Immunobinding Assay for Detection of Mycoplasma bovis in Milk Fidel Infante Martinez, Donald E. Jasper, Jeffrey L. Stott, James S. Cullor and Jon D. Dellinger
ABSTRACT
INTRODUCTION
An immunobinding dot-blot assay (IBA) was developed for the detection of mycoplasma in milk. The test was highly species specific when monoclonal antibody preparations were employed in the assay system. Reactions were obtained with all mycoplasma species tested when polyclonal antisera preparations were used. Preincubation for 48-72 hours was necessary with milk samples containing only a few mycoplasma. Time from sample receipt to diagnosis in most positive samples could be reduced from several days by culture to a few hours by the IBA, thus enabling control procedures to be quickly initiated.
Diagnosis of mycoplasma infection in milk has depended primarily upon microbiological culture of udder secretions (1-3). Although the reliability of this method is reasonably satisfactory, it is time-consuming. Inoculated plates must be incubated at least two to three days before colonies may be observed and should not be considered negative prior to evaluation after seven days of incubation (1-3). Determination of species has required further study which may be biochemical or immunological (3). The most satisfactory method of routine determination of species has been by immunofluorescence (4). The combined microbiological and fluorescent antibody methods for detection and identification of mycoplasma species are sensitive and very specific; however they are slow and expensive in time and equipment. Immunobinding procedures are sensitive, rapid, very specific and do not require expensive equipment. Such an assay has been described for mycoplasma colonies and for mycoplasma from broth or tissue cultures (5-7), but has not been described for use in materials of high fat content such as milk. The purpose of this research was to develop an immunobinding assay (IBA) for detection of Mycoplasma bovis in naturally infected milk and to reduce the time required to do the IBA.
RESUME Une epreuve immunologique afin de detecter la presence de mycoplasme 'a partir du lait a ete developpee. Lorsque des anticorps monoclonaux ont ete utilises, cette technique a demontre une tres grande specificite car des liaisons ont ete obtenues avec toutes les souches de mycoplasme ayant reagi avec des anticorps polyclonaux. Une periode d'incubation de 48 'a 72 h etait cependant necessaire si le lait ne contenait que quelques colonies bacteriennes. Cette technique immunologique de detection a l'avantage de pouvoir reduire de quelques jours a quelques heures le delai d'attente avant de poser un diagnostique positif de linfection.
MATERIALS AND METHODS MYCOPLASMA SPECIES
Mycoplasma and acholeplasma species used in this research were obtained from bovine milk or were American Type Culture Collection (ATCC) strains. Species identity of all cultures was confirmed by immunofluorescence (4). ANTIGEN PRODUCTION FOR THE IMMUNOBINDING ASSAY
Mycoplasmas used for antigen in the immunobinding assays were grown separately in modified Hayflick broth medium containing 15% horse serum (2). After 72 h at 370 C, cultures were washed three times with phosphate buffered saline, pH 7.4 (PBS), adjusted to 1 mg protein per mL in PBS as determined by the method of Bradford (9), and stored at -20° C. This preparation was used as an antigen in modified Hayflick broth or mixed with pasteurized commercial milk containing no fat, 2% or 3.5% homogenized fat, or with nonpasteurized, nonhomogenized natural milk. ANTIGEN PRODUCTION FOR RABBIT IMMUNIZATION
Mycoplasmas (M. bovis, California 201) for antigen were grown for 48 h at 370C in 500 mL of medium, centrifuged at 27,000 g for 30 min and washed three times with PBS, the volume being finally adjusted to 6 mL and stored at -20°. Before use, antigen was thawed, sonicated (model 375, Heat Ultrasonic Inc., Plainview, New
Department of Clinical Pathology (Infante, Jasper, Cullor, Dellinger) and Department of Microbiology and Immunology (Stott), School of Veterinary Medicine, University of California, Davis, California 95616. Reprint requests to Dr. D.E. Jasper. From a thesis submitted by the senior author in partial fulfillment of requirements for the Ph.D. degree. Supported in part by grants from the California Milk Producers Advisory Board and the USDA Endemic Diseases Fund. Submitted July 13, 1989.
