Method for simultaneous detection of multiple microbial antigens in biological specimens from mastitic animals

Abstract

The present invention involves a method and an immuno-analytical device for the rapid and simultaneous detection of multiple micro-organisms in the biological fluids from milk-producing animals suffering from mastitis. This method is based on a lateral flow immuno-assay technique performed to detect antigens specific for multiple infectious agents which are known to cause and/or be encountered in cases of mastitis. Mastitis is an inflammatory condition affecting the udders of milk-producing animals as a result of microbial infections.

What is claimed is:  
     
         1 . A method for detecting a plurality of different pathogens in a milk sample of a mammal, comprising: 
 exposing said milk sample to a plurality of different antibodies specific to an antigen from a pathogen of said mammal; and    identifying whether said specific antibodies bind to one or more of said antigens, wherein said antigens are from a plurality of different pathogens.    
     
     
         2 . The method of  claim 1  wherein the milk sample is allowed to settle into two phases before exposing and wherein the top “clear” phase is exposed.  
     
     
         3 . The method of  claim 1  wherein the milk sample is diluted before exposing.  
     
     
         4 . The method of  claim 1  wherein the cream is removed from the top of the milk before exposing.  
     
     
         5 . The method of  claim 1  wherein said milk sample is treated to remove at least about 50% of the fat and/or the casein.  
     
     
         6 . The method of  claim 5  wherein said treatment comprises adding a detergent to remove the fat globules and precipitating the casein with acid.  
     
     
         7 . The method of  claim 1  wherein said antibodies are specific for a pathogen selected from the group consisting of a bacteria, a virus, and a fungus.  
     
     
         8 . The method of  claim 1  wherein said milk-producing animal is a cow, a sheep or a goat.  
     
     
         9 . The method of  claim 7  wherein said 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.  
     
     
         10 . The method of  claim 7  wherein said pathogen is a yeast or a mold selected from the group consisting of: Nocardia spp. and Prototheca.  
     
     
         11 . The method of claim wherein said pathogen is selected from the group consisting of:  S. agalactiae, S. dysgalactiae , and  S. uberis.    
     
     
         12 . The method of  claim 7  wherein said pathogen is  Staphylococcus aureus.    
     
     
         13 . The method of  claim 7  wherein said pathogen is a. coagulase-negative Staphylococcus.  
     
     
         14 . The method of  claim 7  wherein said mycoplasma is selected from the group consisting of  M. bovis, M. californicum , and  M. bovigenitaliae.    
     
     
         15 . The method of  claim 1  wherein said antibodies are specific to antigens from at least 3 different pathogens.  
     
     
         16 . The method of  claim 1  wherein said antibodies are specific to 7 pathogens.  
     
     
         17 . The method of  claim 1  wherein said antibodies are specific to 10 pathogens.  
     
     
         18 . The method of  claim 15  wherein said 3 pathogens are  Streptococcus agalactiae, Staphylococcus aureus , and  Mycoplasma bovis.    
     
     
         19 . The method of  claim 16  wherein said 7 pathogens are  Streptococcus agalactiae, Staphylococcus aureus, Mycoplasma bovis, Escherichia coli , coagulase-negative Staphylococci,  M. californicum , and  M. bovigenitaliae.    
     
     
         20 . The method of  claim 1  wherein said pathogens are selected from the group consisting  Staphylococcus aureus , non-hemolytic Staphylococci,  Mycoplasma capricolu, M. mycoides  subspecies mycoides,  M. putrefaciens, Mycoplasma agalactiae, M. arginini, M. conjunctivae, M. ovipnuemoniae , and Mycoplasma strain F38.  
     
     
         21 . The method of  claim 1  further comprising a lateral flow test format.  
     
     
         22 . The method of  claim 1 , further comprising: 
 immobilizing said antibodies on one or more test membranes; and    identifying the presence of the pathogen using a second antibody which is free to move by capillary action within the membrane.    
     
     
         23 . The method of  claim 22 , wherein said second antibody is conjugated to a marker.  
     
     
         24 . The method of  claim 23 , wherein said marker is a color-producing agent.  
     
     
         25 . The method of  claim 24 , wherein said color-producing agent is colloidal gold or a colored latex micro-sphere.  
     
     
         26 . The method of  claim 22 , wherein said more than one test membrane is arranged longitudinally side by side.  
     
     
         27 . The method of  claim 22 , wherein said method allows concurrent visualization of test results.  
     
     
         28 . The method of  claim 1 , wherein said antibodies are polyclonal, monoclonal, or parts thereof.  
     
     
         29 . A kit for the detection of a plurality of pathogens in milk, comprising: 
 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 said antigen.    
     
     
         30 . The kit of  claim 29 , further comprising a container which allows the milk to be collected.  
     
     
         31 . The kit of  claim 29 , further comprising cleaning products for the removal of organisms from the outside of a milk-producing organ.  
     
     
         32 . The kit of  claim 29 , further comprising at least one antibody specific to antigens from granulocytes.  
     
     
         33 . The kit of  claim 32  wherein said granulocytes are neutrophils.  
     
     
         34 . The method of  claim 1 , further comprising identifying the presence of ganulocytes as a general indicator of mastitis.  
     
     
         35 . The method of  claim 34 , wherein said identifying comprises exposing said milk sample to at least one antibody specific to antigens from neutrophils.