clutches, fluid passages communicated to the plural clutches are formed in a transmission shaft on which the plural clutches are mounted. The fluid passages in the transmission shaft will rotate together with the rotation of the transmission shaft. Thus, rotary joints are required for connecting the fluid passages in the transmission shaft to stationary fluid passages. In a transmission assembly in which two fluid-operated speed change mechanisms are provided, such rotary joints are formed in outer circumferences of two transmission shafts, on which respective plural fluid-operated clutches of the respective change mechanisms are mounted, such that two sets of the rotary joints are placed within two separate bearing support members which support the two transmission shafts, as shown, for example, in U.S. Pat. No. 6,044,720. Each of the bearing support members is made thick in a longitudinal direction of the vehicle because of the plural rotary joints included therein. Thus, the two separate bearing support members each of which is thick will lengthen the transmission assembly and, therefore, the vehicle.
Accordingly, a primary object of the present invention is to provide a novel transmission assembly for a working vehicle in which two supplementary speed change mechanisms are provided in a front housing, having an engine flywheel at a front end portion thereof, with preventing the front housing from being lengthened by taking measures how to form the rotary joints between rotatable and stationary fluid passages.
An attendant object of the invention is to provide a novel transmission assembly which is as simple in structure as possible.
Another attendant object is to provide a novel transmission assembly which is easy in assembling.
The present invention relates to a transmission assembly in a working vehicle comprising a front housing (1) and a transmission case (2) which are arranged in series in a longitudinal direction of the vehicle and are fastened together. The front housing includes at its front end portion an engine flywheel (6), and the transmission case includes at least one speed change mechanism (14, 15). The transmission assembly according to the present invention is characterized in:
that there are provided within the front housing (1) two fluid-operated supplementary speed change mechanisms (9, 10) which are disposed before and behind a bearing support wall (4) and are connected to each other in series, the bearing support wall dividing the inside of the front housing into front and rear chambers, one of the supplementary speed change mechanisms being formed into a direction-reversing mechanism and the other of the supplementary speed change mechanisms being formed into a high/low speed-selecting mechanism, and
that two transmission shafts (8, 12), on which fluid-operated clutches (38F, 38R, 45, 46) of the supplementary speed change mechanisms (9, 10) are mounted, are disposed non-coaxially with each other and are supported by the bearing support wall (4), rotary joints (54F, 54R, 54L, 55H, 55L) for connecting fluid passages (51F, 51R, 51L, 52H, 52L), which are formed in the two transmission shafts and are communicated with the fluid-operated clutches, to stationary fluid passages being formed in outer circumferences of the two transmission shafts (8, 12) at an inside of the bearing support wall (4).
In the transmission assembly in which two supplementary speed change mechanisms are provided within the front housing before and behind a bearing support wall which divides the inside of the front housing into front and rear chambers, the two transmission shafts on which fluid-operated clutches are mounted are disposed, according to the present invention, non-coaxially with each other and the rotary joints (54F, 54R, 54L, 55H, 55L) for connecting fluid passages in the two transmission shafts are particularly formed in the outer circumferences of the two transmission shafts (8, 12) at an inside of the single bearing support wail (4). Thus, only the single support wall (4) is used for forming the rotary joints, so that the front housing and, therefore, the vehicle are prevented from being lengthened.
For simplifying the structure of the transmission assembly, it is preferred that one of the two fluid-operated clutches (45) of the high/low speed-selecting mechanism (10) is formed into a fluid-actuated clutch which is actuated by the action of fluid pressure and is disactuated by the action of a spring (45a) whereas the other of the two fluid-operated clutches (46) of the high/low speed-selecting mechanism is formed into a spring-actuated clutch which is actuated by the action of a spring (46a) and is disactuated by the action of fluid pressure, the two fluid-operated clutches (45, 46) being adapted to be supplied with fluid pressure at a same time through a single fluid passage (52H) formed in the transmission shaft (12) on which the two fluid-operated clutches are mounted. According to this structure, an operating fluid passage to be formed in the above-referenced transmission shaft can be made single.