Can J Vet Res 1990; 54: 251-255
251
York) twice for 1 min at 20 s intervals and diluted 1:200 in PBS. Two mL of antigen were mixed with 2 mL of Freund's complete adjuvant for use in immunizing rabbits. RABBIT POLYCLONAL MYCOPLASMA ANTIBODY PRODUCTION
Antigen injections for polyclonal antibody were made in rabbits over a nine week period. On day 1, 0.2 mL of antigen with adjuvant was given intradermally (ID) in each hind foot pad, 0.6 mL ID at three to four sites along the back, 0.6 mL intramuscularly (IM) in each hind leg and 1 mL IM in the shoulder muscle of each front leg. The second injection (week 3) consisted of 0.5 mL IM in each hind leg and 0.2 mL of antigen without adjuvant intravenously (IV). At weeks 6, 7, 8 and 9, 0.2 mL of antigen without adjuvant was given IV. Rabbits were bled at weeks 6, 7, 8 and 9. After two months rest, injections and bleeding were repeated beginning with the third week of the schedule. The procedures followed the guidelines of the "Guide to the Use and Care of Experimental Animals" of the Canadian Council on Animal Care. Polyclonal antibody was used to bind mycoplasma in the IBA and conjugated to fluorescein (8) to confirm species identification of mycoplasma by immunofluorescence (4). MOUSE MONOCLONAL MYCOPLASMA ANTIBODY PRODUCTION
The mouse monoclonal antibody to M. bovis (California 201) used in this study was prepared by John Boothby (10) and contained 10 mg of globulin/ mL. It was used to confirm species identification of mycoplasma by immunofluorescence (4) and to bind with mycoplasma in the IBA. CONJUGATES
The following conjugates were used in the IBA procedures: Biotin goat antirabbit IgG (H & L fragments) conjugate (Zymed Laboratories, San Francisco, California), Biotin F (ab)2 fragment, goat antimouse (H & L fragments) conjugate (Zymed Laboratories) and streptavidin peroxidase conjugate (Zymed Laboratories). BUFFERS
Initially, PBS was used in the IBA development trials. Subsequently, 252
Tris (Sigma Chemical Company, St. on a small area of the NCP under Louis, Missouri) buffered saline vacuum. (TBS), pH 7.5, was used as the BLOTTING preferred buffer system. Blotting was concentrated on the BLOCKING (WASHING) SOLUTION NCP by using the manifold and 15 to Blocking solution consisted of 18 mm of Hg vacuum to draw 500 ,uL 0.05% Tween 20 (Sigma Chemical of antigen-containing fluid through Company) with 1% gelatin, initially in the NCP prewet with buffer. In the PBS and later in TBS. final procedures adopted for blotting, 500 AL of milk was placed directly on NITROCELLULOSE FILTER PAPER the prewashed NCP (dot-blotting). Nitrocellulose paper (NCP) (Applied Scientific Company, Richmond, DEVELOPMENT OF IMMUNOBINDING California) pore size 0.45, prewashed ASSAY PROCEDURES FOR with TBS for 5 min, was used to bind MYCOPLASMA IN MODIFIED antigenic proteins to enable visualiza- HAYFLICK BROTH MEDIUM tion using peroxidase-labeled Procedures based on those of antibody. Kotani and McGarrity (5) were tested, using the manifold for blotting, and SUBSTRATE modified step-by-step until satisfacThe substrate or developing solu- tory blotting dilutions, incubation tion was prepared just before use by times, and other factors were estabcombining Solution A, consisting of 2 lished for use with mycoplasma in mL of ice cold methanol with 60 mg of modified Hayflick broth medium 4-chloronaphtol (Sigma Chemical (Table I, Method A). Factors investiCompany), with Solution B, consist- gated included: a) use of TBS versus ing of 100 mL TBS with 60 1AL of 30% PBS for all dilutions, blocking hydrogen peroxide. solutions, and washing steps; b) use of a gentle wash with distilled water after WASHING SOLUTION each incubation with the conjugates Washing solution consisted of TBS and before using the washing solution with 0.5% Tween 20 (Sigma Chemical versus no wash; c) use of whole-cell Company) prepared in advance and versus sonicated M. bovis antigen, refrigerated until use. prepared as for rabbit inoculation, with rabbit polyclonal and monocloMANIFOLD nal antibodies; d) optimization of The Manifold Model II (Schleicher antibody and antigen working diluand Schull, Keene, New Hampshire) tions by using polyclonal and monocwas used in initial experiments to lonal antibody in 12 doubling diluconcentrate antigen from test material tions in blocking solution from 1:500 TABLE I. Procedures for the IBA as performed using Methods A, B and C Procedure 01 Strip blotting 02 Strip blocking 03 Strip washing 04 Incubate with Abb 05 Strip washing 06 Incubate second Ab 07 Repeat step 5 08 Incubate peroxidase 09 Repeat step 5 10 Develop 11 Repeat step 5 Time required alncubation at 37°C in Method A. temperature bAb = antibody
Method A Method B Method C Dot method 5 jiL Dot method Dot method 120 mina 60 min 20 min 0 min 3 x 10 min 1 x 4 min 60 min 60 min 120 min 4 min 2 min 30 min 60 min 30 min 30 min 4 min 2 min 30 min 30 min 30 min 60 min 4 min 2 min 30 min 10 min 10 min 10 min 4 min 2 min 30 min 158 min 210 min 520 min All other procedures in Methods A, B and C were done at room
to 1:512,000, and whole-cell M. bovis antigen in TBS in seven tenfold serial dilutions from 1:10 to 1:106; e) immunological sensitivity by doing colony plate counts and running the IBA on seven tenfold serial dilutions of 11 different broth cultures of M. bovis in TBS using polyclonal and monoclonal antibodies.
e) Immunological specificity of IBA for mycoplasma species in milk was determined in artificially infected normal whole milk and using polyclonal and monoclonal antibodies, for four acholeplasma, seven M. bovis isolates and ten other mycoplasma species as was previously done in broth.
DETERMINATION OF IMMUNOLOGICAL SPECIFICITY FOR MYCOPLASMA IN BROTH
EVALUATION OF IBA PROCEDURES FOR DETECTION OF M. BOVIS IN NATURALLY INFECTED MILK
Immunological specificity of the IBA was determined by testing sevenfold broth dilutions of four acholeplasma strains, seven isolates of M. bovis and ten other isolates of mycoplasma species in duplicate against polyclonal antibodies at a dilution of 1:8000 and monoclonal antibodies at a dilution of 1:1000.
