Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2001-10-11
2004-04-13
Nguyen, Bao-Thuy L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007330, C435S007340, C435S007100, C435S007920, C435S007930, C435S007940, C435S007950, C435S287100, C435S287700, C435S970000, C435S973000, C436S514000, C436S518000, C436S810000, C436S823000, C424S237100, C424S243100, C424S244100, C424S264100
Reexamination Certificate
active
06720160
ABSTRACT:
FIELD OF THE INVENTION
The invention relates generally to the field of immuno-assays. More specifically, the invention relates to a simple, rapid inexpensive pathogen-specific immuno-assay method for simultaneously screening multiple infectious agents associated with mastitis, and inflammatory disease of the udder in milk-producing animals.
BACKGROUND OF THE INVENTION
Mastitis is a disease of cattle and other ruminants that, if not detected early, has adverse economic consequences to the dairy farmer. Estimated annual losses amount to $150-300 per cow per year with total annual losses in the U.S. ranging from $1.5 to $3.0 billion.
Untreated mastitis results in economic losses due to costs arising from reduced milk production, discarded milk, treatment and premature culling or death. This disease is found in four forms: acute, chronic, clinical, and sub-clinical. Sub-clinical mastitis, which shows no visible signs of disease, causes the greatest financial loss to dairy farmers. For every clinical case, there will be 15 to 40 sub-clinical cases, almost all of which progress to clinical cases. Chronic cases serve as a constant reservoir of pathogens causing mastitis.
Screening and evaluation of mastitis has been routinely performed utilizing the following procedures: California Mastitis Test (CMT, a cow-side test), Wisconsin Mastitis Test (WMT, a lab variant of the CMT), somatic cell count (SCC), strip test (cow-side test on foremilk), and milk cultures (a lab test). All but the last of these tests are non-specific indicators of inflammation and do not detect specific infectious agents. They are based on estimating or counting abnormal numbers of somatic cells, mostly white blood cells and epithelial cells found in milk suggestive of infections or injury. CMT test results can be elevated due to malfunctioning or misused milking equipment unrelated to bacterial infection. SCC requires expensive equipment and does not pinpoint infected quarters of the udder. Infectious agents, mostly bacteria, are only sought for and identified utilizing the milk culture procedure. It is laborious, expensive, and takes from 24 hours to 10 days to complete. It also requires samples collected aseptically which are difficult to obtain under field conditions where there are numerous sources of contamination.
In the patent literature, only one same-day test for bacterial identification in mastitis has been described. This is covered under U.S. Pat. Nos. 4,849,341 (Adams, et.al. Jul. 1989) and U.S. Pat. No. 5,132,210 (Adams, et.al. July 1992), and comprises a diagnostic test for detecting antibodies to
Staphylococcus aureus
utilizing a classic ELISA test format. Antibody detection is an indirect test and not necessarily specific for current infection. In addition, although it is one of the most important pathogens causing mastitis,
Staphylococcus aureus
is only one among many encountered. Similar test formats, mostly for antibody detection from single infectious agents found in mastitic milk, are also found in veterinary research publications with no mention of a simultaneous detection format for two or more organisms.
The following issues and problems summarize the current state of mastitis testing and unnecessary costs to the dairy farmer in lost time and resources.
1. Currently used CMT, WMT, SCC and strip tests do not identify pathogens. They are insensitive and non-specific cow-side testing methods. Mixed species of infectious agents, whose identification is essential to diagnosis of mastitis, are not identified on a timely basis using these presently popular techniques.
2. Culture tests for identifying pathogens take at least 24 hours and up to 10 days to obtain results. Such long turnaround on lab results does not allow timely identification of cows with poor (volume and quality) milk output and hence does not ameliorate economic losses.
3. Culture tests are laborious, expensive, and require aseptically collected milk to minimize occurrence of contaminants.
4. There is currently no pathogen-specific test for field (cow-side) use on the market. A simple and rapid test that can be used by untrained personnel and that can be stored under ambient conditions is badly needed.
5. There is no pathogen-specific field test to evaluate infections in all four quarters of the udder to pinpoint the source of poor quality milk or poor milk yield per cow.
6. There is no simple and rapid pathogen-specific test that can aid buyers and sellers of herds in identifying healthy and economically useful animals.
7. Currently available, non-pathogen specific methods do not provide any information on an appropriate therapeutic course that can be instituted in a timely manner.
Therefore, there is a need for a simple, low-cost test which will identify the presence of a variety of pathogens associated with mastitis.
SUMMARY OF THE INVENTION
A method for the simultaneous and rapid detection of multiple pathogens in the milk of animals with suspected mastitis is disclosed herein. The method uses pathogen-specific antibodies as detection tools. One embodiment of the method uses a lateral flow test format, allowing for at least 10 simultaneous tests on one card. Up to 10 lateral flow strips can be arranged on a typically-sized card. This embodiment allows the testing of animals in the field with almost immediate results.
One embodiment is a method for detecting a plurality of different pathogens in a milk sample of a mammal by exposing the milk sample to a plurality of different antibodies specific to an antigen from a pathogen of said mammal; and identifying whether the specific antibodies bind to one or more of the antigens, wherein the antigens are from a plurality of different pathogens. In a further embodiment, the milk sample is allowed to settle into two phases before exposing and wherein the top “clear” phase is exposed. Alternatively, the milk sample is diluted before exposing. The cream may be removed from the top of the milk before exposing. In one embodiment, the milk sample is treated to remove at least about 50% of the fat and/or the casein. The treatment may be by adding a detergent to remove the fat globules and/or by precipitating the casein with acid. The antibodies are specific for a pathogen which may be a bacteria, a virus, and a fungus. The milk-producing animal may be a cow, a sheep or a goat.
In one embodiment, the pathogen is a bacteria selected from the group consisting of: Streptococcus spp., Enterococcus spp., Staphylococcus spp., Micrococcus spp.,
Escherischia coli,
Klebsiella spp. Enterobacteria, Serratia spp., Pseudomonas spp., Proteus spp., Pasteurella spp.,
Corynebacterium bovis, Arcanobacterium pyogenes,
Mycobacterium spp., Bacillus spp., and Mycoplasma spp.
In a further embodiment, the pathogen is a yeast or a mold selected from the group consisting of: Nocardia spp. and Prototheca. The pathogen may be selected from the group consisting of:
S. agalactiae, S. dysgalactiae,
and
S. uberis
or
Staphylococcus aureus.
The pathogen may be a. coagulase-negative Staphylococcus. The mycoplasma may be selected from the group consisting of
M. bovis, M.californicum,
and
M.bovigenitaliae.
In one embodiment, the antibodies are specific to antigens from at least 3 different pathogens. In a further embodiment the antibodies are specific to 7 pathogens. In a further embodiment the antibodies are specific to 7 pathogens. In one embodiment, the 3 pathogens are
Streptococcus agalactiae, Staphylococcus aureus,
and
Mycoplasma bovis.
In a further embodiment, the 7 pathogens are
Streptococcus agalactiae, Staphylococcus aureus, Mycoplasma bovis, Escherichia coli,
coagulase-negative Staphylococci,
M. californicum,
and
M. bovigenitaliae.
In one embodiment, the method is a lateral flow test format.
A further embodiment is a kit for the detection of a plurality of pathogens in milk, having a plurality of antibodies, wherein said antibodies are specific to an antigen from a pathogen of milk, wherein said antigens are from a plurality of pathogens; and a marker for the binding of said antibodies to
Helica BioSystems, Inc.
Knobbe Martens Olson & Bear LLP
Nguyen Bao-Thuy L.
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