For an easy assembly, it is preferred that the bearing support wall (4) is detachably secured to the front housing (1).
For an easy assembly, too, it is preferred that there is detachably secured to an outer wall of the front housing (1) a fluid passage-forming plate (57) including fluid passages (61F, 61R, 61L, 62H, 621) which are in fluid communication with the rotary joints (54F, 54R, 54L, 55H, 55L), the fluid passages and the rotary joints being connected through pipes (63F, 63R, 63L, 64H, 64L) which extend through openings (65, 66) formed in the outer wall of the front housing (1).
For simplifying the structure of the transmission assembly set forth above, it is preferred that a directional control valve (90) for the direction-reversing mechanism (9) and a directional control valve (76) for the high/low speed-selecting mechanism (10) are attached to the fluid-passage-forming plate (57). The directional control valves (90, 76) are provided at locations as near to the supplementary speed change mechanisms (9, 10) as possible, so that lowering of fluid pressure between the directional control valves and the two supplementary speed change mechanisms is small.
It is often the cases that the fluid-operated clutches of the direction-reversing mechanism are selectively engaged in a slipping condition by selectively reducing fluid pressure applied thereto so as to achieve running of the vehicle at a reduced speed. According to a preferred embodiment of the present invention, the transmission shaft (8) of the direction-reversing mechanism (9), which is disposed at an upstream side of the high/low speed-selecting mechanism (10), is arranged at a level higher than the level of the transmission shaft (12) of the high/low speed-selecting mechanism. According to this structure, the transmission shaft (8), on which the fluid-operated clutches (38F, 38R) of the direction-reversing mechanism are mounted, can be arranged at such a high level that the fluid-operated clutches are not immersed in lubricant oil which is accomodated in a low level within the front housing. Thus, the control for reducing fluid pressure applied to the fluid-operated clutches is not damaged by lubricant oil which might otherwise exist between frictional elements of the fluid-operated clutches, so that the fluid pressure-reducing control can be performed reliably.
According to another preferred embodiment of the present invention, the bearing support wall (4) is detachably secured to the front housing (1) and another bearing support wall (5) is detachably secured to the front housing at a location spaced from and behind the bearing support wall, one of the supplementary speed change mechanisms (9) being disposed before the bearing support wall (4) whereas the other of the supplementary speed change mechanisms (10) being disposed between the bearing support wall (4) and the another bearing support wall (5). According to this structure, the fluid-operated direction-reversing mechanism and high/low speed-selecting mechanism both requiring a high accuracy can be assembled into the front housing as a package for which the bearing support wall and another bearing support wall are used as support members for the supplementary speed change mechanisms.

Transmission assembly for working vehicles


A fusion protein which comprises the B subunit of the labile toxin (LT-B) of E. coli and part of the flagellin (flaA) protein of C. jejuni is antigenic and is useful for decreasing colonization in chickens by Campylobacter species. The protein is produced by E. coli cells, transformed by the plasmid pBEB into which DNA sequences encoding the novel protein have been introduced.

1. Field of the Invention
Approximately 3 billion chickens are raised in the United States every year, and virtually all are contaminated with Campylobacter jejuni (C. jejuni). Currently, there is no vaccine or any other method available to the poultry industry for the prevention of colonization, therefore there exists a need to develop a product which will provide protection from C. jejuni contamination. This invention relates to a novel fusion protein which, upon administration to a poultry population, will decrease the incidence of colonization by C. jejuni. The protein is relatively simple to produce and purify, and it is expressed in large quantities and can be used without further treatment (beyond purification) for vaccination.