Frozen (-20° C) composite milk samples from 120 cows of unknown infection status in two herds with mycoplasma mastitis were obtained. Enriched samples were prepared by adding 200 ,uL of each thawed milk sample to 3 mL of mycoplasma broth and incubating for 48 h to increase numbers of M. bovis, if present, thereby increasing diagnostic sensitivity. All samples, before and after enrichment, were streaked in 0.01 mL amounts on modified Hayflick medium (3), incubated for seven days and examined for colony growth. Samples yielding mycoplasma colonies were considered positive. Species was determined on colonies by immunofluorescence (4). The IBA (Table I, Method A), was also performed on all samples, enriched or not. Because false-negative IBA results were obtained on two enriched samples of incubated milk, 200 ,uL from these enriched samples were reincubated in 3 mL of Hayflick broth medium (3) at 370C for an additional 24 h and then tested using the IBA.
DEVELOPMENT OF IBA PROCEDURES FOR DETECTION OF M. BOVIS IN ARTIFICIALLY INFECTED MILK
Commercially available pasteurized nonfat milk, homogenized 2% and 3.5% fat milk, and fresh nonpasteurized whole milk were used. Two mL of 48 h broth culture of M. bovis was added to 8 mL of each type of milk. From these, tenfold serial dilutions (1:10, 1:100, 1:1000) in TBS were prepared from each undiluted sample. These four preparations, constituting Set 1, were the basic samples used for testing blotting, dot-blotting and IBA procedures in a series of experiments (a-e). a) Undiluted milk and three dilutions (Set 1) were blotted in 500 ,uL amounts on NCP strips using the manifold under vacuum to observe efficacy of blotting. b) To reduce protein and fat concentration, 5 ,uL of each sample were transferred to 9.95 mL of TBS (Set 2). Strips of NCP were blotted, using the manifold, by transfer of 500 ,uL from each tube in Set 2. c) A dot-blot using 5 ,uL from each tube in Set 1 was placed directly on the NCP strip. Speed of disappearance of the sample from the surface of the NCP and final results from the IBA were noted. The experiment was repeated three times. d) Artificially infected milk (5 ,uL) from each tube in Set 1 was dotblotted on NCP and submitted to the IBA test, Method A, Table I, using polyclonal and monoclonal antibody preparations.
REDUCTION OF ASSAY TIME FOR THE IMMUNOBINDING ASSAY
Without changing the sequence of steps for the IBA, times allocated for the steps were reduced from a total of 8 h and 40 min, to 3 h and 30 min in one trial and to 2 h and 38 min in another trial (Table I, Methods A, B, C). The 120 composite samples of milk were retested using methods A, B, and C.
b)
c)
d)
e)
more uniform reactions and became standard procedure. Gentle washing of the blots with distilled water after each incubation with conjugate and before using washing solution improved clarity of results and became standard procedure. With modifications (a) and (b) above, the standard procedures for the IBA during other observations became Method A (Table I). Polyclonal antibodies reacted with both sonicated and whole cell M. bovis antigens. Monoclonal antibodies reacted only with whole-cell antigens. Polyclonal antibodies gave generally strong reactions at all antibody dilutions from 1:500 to 1:128,000 and at antigen dilutions of 1:10 to 1:100. Reactions were weak at antibody dilution 1:256,000 and negative at 1:512,000. A working antibody dilution of 1:8000 was chosen which provided a clear and strong positive reaction with very little background staining. Monoclonal antibodies gave strong reactions up to a dilution of 1:512,000. A working dilution of 1:1000 was chosen because it gave strong positive reactions which did not fade. Sensitivity of the IBA using polyclonal or monoclonal antibodies was the same, giving positive reactions to antigen concentrations in a range of 4.8 to 5 x 103 CFU/ mL.
DETERMINATION OF IMMUNOLOGICAL SPECIFICITY OF THE IBA FOR MYCOPLASMA IN BROTH
Polyclonal M. bovis antibody gave positive reactions with all the strains of M. bovis as well as with the acholeplasmas and the other mycoplasmas, except for the type strain of M. arginini (Table II). In tests with artificially infected whole milk, polyclonal M. bovis antibody reacted positively also with M. arginini (Table II). RESULTS Monoclonal antibody reacted specifDEVELOPMENT OF GENERAL ically with all strains of M. bovis. It did IMMUNOBINDING ASSAY not react with mycoplasmas other than PROCEDURES FOR MYCOPLASMA IN M. bovis or with acholeplasmas. MODIFIED HAYFLICK BROTH MEDIUM Consequently, monoclonal antibodies a) Use of TBS, pH 7.5, at all times exhibited high specific immunoreactivinstead of PBS, pH 7.4, provided ity without cross-reactions (Table II). 253
TABLE II. Results of the test for IBA specificity using M. bovis polyclonal and monoclonal antibodies on strips prepared with broth or with natural whole milk artifically infected with four Acholeplasma and 17 Mycoplasma species Reaction Polyclonal Monoclonal + +
Strip Microorganism 01 M. bovigenitalium 02 M. bovoculi + 03 unknown + + 04 M. bovis + 05 A. axanthum + 06 A. modicum + A. laidlawii 07 + M. bovirhinus 08 M. bovis + + 09 + A. granularum 10 + M. arginini Ila + + M. bovis 12 + + M. bovis 15 + 16 unknown + 17 M. bovigenitalium + 18 M. alkalescens + 19 M. californicum + 21 unknown + + 22 M. bovis + + 23 M. bovis + + 25 M. bovis aPolyclonal antiserum was negative for M. arginini in broth but was positive for M. arginini in milk
DEVELOPMENT OF IBA PROCEDURES FOR DETECTION OF M. BOVIS IN ARTIFICIALLY INFECTED MILK
a) Neither milk nor any of the dilutions (1:10, 1:100, 1:1000) passed satisfactorily through the NCP using the manifold procedures. b) Although some blotting occurred from all dilutions, the manifold procedure was deemed erratic and unsatisfactory. c) When 5 ,uL of the original milk or its dilutions (Set 1) were applied directly as dots to the NCP strips, blotting occurred quickly (within 10 min) on all samples. Direct dot-
TABLE III. Results using the dot-blotting procedure on strips prepared using different combinations of commercial milk and natural whole milk artifically infected with M. bovis and either polyclonal or monoclonal antibody
Strip preparation Nonfat milk 2% fat milk 3.5%fat milk Whole milk Negative control Positive control aWeak positive
254
blotting of 5 ,uL of milk was adopted as the routine blotting procedure for milk. d) The IBA using artificially infected normal whole milk or commercial homogenized milk of differing antigen and fat content gave identical results using the dot-blotting technique and either polyclonal or monoclonal antibody (Table III). e) Polyclonal M. bovis antibody reacted with all mycoplasma and acholeplasma species, including M. arginini, but monoclonal antibody reacted only with M. bovis antigen (Table II).
Antigen dilution + + + +
+ + + +
-
+
+
+
wa
When fresh milk from 120 cows was examined by using Method A (Table I), 43 samples were positive by culture and 22 by IBA. After enrichment for 48 h, 46 samples were positive by culture and 44 by IBA. After additional enrichment for 24 h, the same 46 samples were positive by each method (Table IV). The presence of gramnegative or gram-positive bacteria did not interfere with IBA results. REDUCTION OF TIME REQUIRED FOR IMMUNOBINDING ASSAY
Results from using three different assay times on 120 enriched composite milk samples were identical (Fig. 1). Method C required only 2 h and 38 min, in contrast to 8 h and 40 min required for Method A and 3 h and 30 min for Method B (Table I). Method C is therefore suggested for routine use of the IBA for the detection of mycoplasma in whole or diluted bovine milk.
DISCUSSION The IBA was shown during these experiments to be very effective in detecting M. bovis in artificially or naturally infected bovine milk. The test was highly specific to the species level when monoclonal antibodies were employed in the assay system. Polyclonal antibodies recognized common antigens and were not species-specific. Sensitivity was about 5 x 103 organisms, which appeared to be somewhat more sensitive than for the enzyme-linked immunosorbent
TABLE IV. Results of the IBA on 120 milk samples using monoclonal antibodies at a working dilution of 1:1000
Undiluted 1:10 1:100 1:1000 + + + +
EVALUATION OF IBA PROCEDURES FOR DETECTION OF M. BOVIS IN NATURALLY INFECTED MILK
Positive
% Specificity % Sensitivity compared to culture
Milk not preincubated Number positive IBA Culture 22 43 100 100
Milk preincubated 72 hours
Milk preincubated 48 hours Number positive IBA Culture
Number positive IBA Culture
44
46
46
46
100
100
100
100
51.16
95.65
100
82.50
98.33
100
% Agreement with culture
REFERENCES
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Fig. 1. Sample results from immunobinding assay for M. bovis used on 120 enriched composite milk samples using Methods A, B and C (top to bottom). Four samples were negative and six were positive, although two samples were near test sensitivity limits.
assay (10). Method C (Table I) appeared to be the method of choice because it required less time to complete. Detection of low numbers of M. bovis in milk could be enhanced by incubation of the milk for 48 to 72 h (Table IV), after which results were comparable to detection by culture. Time from sample receipt to diagnosis in most positive samples could be reduced from several days by culture to a few hours by IBA. However, negative samples would need to be enriched by incubation and
retested before a negative result was reported. Use of the IBA, even with enrichment, would require less total test time than that required for culture methods. In the meantime, mycoplasma infection in a herd could be confirmed, species determined and many of the infected cows identified within a few hours thereby enabling control procedures to be quickly initiated. The test should be useful for herd tests in which milk of individual cows is tested and in survey tests of bulk tank milk.
1. GOURLAY RN, HOWARD CJ. Recovery and identification of bovine mycoplasmas. In: Tully CJ, Razin S, eds. Methods in Mycoplasmology. Vol 2. New York: Academic Press, 1983: 81-89. 2. FABRICANT J, BARBER TL. The laboratory diagnosis of mycoplasma infection. Proc US Anim Health Assoc 73rd Meet 1969: 573-581. 3. JASPER DE. Bovine mycoplasmal mastitis. Adv Vet Sci Comp Med 1981; 25: 121159. 4. BASS EJ, JASPER DE. Agar block technique for identification of mycoplasma by use of fluorescent antibody. Appl Microbiol 1972; 23: 1097-1100. 5. KOTANI H, McGARRITY GJ. Rapid and simple identification of mycoplasmas. J Immunol Methods 1985; 85: 257-267. 6. KOTANI H, McGARRITY GJ. Identification of mycoplasma colonies by immunobinding. J Clin Microbiol 1986; 23: 783-785. 7. KOTANI H, HUANG H, McGARRITY GJ. Identification and isolation of mycoplasmas by immunobinding. Isr J Med Sci 1987; 23: 752-758. 8. DEDMON RE, HOLMES AW, DE1HARDT F. Proportion of fluorescein isothiocyanate labeled gamma globulin by dialysis, gel filtration, and ion chromatography in combination. J Bacteriol 1965; 89: 734-739. 9. BRADFORD HH. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye finding. Biochemistry 1976; 72: 143-151. 10. BOOTHBY JT, MUELLER R, JASPER DE. Detecting Mycoplasma bovis in milk by enzyme-linked immunosorbent assay using monoclonal antibodies. Am J Vet Res 1986; 47: 1082-1084.