2. Description of the Prior Art
In recent years, C. jejuni has been recognized as a major human enteropathogen and is the species implicated in more than 95% of the cases of campylobacteriosis in the United States (Karmali et al. 1983. J. Infect. Dis., vol. 147, pp. 243-246). C. jejuni has also been recognized as a common cause of gastroenteritis worldwide (Georges-Courbot et al. 1989. Res. Microbiol. vol. 140, pp. 292-296) with two-thirds of the reported cases found in the United States (Bokkenheuser and Sutter. 1981. pp.301-310. In Diagnostic Procedures for Bacterial, Mycotic and Parasitic Infections. Ed. Balows and Hausler. 6th edition, American Public Health Association, Washington, D.C.). Several reports indicate that C. jejuni enteritis is associated with eating in restaurants (Genigeorgis, C. 1987. pp. 111-145. In Elimination of Pathogenic Organisms from Meat and Poultry. Ed. F. J. M. Smulders, Elsevier, Amsterdam), drinking raw milk or unchlorinated water (Hutchinson et al. J. Hyg. Cam., vol. 94, pp. 204-215; Schmid et al. 1987. J. Infect. Dis., vol, 156, pp. 218-222), eating under-cooked poultry meat (Harris et al. 1986. Am. J. Pub. Health, vol. 76, pp. 407-411; Izat and Gardner. 1986. Poultry Science, vol. 67, pp. 1431-1435), and living in a household with pets (Genigeorgis, supra; Vandenberghe et al. 1982. Br. Vet. J., vol. 138, pp. 356-361). C. jejuni is common in the intestine of most domestic and many wild animals and is present in high numbers in most birds (Bokkenheuser, supra).
Species of Camplyobacter enter into a non-pathological, commensal relationship in the intestine of the chicken (Juven et al. 1991. Eur. J. Clin. Microbiol., v. 70, pp. 95-103). Chickens carry the organism as part of the indigenous intestinal flora, and for this reason they have been suspected as an important vehicle in the transmission or Campylobacter spp. to humans (Izat and Gardner, supra; Juven et al., supra). Poultry has been implicated as the major reservoir of human campylobacteriosis in the developed world (Genigeorgis, supra). As many as 90% of broiler chickens may harbor this organism (Lam et al. 1992. Av. Dis., v. 36, pp. 359-363). The pathogen can survive the chicken processing procedures and may be present on the product in retail outlets (Hood et al. 1988. Epidem. Inf., vol. 100, pp. 17-25). In a study at a live poultry market in New York City, more than 80% of broiler chickens sold yielded the organism. In another study, 68% of the retail broiler carcasses tested had C. jejuni (Izat et al., supra). Undercooked or improperly handled poultry has been implicated in 50 to 70% of Campylobacter spp. infections in humans (Hood et al., supra). In a study on university students in Georgia infected with Campylobacter, 70% of the cases were accounted for by eating chicken, often undercooked or raw, and 30% by contact with cats Tauxe et al. 1988. CDC Surveillance Summaries, vol. 37(SS-2), pp. 1-13!.
In a five year study conducted by the CDC between 1982 and 1986, 41,343 isolates of Campylobacter were reported. This yielded an annual isolation rate of 5.5 per 100,000 persons (Tauxe et al., supra). C. jejuni represented 99% of the reported species.
Campylobacter is believed to establish itself in the host by reaching and colonizing the mucosal surface of the intestinal tract. Enteropathogenic bacteria must overcome a number of host defense mechanisms to establish infection or to colonize. As part of the host defense mechanism, the mucous layer quickly eliminates unattached organisms from the gastrointestinal tract. As a virulence factor, the motility of the bacterium allows it to traverse the mucous layer and attach to or invade the epithelial cells of the intestinal tract (Newell and McBride. 1985. J. Hyg. Camb., vol. 95, pp. 217-227). Motility and chemotaxicity are considered important factors in the mechanism of association of C. jejuni with the intestinal epithelium (Griffiths and Park. 1990. J. Appl. Bacteriol., vol. 69, pp. 281-301). Field et al. (1981. Infect. Immun., vol. 33, pp. 884-892) showed, by scanning electron microscopy, that C. jejuni were present on, in, and below the mucous gel in the lower ileum of infected neonatal mice 2 h after inoculation. The ability to swim through environments of high viscosity, such as mucous, depends on the possession of a specialized type of motility.
To establish the role of flagella in the virulence of C. jejuni, Newell and McBride (supra) used a wild type strain and two non-motile variants (one flagellate and one non-flagellate) to conduct some colonization experiments. Their results showed that the aflagellate variant colonized the intestinal tract poorly. This occurred because the organisms were rapidly eliminated from the gut. The nonmotile flagellate colonized the gut as successfully as the wild type strain in some cases. These results suggest that flagella, active or inactive, are necessary for the efficient colonization of the gastrointestinal tract.
In a similar study (Morooka et al. 1985. J. Gen. Microbiol., vol. 131, pp. 1973-1980), the colonization of the intestinal tract by several mutant strains differed strikingly according to their motility. The wild type strain colonized well, while the aflagellated mutants where cleared. A poorly motile mutant, which had short flagellar filaments, colonized mice better than the non-motile flagellated mutants. These observations confirmed Newell's conclusion, but further suggested that motility was a necessary factor for the intestinal colonization by this pathogen.
Bacterial colonization of mucosal surfaces depends on the bacteria being able to maintain close proximity to the mucosa and to attach so as to avoid being swept away (Griffiths and Park, supra). C. jejuni colonizes the small intestine, mainly the ileum (Griffiths and Park, supra), but it may also colonize the colon. Invasiveness, enterotoxin, and cytotoxin production have all been implicated in causing campylobacteriosis (Genigeorgis, supra).
During infection, antibodies are made to a variety of Campylobacter surface structures, e.g. outer membrane proteins, and lipopolysaccharides (McSweegan et al. 1987. Infect. Immun., vol. 55, pp. 1431-1435). Nachamkin and Hart (1985. J. Clin Microbiol, vol. 21, pp. 33-38) did some Western blot analysis of the human antibody response to C. jejuni cellular antigens during gastrointestinal infections. They used acute and convalescent phase sera from patients, and they analyzed the antibody activity against their homologous infecting strains and heterologous clinical isolates. Their results showed that with acute phase sera, 3 major bands were recognized, one of which corresponded to the flagellar antigen. Convalescent phase sera recognized many more proteins and the Campylobacter flagellin was the major immunodominant component in all sera tested. The flagellin was not the major protein however in Coomassie blue stained gels.
Winsor et al. (1985. Gastroenterology, vol. 90, pp. 1217-1222) carried on some experiments to determine which C. jejuni outer membrane antigens elicited secretory IgA (sIgA) by using Western blot analyses of fecal extracts in patients with naturally acquired campylobacteriosis. Seven out of the eight patients elicited sIgA titres. The antigen to which the immunoglobulin reacted very strongly was the 63 kd flagellar antigen.
The flagellum is a major antigen of the Campylobacter cell (Harris et al. 1987. Am. J. Pub. Health, vol. 76, pp. 407-411), and it is the immunodominant antigen recognized during an infection in humans (Pavloskis et al. 1991. Infect. Immun., vol. 59, pp. 1159-1164). It has been reported that there were various classes of antibodies against the flagellar protein in convalescent sera (Ueki et al. 1988. Microbiol. Immunol., vol. 32, pp. 327-337). Herbrink et al. (1988. Eur. J. Clin. Microbiol. Infect. Dis., vol. 7, pp. 388-393) investigated the IgG, IgA, and IgM immune response against C. jejuni at various timepoints during and after infection in humans. Their results showed that IgG antibody titers generally remained at a constant level for more than 50 days, where IgA and IgM titers declined more rapidly to normal values within 30 to 50 days after onset of clinical symptoms.
When an isogenic aflagellar mutant was used to challenge a rabbit, the campylobacters were cleared in less than 24 h. There was no significant IgA response, and the non-flagellar mutant did not protect the rabbit against challenge with the parent strain (Pavloskis et al., supra).