ABSTRACT
INTRODUCTION
An immunobinding dot-blot assay (IBA) was developed for the detection of mycoplasma in milk. The test was highly species specific when monoclonal antibody preparations were employed in the assay system. Reactions were obtained with all mycoplasma species tested when polyclonal antisera preparations were used. Preincubation for 48-72 hours was necessary with milk samples containing only a few mycoplasma. Time from sample receipt to diagnosis in most positive samples could be reduced from several days by culture to a few hours by the IBA, thus enabling control procedures to be quickly initiated.
Diagnosis of mycoplasma infection in milk has depended primarily upon microbiological culture of udder secretions (1-3). Although the reliability of this method is reasonably satisfactory, it is time-consuming. Inoculated plates must be incubated at least two to three days before colonies may be observed and should not be considered negative prior to evaluation after seven days of incubation (1-3). Determination of species has required further study which may be biochemical or immunological (3). The most satisfactory method of routine determination of species has been by immunofluorescence (4). The combined microbiological and fluorescent antibody methods for detection and identification of mycoplasma species are sensitive and very specific; however they are slow and expensive in time and equipment. Immunobinding procedures are sensitive, rapid, very specific and do not require expensive equipment. Such an assay has been described for mycoplasma colonies and for mycoplasma from broth or tissue cultures (5-7), but has not been described for use in materials of high fat content such as milk. The purpose of this research was to develop an immunobinding assay (IBA) for detection of Mycoplasma bovis in naturally infected milk and to reduce the time required to do the IBA.
RESUME Une epreuve immunologique afin de detecter la presence de mycoplasme 'a partir du lait a ete developpee. Lorsque des anticorps monoclonaux ont ete utilises, cette technique a demontre une tres grande specificite car des liaisons ont ete obtenues avec toutes les souches de mycoplasme ayant reagi avec des anticorps polyclonaux. Une periode d'incubation de 48 'a 72 h etait cependant necessaire si le lait ne contenait que quelques colonies bacteriennes. Cette technique immunologique de detection a l'avantage de pouvoir reduire de quelques jours a quelques heures le delai d'attente avant de poser un diagnostique positif de linfection.
MATERIALS AND METHODS MYCOPLASMA SPECIES
Mycoplasma and acholeplasma species used in this research were obtained from bovine milk or were American Type Culture Collection (ATCC) strains. Species identity of all cultures was confirmed by immunofluorescence (4). ANTIGEN PRODUCTION FOR THE IMMUNOBINDING ASSAY
Mycoplasmas used for antigen in the immunobinding assays were grown separately in modified Hayflick broth medium containing 15% horse serum (2). After 72 h at 370 C, cultures were washed three times with phosphate buffered saline, pH 7.4 (PBS), adjusted to 1 mg protein per mL in PBS as determined by the method of Bradford (9), and stored at -20° C. This preparation was used as an antigen in modified Hayflick broth or mixed with pasteurized commercial milk containing no fat, 2% or 3.5% homogenized fat, or with nonpasteurized, nonhomogenized natural milk. ANTIGEN PRODUCTION FOR RABBIT IMMUNIZATION
Mycoplasmas (M. bovis, California 201) for antigen were grown for 48 h at 370C in 500 mL of medium, centrifuged at 27,000 g for 30 min and washed three times with PBS, the volume being finally adjusted to 6 mL and stored at -20°. Before use, antigen was thawed, sonicated (model 375, Heat Ultrasonic Inc., Plainview, New
Department of Clinical Pathology (Infante, Jasper, Cullor, Dellinger) and Department of Microbiology and Immunology (Stott), School of Veterinary Medicine, University of California, Davis, California 95616. Reprint requests to Dr. D.E. Jasper. From a thesis submitted by the senior author in partial fulfillment of requirements for the Ph.D. degree. Supported in part by grants from the California Milk Producers Advisory Board and the USDA Endemic Diseases Fund. Submitted July 13, 1989.