Flagellar filament seems to carry some of the serogroup-specific epitopes, since non-flagellated mutants lose their capacity to be serotyped by the Lior procedure. For most LIO serogroups however, the contribution of the flagellum to serotypic specificity has yet to be determined (Harris et al., J. Bacteriol., supra). Flagella are the locomotory organelles of bacteria (Power et al. 1992. J. Bacteriol., vol. 174, pp. 3874-3883). They are reversible rotary devices, driven by protonmotive force that propel the bacteria through liquid environments (Macnab et al. 1991. Trends in Genetics, vol. 7, pp. 196-200). At a gross level, the known features of the flagellar apparatus are a filament, a hook, and a basal body. This structure is called the "filament hook basal-body complex" (Macnab and De Rosier. 1988. Can. J. Microbiol., vol. 34, pp. 442-451). The locations of the flagellar components fall into five compartments: the cytoplasmic face of the cell membrane, the cell membrane itself, the periplasmic space, the outer membrane, and the cell exterior. The hook is attached to the basal body. The hook and filament are both external to the cell. The flagellar filament is the portion that performs the hydrodynamic work on the cell's environment.
A flagellar filament is a long helical thread of uniform thickness. Its thickness is around 20 nm and its length is 15 .mu.m. Heating of flagellar filaments at 56.degree. C. for 15 min disintegrated them and released a single protein called flagellin (Iino, T. 1985. In Molecular Cytology of Escherichia coli, Academic Press, London, pp. 9-37). The MW of the flagellin monomer differs among different bacterial species, ranging from 40,000 to 63,000. C. jejuni flagellin monomer has a MW of 63,000 (Ueki et al. 1987. Microb. Immunol., vol. 31, pp. 1161-1171). The flagellin monomers, which formed globular units, are lined in 11 longitudinal rows, alternate with each other in adjacent rows, and form as a whole a tubular structure. Flagellin monomers at high concentration assemble by themselves and form filaments in vitro. The reaction is reversible, and the binding among the monomers is thought to be hydrophobic (Iino, supra).
The flagellar systems of similar bacteria, i.e. Escherichia coli (E. coli) and Salmonella typhimurium (S. typhimurium), are encoded by at least 40 genes organized into three regions on the chromosome (Mcnab, 1991, supra; Muller et al. 1992. J. Bacteriol., vol. 174, pp. 2298-2304). However, more than 60 genes are known to be involved in motility and chemotaxis (Macnab, R. M. 1987. In Escherichia coli and Salmonella typhimurium Cellular and Molecular Biology, Vol. 1., Eds. Neidhardt et al. American Society for Microbiology, Washington, D.C., pp. 732-759). The genes associated with the motile behavior are divided into three groups (Macnab, 1991, supra). Genes whose products are essential for the assembly of the flagella are given the symbol `fla`. Genes whose products are not necessary for the flagellar assembly, but essential for motor rotation, are given the symbol `mot`. The group of genes whose products are responsible for chemotactic responsiveness and control of switching between clockwise and anticlockwise direction of rotation, are given the symbol `che`. Nearly all of the flagellar, motility and chemotaxis genes are located in four clusters on the E. coli. chromosome (Macnab, 1991, supra).
The genes are organized into a number of operons, so regulation is especially critical with regards to the flagellin structural gene (Macnab and Aizawa. 1984. Ann. Rev. Biophys. Bioeng., vol. 13, pp. 51-83). A flagellar filament of typical length contains about 20,000 subunits. Synthesis of proteins in such large quantities is very wasteful if the bacterium cannot incorporate the proteins into the flagellum, due to a basal body defect for example. The majority of the regulatory mechanisms operate at the transcriptional level (Macnab, 1991, supra). They regulate expression of the flagellar genes in a hierarchy that parallels their roles in the assembly pathway. Operons coding for proteins needed in the initial steps of the assembly, i.e. switch, basal body, and export apparatus components, are expressed early. Genes for filament structure, motor rotation and chemotactic signaling, whose products are needed only when the basal body-hook complex is complete, are expressed late. All of the early genes must be expressed to obtain transcription of the late genes. A functional defect in any of the early genes can prevent expression of the late genes (Macnab 1991, supra).