Can J Vet Res 1990; 54: 251-255
251
York) twice for 1 min at 20 s intervals and diluted 1:200 in PBS. Two mL of antigen were mixed with 2 mL of Freund's complete adjuvant for use in immunizing rabbits. RABBIT POLYCLONAL MYCOPLASMA ANTIBODY PRODUCTION
Antigen injections for polyclonal antibody were made in rabbits over a nine week period. On day 1, 0.2 mL of antigen with adjuvant was given intradermally (ID) in each hind foot pad, 0.6 mL ID at three to four sites along the back, 0.6 mL intramuscularly (IM) in each hind leg and 1 mL IM in the shoulder muscle of each front leg. The second injection (week 3) consisted of 0.5 mL IM in each hind leg and 0.2 mL of antigen without adjuvant intravenously (IV). At weeks 6, 7, 8 and 9, 0.2 mL of antigen without adjuvant was given IV. Rabbits were bled at weeks 6, 7, 8 and 9. After two months rest, injections and bleeding were repeated beginning with the third week of the schedule. The procedures followed the guidelines of the "Guide to the Use and Care of Experimental Animals" of the Canadian Council on Animal Care. Polyclonal antibody was used to bind mycoplasma in the IBA and conjugated to fluorescein (8) to confirm species identification of mycoplasma by immunofluorescence (4). MOUSE MONOCLONAL MYCOPLASMA ANTIBODY PRODUCTION
The mouse monoclonal antibody to M. bovis (California 201) used in this study was prepared by John Boothby (10) and contained 10 mg of globulin/ mL. It was used to confirm species identification of mycoplasma by immunofluorescence (4) and to bind with mycoplasma in the IBA. CONJUGATES
The following conjugates were used in the IBA procedures: Biotin goat antirabbit IgG (H & L fragments) conjugate (Zymed Laboratories, San Francisco, California), Biotin F (ab)2 fragment, goat antimouse (H & L fragments) conjugate (Zymed Laboratories) and streptavidin peroxidase conjugate (Zymed Laboratories). BUFFERS
Initially, PBS was used in the IBA development trials. Subsequently, 252
Tris (Sigma Chemical Company, St. on a small area of the NCP under Louis, Missouri) buffered saline vacuum. (TBS), pH 7.5, was used as the BLOTTING preferred buffer system. Blotting was concentrated on the BLOCKING (WASHING) SOLUTION NCP by using the manifold and 15 to Blocking solution consisted of 18 mm of Hg vacuum to draw 500 ,uL 0.05% Tween 20 (Sigma Chemical of antigen-containing fluid through Company) with 1% gelatin, initially in the NCP prewet with buffer. In the PBS and later in TBS. final procedures adopted for blotting, 500 AL of milk was placed directly on NITROCELLULOSE FILTER PAPER the prewashed NCP (dot-blotting). Nitrocellulose paper (NCP) (Applied Scientific Company, Richmond, DEVELOPMENT OF IMMUNOBINDING California) pore size 0.45, prewashed ASSAY PROCEDURES FOR with TBS for 5 min, was used to bind MYCOPLASMA IN MODIFIED antigenic proteins to enable visualiza- HAYFLICK BROTH MEDIUM tion using peroxidase-labeled Procedures based on those of antibody. Kotani and McGarrity (5) were tested, using the manifold for blotting, and SUBSTRATE modified step-by-step until satisfacThe substrate or developing solu- tory blotting dilutions, incubation tion was prepared just before use by times, and other factors were estabcombining Solution A, consisting of 2 lished for use with mycoplasma in mL of ice cold methanol with 60 mg of modified Hayflick broth medium 4-chloronaphtol (Sigma Chemical (Table I, Method A). Factors investiCompany), with Solution B, consist- gated included: a) use of TBS versus ing of 100 mL TBS with 60 1AL of 30% PBS for all dilutions, blocking hydrogen peroxide. solutions, and washing steps; b) use of a gentle wash with distilled water after WASHING SOLUTION each incubation with the conjugates Washing solution consisted of TBS and before using the washing solution with 0.5% Tween 20 (Sigma Chemical versus no wash; c) use of whole-cell Company) prepared in advance and versus sonicated M. bovis antigen, refrigerated until use. prepared as for rabbit inoculation, with rabbit polyclonal and monocloMANIFOLD nal antibodies; d) optimization of The Manifold Model II (Schleicher antibody and antigen working diluand Schull, Keene, New Hampshire) tions by using polyclonal and monocwas used in initial experiments to lonal antibody in 12 doubling diluconcentrate antigen from test material tions in blocking solution from 1:500 TABLE I. Procedures for the IBA as performed using Methods A, B and C Procedure 01 Strip blotting 02 Strip blocking 03 Strip washing 04 Incubate with Abb 05 Strip washing 06 Incubate second Ab 07 Repeat step 5 08 Incubate peroxidase 09 Repeat step 5 10 Develop 11 Repeat step 5 Time required alncubation at 37°C in Method A. temperature bAb = antibody
Method A Method B Method C Dot method 5 jiL Dot method Dot method 120 mina 60 min 20 min 0 min 3 x 10 min 1 x 4 min 60 min 60 min 120 min 4 min 2 min 30 min 60 min 30 min 30 min 4 min 2 min 30 min 30 min 30 min 60 min 4 min 2 min 30 min 10 min 10 min 10 min 4 min 2 min 30 min 158 min 210 min 520 min All other procedures in Methods A, B and C were done at room
to 1:512,000, and whole-cell M. bovis antigen in TBS in seven tenfold serial dilutions from 1:10 to 1:106; e) immunological sensitivity by doing colony plate counts and running the IBA on seven tenfold serial dilutions of 11 different broth cultures of M. bovis in TBS using polyclonal and monoclonal antibodies.
e) Immunological specificity of IBA for mycoplasma species in milk was determined in artificially infected normal whole milk and using polyclonal and monoclonal antibodies, for four acholeplasma, seven M. bovis isolates and ten other mycoplasma species as was previously done in broth.
DETERMINATION OF IMMUNOLOGICAL SPECIFICITY FOR MYCOPLASMA IN BROTH
EVALUATION OF IBA PROCEDURES FOR DETECTION OF M. BOVIS IN NATURALLY INFECTED MILK
Immunological specificity of the IBA was determined by testing sevenfold broth dilutions of four acholeplasma strains, seven isolates of M. bovis and ten other isolates of mycoplasma species in duplicate against polyclonal antibodies at a dilution of 1:8000 and monoclonal antibodies at a dilution of 1:1000.
Frozen (-20° C) composite milk samples from 120 cows of unknown infection status in two herds with mycoplasma mastitis were obtained. Enriched samples were prepared by adding 200 ,uL of each thawed milk sample to 3 mL of mycoplasma broth and incubating for 48 h to increase numbers of M. bovis, if present, thereby increasing diagnostic sensitivity. All samples, before and after enrichment, were streaked in 0.01 mL amounts on modified Hayflick medium (3), incubated for seven days and examined for colony growth. Samples yielding mycoplasma colonies were considered positive. Species was determined on colonies by immunofluorescence (4). The IBA (Table I, Method A), was also performed on all samples, enriched or not. Because false-negative IBA results were obtained on two enriched samples of incubated milk, 200 ,uL from these enriched samples were reincubated in 3 mL of Hayflick broth medium (3) at 370C for an additional 24 h and then tested using the IBA.