The alteration of the transcriptional specificity of the RNA polymerase by the synthesis of alternative sigma factors provides a powerful way of controlling gene expression (Helmann, J. D. 1991. Molec. Microbiol., vol. 5, pp. 2875-2882). The flagellin protein accounts for greater than 98% of the mass of the bacterial flagellum. C. jejuni, among other enteric bacteria, was found to have a sigma-28-like promoter element preceding the flagellin genes (Helmann, supra). Another alternative sigma factor (sigma-54) was found to control flagellin expression in some bacteria. Campylobacters have two flagellin genes, flaA and flaB. A sigma-54-like promoter element was found upstream of the flaB gene, although only the sigma-28-dependent flaA protein is required for motility (Galan et al. 1990. Gene, vol. 94, pp. 29-35). The sigma factor is part of the control mechanisms over flagellin expression, the other mechanisms are still unknown.
The flagellin antigen is highly immunogenic (Khawaja et al. 1992. Curr. Microbiol., vol. 24, pp. 213-221). The flaA flagellin protein has been divided into three distinct regions consisting of two common and one variable regions (Fisher and Nachamkin. 1991. Molec. Microbiol., vol. 5, pp. 1151-1158). The two common regions, C1 and C2, comprising the N-terminal 170 amino acids and C-terminal 100 amino acids, showed 94% and 96% identity to Campylobactyer coli (C. coli) common flagellin regions, respectively. The variable V1 region, comprising the middle of the protein, shows 61% identity to C. coli residues. Comparison of these regions with the sequence of other bacteria, E. coil and Salmonella, showed a similar pattern but with much less identity.
The amino acid sequence of the flagellin N-terminal region, mainly the first 20 residues, has been shown to be homologous in all C. jejuni strains tested to date (Fisher and Nachamkin, supra). This part of the flagellin is essential for filament assembly. During assembly of the flagellum, flagellin subunits are transported through the center of the filament and polymerize at its tip (Nuitjen et al. 1990. J. Biochem. Chem., vol. 265, pp. 17798-17804). Both termini of the flagellum are important to the extension of the filament, and the amino terminus is necessary for the transport. By deletion analysis (Logan et al. 1989. J. Bacteriol., vol. 171, pp. 3031-3038), it was shown that the smallest E. coli flagellin capable of forming flagellar filament required the N-terminal 193 residues and the C-terminal 117 residues. The exposed antigenic regions are less restricted and susceptible to mutations, some of which are advantageous to the organism (Khawaja et al., supra).
Two copies of the flagellin gene of C. jejuni have been identified which are 95% identical (King et al. 1991. Microb. Ecol. Health Dis., vol. 4, pp. 135-140). Flagellar expression is subject to both phase and antigenic variation in Campylobacter species (Logan et al., supra), probably as an adaptation to the environment and the immune response of the host (Nuitjen et al. 1991. Infect. Immun., vol. 59, pp. 1100-1105). Phase variation refers to the ability of some strains to exhibit a bidirectional transition between flagellated and nonflagellated states (Guerry et al. 1990. J. Bacteriol., vol. 172, pp. 1853-1860). Antigenic variation refers to the ability of some strains to synthesize alternate flagellin protein that are distinguishable antigenically and that have different molecular weights. The immunogenicity and antigenic diversity of campylobacter flagella makes them important antigens in serotyping schemes based on the heat-labile antigens like the Lior scheme (Logan et al. 1987. J. Bacteriol., vol. 169, pp. 5072-5077). In some of the LIO serotypes the use of nonflagellated organisms has shown that the flagella can carry the serotype specific determinant (Logan et al., 1987, supra).
The two flagellin genes of C. jejuni 81116 were identified, cloned, and sequenced (Nuitjen et al., 1990, supra). The two copies of the flagellin genes were called flaA and flaB. Both genes are 1,731 base pairs each, they occurred as tandem repeats, and were 95% identical. They have the same orientation, and they are separated by a 173-bp intergenic region. The calculated moledular weights of flagellin A and B were 59,538 and 59,909, respectively. The estimated weight from polyacrylamide gels is 62,000; this difference is probably due to post translational modifications.
Nuitjen et al. (1990, supra) used two specific oligonucleotide probes to discriminate between the mRNA of flagellin A and B. In motile bacteria only mRNA transcribed from flagellin A was detected as a monocistronic messenger of about 1800 nucleotides. By carrying out primer extension studies on the mRNA, they located the start of transcription 43 nucleotides upstream of the ATG start codon. C. coli (Guerry et al. 1991. J. Bacteriol., vol. 173, pp. 4757-4764) also have two copies of the fla gene, flaA and flaB. The two genes share 91.9% sequence identity. Both products are expressed and are required for a fully active flagella (Wassenaar et al. 1991. EMBO J., vol. 10, pp. 2055-2061).
Harris et al. (1987, supra) showed that the flagella of certain strains of C. jejuni and C. coli undergo antigenic variation. C. jejuni 81116 expressed one of two flagellin proteins, one with a MW of 61,500 and the other with a MW of 60,000. A reversible DNA rearrangement has been detected in a C. coli strain, but not in C. jejuni (Harris, 1987, supra). King et al. (supra) studied the expression of flagellin with isolates associated with a milk-borne outbreak of campylobacteriosis. They found that the milk isolates expressed a flagellin with a MW of 62,000 while the human isolates expressed a 58,000 flagellin. They speculated that this antigenic variation gave a virulence advantage for the phenotype.
Very few C. jejuni genes have been cloned and expressed in E. coli. This is due mainly to the lack of genetic markers, the absence of a developed natural gene transfer mechanism, and possibly due to some distinct differences in the regulatory sequences of these two bacteria (Chan et al. 1988. Gene, vol. 73, pp. 185-191). Two genes that have been expressed in E. coli are proB (gamma-glutamylkinase) and proA (gamma-glutamylphosphate-reductase). These genes were isolated by complementation of pro mutant E. coli. It is speculated that these genes were expressed only because the host cells were under pressure. Some of the genes identified in C. jejuni are glyA gene (serine hydroxymethyltransferase) (Chan and Bingham. 1990. Gene, vol. 101, pp. 51-58; Chan, 1988, supra), lysyl-tRNA sypthetase gene (Chan and Bingham. 1992. J. Bacteriol., vol. 174, pp. 695-701), and the 5S, 16S and 23S ribosomal RNA (Ouellette et al. 1987. Antimicrob. Agents Chemother., vol. 31, pp. 1021-1026).
Wang and Taylor (1990. J. Bacteriol., vol. 172, pp. 949-955) reported that growing cells of C. jejuni and C. coli could be naturally transformed by naked DNA without the requirement for any special treatment. Maximum competence was found in early log phase of growth. The cells took up their own DNA much better than E. coli DNA.
Recently Labine-Roussel et al. (1987. J. Bacteriol., vol. 169, pp. 5320-5323) constructed a shuttle cloning vector which can be mobilized from E. coli to C. jejuni, C. coli, and Campylobacter fetus (C. fetus). This vector was used to carry on gene disruption and replacement via homologous recombination (Labigne-Roussel et al. 1988. J. Bacteriol., vol. 170, pp. 1704-1708).
The host responses to intestinal microbial infections involves a complex interplay of soluble factors or mediators, leukocytes, epithelial and endothelial cells of the gut-associated lymphoid tissue (GALT). The GALT is one component of the mucosa-associated lymphoid tissue (MAST), which also includes the bronchial, salivary, nasopharyngeal and genitourinary lymphoid tissues. The GALT consists of discrete lymphoid follicles scattered along the wall of the small intestine (Mesteky and McGhee. 1987. Adv. Immunol., vol. 40, pp. 153-229).