DEVELOPMENT OF IBA PROCEDURES FOR DETECTION OF M. BOVIS IN ARTIFICIALLY INFECTED MILK
Commercially available pasteurized nonfat milk, homogenized 2% and 3.5% fat milk, and fresh nonpasteurized whole milk were used. Two mL of 48 h broth culture of M. bovis was added to 8 mL of each type of milk. From these, tenfold serial dilutions (1:10, 1:100, 1:1000) in TBS were prepared from each undiluted sample. These four preparations, constituting Set 1, were the basic samples used for testing blotting, dot-blotting and IBA procedures in a series of experiments (a-e). a) Undiluted milk and three dilutions (Set 1) were blotted in 500 ,uL amounts on NCP strips using the manifold under vacuum to observe efficacy of blotting. b) To reduce protein and fat concentration, 5 ,uL of each sample were transferred to 9.95 mL of TBS (Set 2). Strips of NCP were blotted, using the manifold, by transfer of 500 ,uL from each tube in Set 2. c) A dot-blot using 5 ,uL from each tube in Set 1 was placed directly on the NCP strip. Speed of disappearance of the sample from the surface of the NCP and final results from the IBA were noted. The experiment was repeated three times. d) Artificially infected milk (5 ,uL) from each tube in Set 1 was dotblotted on NCP and submitted to the IBA test, Method A, Table I, using polyclonal and monoclonal antibody preparations.
REDUCTION OF ASSAY TIME FOR THE IMMUNOBINDING ASSAY
Without changing the sequence of steps for the IBA, times allocated for the steps were reduced from a total of 8 h and 40 min, to 3 h and 30 min in one trial and to 2 h and 38 min in another trial (Table I, Methods A, B, C). The 120 composite samples of milk were retested using methods A, B, and C.
b)
c)
d)
e)
more uniform reactions and became standard procedure. Gentle washing of the blots with distilled water after each incubation with conjugate and before using washing solution improved clarity of results and became standard procedure. With modifications (a) and (b) above, the standard procedures for the IBA during other observations became Method A (Table I). Polyclonal antibodies reacted with both sonicated and whole cell M. bovis antigens. Monoclonal antibodies reacted only with whole-cell antigens. Polyclonal antibodies gave generally strong reactions at all antibody dilutions from 1:500 to 1:128,000 and at antigen dilutions of 1:10 to 1:100. Reactions were weak at antibody dilution 1:256,000 and negative at 1:512,000. A working antibody dilution of 1:8000 was chosen which provided a clear and strong positive reaction with very little background staining. Monoclonal antibodies gave strong reactions up to a dilution of 1:512,000. A working dilution of 1:1000 was chosen because it gave strong positive reactions which did not fade. Sensitivity of the IBA using polyclonal or monoclonal antibodies was the same, giving positive reactions to antigen concentrations in a range of 4.8 to 5 x 103 CFU/ mL.
DETERMINATION OF IMMUNOLOGICAL SPECIFICITY OF THE IBA FOR MYCOPLASMA IN BROTH
Polyclonal M. bovis antibody gave positive reactions with all the strains of M. bovis as well as with the acholeplasmas and the other mycoplasmas, except for the type strain of M. arginini (Table II). In tests with artificially infected whole milk, polyclonal M. bovis antibody reacted positively also with M. arginini (Table II). RESULTS Monoclonal antibody reacted specifDEVELOPMENT OF GENERAL ically with all strains of M. bovis. It did IMMUNOBINDING ASSAY not react with mycoplasmas other than PROCEDURES FOR MYCOPLASMA IN M. bovis or with acholeplasmas. MODIFIED HAYFLICK BROTH MEDIUM Consequently, monoclonal antibodies a) Use of TBS, pH 7.5, at all times exhibited high specific immunoreactivinstead of PBS, pH 7.4, provided ity without cross-reactions (Table II). 253
TABLE II. Results of the test for IBA specificity using M. bovis polyclonal and monoclonal antibodies on strips prepared with broth or with natural whole milk artifically infected with four Acholeplasma and 17 Mycoplasma species Reaction Polyclonal Monoclonal + +
Strip Microorganism 01 M. bovigenitalium 02 M. bovoculi + 03 unknown + + 04 M. bovis + 05 A. axanthum + 06 A. modicum + A. laidlawii 07 + M. bovirhinus 08 M. bovis + + 09 + A. granularum 10 + M. arginini Ila + + M. bovis 12 + + M. bovis 15 + 16 unknown + 17 M. bovigenitalium + 18 M. alkalescens + 19 M. californicum + 21 unknown + + 22 M. bovis + + 23 M. bovis + + 25 M. bovis aPolyclonal antiserum was negative for M. arginini in broth but was positive for M. arginini in milk
DEVELOPMENT OF IBA PROCEDURES FOR DETECTION OF M. BOVIS IN ARTIFICIALLY INFECTED MILK
a) Neither milk nor any of the dilutions (1:10, 1:100, 1:1000) passed satisfactorily through the NCP using the manifold procedures. b) Although some blotting occurred from all dilutions, the manifold procedure was deemed erratic and unsatisfactory. c) When 5 ,uL of the original milk or its dilutions (Set 1) were applied directly as dots to the NCP strips, blotting occurred quickly (within 10 min) on all samples. Direct dot-
TABLE III. Results using the dot-blotting procedure on strips prepared using different combinations of commercial milk and natural whole milk artifically infected with M. bovis and either polyclonal or monoclonal antibody
Strip preparation Nonfat milk 2% fat milk 3.5%fat milk Whole milk Negative control Positive control aWeak positive
254
blotting of 5 ,uL of milk was adopted as the routine blotting procedure for milk. d) The IBA using artificially infected normal whole milk or commercial homogenized milk of differing antigen and fat content gave identical results using the dot-blotting technique and either polyclonal or monoclonal antibody (Table III). e) Polyclonal M. bovis antibody reacted with all mycoplasma and acholeplasma species, including M. arginini, but monoclonal antibody reacted only with M. bovis antigen (Table II).