The GALT in chickens consists of the bursa of Fabricius, cecal tonsils (CT), Peyer's patches (PP), and lymphocyte aggregates in the intraepithelium and in the lamina propria (LP) of the gastrointestinal wall. The bursa of Fabricius was considered to be the only site where antibody-forming cells could form (Befus et al. 1980. J. Immunol., vol. 125, pp. 2626-2632). However, surgical ablation of the bursa of Fabricius, even in early embryonic development, does not completely inhibit the production of a humoral response. Thus, other non-bursal lymphoid tissue support some B cell differentiation (Befus et al., supra).
It has been suggested that prevention of infection by C. jejuni can be attained by blocking the colonization factor with specific antibodies (Ueki et al., supra). Wu et al. (1991. Infect. Immun., vol. 59, pp. 2555-2559) showed that the flagellar protein was the major antigen recognized by intestinal lavage IgA in mice infected with C. jejuni.
Serum antibody response to invasive enteric pathogens is very important in protection against systemic infections. The initial immunologic response to enteric infection occurs at the level of the intestinal mucosa. Secretory immunoglobulin A (sIgA) response at the intestinal mucosa is a primary defense against enteric infections (Winsor et al. supra). Stern et al. (1990. Avian Dis., vol. 34, pp. 595-601) found that specific anti-C. jejuni antibodies diminish the ability of the bacterium to colonize the gut of 1-day-old chicks when incubated with the organism as compared with preincubation with phosphate buffered saline.
The flagella of C. jejuni are essential in the colonization of the intestine. Nonflagellated organisms are quickly cleared from the intestine. Chicken polyclonal antiflagellin antibodies as well as monoclonal antiflagellin antibodies have been found to prevent C. jejuni from colonizing the chickens or to increase the dose of bacteria required to colonize the chickens (Carr, unpublished). Flagellar antigens are therefore potential candidates for vaccines as well as suitable antigens for diagnostic purposes, since the flagellin protein is immunodominant during human infections.
Kim et al. (1989. Infect. Immun., vol. 57, pp. 2434-2440) immunized chickens with live E. coli expressing Eimeria acervulina merozoite recombinant antigen. The transformant cells were administered orally. Their results suggested that the recombinant vaccine could elicit antigen-specific humoral and cellular immune responses against the protozoan. Challenge with infective oocysts enhanced both immune responses, implying that the vaccine primed the chicken immune system against this protozoan. The protection, however, was partial. Immunoglobulin and T-cell responses against the recombinant antigen could be detected 7 days after vaccination.
Oral immunization to induce immunity against infectious diseases is convenient, relatively safe, and takes advantage of the mass of lymphoid tissue associated with the gut (Liang et al. 1988. J. Immun., vol. 141, pp. 1495-1501).
The protective role of sIgA is well documented in many experimental models. sIgA neutralizes viruses, toxins, enzymes, inhibits adherence of bacteria to epithelial surfaces (Mesteky and McGhee, supra). sIgA binds to and agglutinates bacteria, but it is not thought to be bateriocidal (McSweegan et al., supra). Thus the induction of specific sIgA is desireable to selectively inhibit and clear colonizing bacteria from the gut. The presence of antibody-antigen complexes in the gut is known to stimulate the production of large quantities of mucus. This flow of mucus will trap the pathogens which will be more readily removed by normal intestinal peristalsis. Moreover, sIgA are better adapted in secretions, being more resistant to denaturation and proteolytic breakdown than IgG antibodies (Pierre et al. 1988. Immunology, vol. 18, pp. 51-56).
The major natural pathway for stimulating the immune system is thus through the GALT, where natural or artificially introduced antigens penetrate through the highly pinocytic and phagocytic M cells and interact with resident accessory and lymphoid cells (Mesteky and McGhee, supra). Precursor IgA B cells leave the site, mature and home back to the lamina propria of the GALT where they differentiate into IgA plasma cells specific for ingested antigens (Mesteky and McGhee, supra).

Campylobacteri jejuni flagellin-escherichia coli LT-B fusion protein

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