Antigen dilution + + + +
+ + + +
-
+
+
+
wa
When fresh milk from 120 cows was examined by using Method A (Table I), 43 samples were positive by culture and 22 by IBA. After enrichment for 48 h, 46 samples were positive by culture and 44 by IBA. After additional enrichment for 24 h, the same 46 samples were positive by each method (Table IV). The presence of gramnegative or gram-positive bacteria did not interfere with IBA results. REDUCTION OF TIME REQUIRED FOR IMMUNOBINDING ASSAY
Results from using three different assay times on 120 enriched composite milk samples were identical (Fig. 1). Method C required only 2 h and 38 min, in contrast to 8 h and 40 min required for Method A and 3 h and 30 min for Method B (Table I). Method C is therefore suggested for routine use of the IBA for the detection of mycoplasma in whole or diluted bovine milk.
DISCUSSION The IBA was shown during these experiments to be very effective in detecting M. bovis in artificially or naturally infected bovine milk. The test was highly specific to the species level when monoclonal antibodies were employed in the assay system. Polyclonal antibodies recognized common antigens and were not species-specific. Sensitivity was about 5 x 103 organisms, which appeared to be somewhat more sensitive than for the enzyme-linked immunosorbent
TABLE IV. Results of the IBA on 120 milk samples using monoclonal antibodies at a working dilution of 1:1000
Undiluted 1:10 1:100 1:1000 + + + +
EVALUATION OF IBA PROCEDURES FOR DETECTION OF M. BOVIS IN NATURALLY INFECTED MILK
Positive
% Specificity % Sensitivity compared to culture
Milk not preincubated Number positive IBA Culture 22 43 100 100
Milk preincubated 72 hours
Milk preincubated 48 hours Number positive IBA Culture
Number positive IBA Culture
44
46
46
46
100
100
100
100
51.16
95.65
100
82.50
98.33
100
% Agreement with culture
REFERENCES
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Fig. 1. Sample results from immunobinding assay for M. bovis used on 120 enriched composite milk samples using Methods A, B and C (top to bottom). Four samples were negative and six were positive, although two samples were near test sensitivity limits.
assay (10). Method C (Table I) appeared to be the method of choice because it required less time to complete. Detection of low numbers of M. bovis in milk could be enhanced by incubation of the milk for 48 to 72 h (Table IV), after which results were comparable to detection by culture. Time from sample receipt to diagnosis in most positive samples could be reduced from several days by culture to a few hours by IBA. However, negative samples would need to be enriched by incubation and
retested before a negative result was reported. Use of the IBA, even with enrichment, would require less total test time than that required for culture methods. In the meantime, mycoplasma infection in a herd could be confirmed, species determined and many of the infected cows identified within a few hours thereby enabling control procedures to be quickly initiated. The test should be useful for herd tests in which milk of individual cows is tested and in survey tests of bulk tank milk.
1. GOURLAY RN, HOWARD CJ. Recovery and identification of bovine mycoplasmas. In: Tully CJ, Razin S, eds. Methods in Mycoplasmology. Vol 2. New York: Academic Press, 1983: 81-89. 2. FABRICANT J, BARBER TL. The laboratory diagnosis of mycoplasma infection. Proc US Anim Health Assoc 73rd Meet 1969: 573-581. 3. JASPER DE. Bovine mycoplasmal mastitis. Adv Vet Sci Comp Med 1981; 25: 121159. 4. BASS EJ, JASPER DE. Agar block technique for identification of mycoplasma by use of fluorescent antibody. Appl Microbiol 1972; 23: 1097-1100. 5. KOTANI H, McGARRITY GJ. Rapid and simple identification of mycoplasmas. J Immunol Methods 1985; 85: 257-267. 6. KOTANI H, McGARRITY GJ. Identification of mycoplasma colonies by immunobinding. J Clin Microbiol 1986; 23: 783-785. 7. KOTANI H, HUANG H, McGARRITY GJ. Identification and isolation of mycoplasmas by immunobinding. Isr J Med Sci 1987; 23: 752-758. 8. DEDMON RE, HOLMES AW, DE1HARDT F. Proportion of fluorescein isothiocyanate labeled gamma globulin by dialysis, gel filtration, and ion chromatography in combination. J Bacteriol 1965; 89: 734-739. 9. BRADFORD HH. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye finding. Biochemistry 1976; 72: 143-151. 10. BOOTHBY JT, MUELLER R, JASPER DE. Detecting Mycoplasma bovis in milk by enzyme-linked immunosorbent assay using monoclonal antibodies. Am J Vet Res 1986; 47: 1082-1084.
