The Indirect Hemagglutination Test for the Detection of Antibodies in Cattle Naturally Infected with Mycoplasmas H. J. Cho, H. L. Ruhnke and E. V. Langford*

ABSTRACT

Stable mycoplasma antigens for the indirect hemagglutination test (IHA) were prepared employing glutaraldehyde treated sheep erythrocytes sensitized with Mycoplasma agalactiae subsp. bovis and Mycoplasma bovigenitalium antigens. Employing these antigens mycoplasma antibodies were detected in sera from cattle which had mastitic symptoms due to natural infection with either M. agalactiae subsp. bovis or M. bovigenitalium. A total of 200 cows from four herds were examined at varying intervals for the presence of M. agalactiae subsp. bovis and for the detection of antibody using growth inhibition and IHA tests. Mycoplasmas were isolated from 37 animals. Growth inhibiting antibody was detected from 56 of the 200 animals. In the IHA tests, antibody titers _ 1:80 were detected in 148 animals, 76 of these having antibody titers _ 1:160, while sera of 116 normal control animals had no growth inhibiting antibody and none had IHA antibody titers greater than 1:40. M. bovigenitalium was isolated from the milk of three of 26 animals in a fifth herd during an outbreak of mastitis. Growth inhibiting antibodies were demonstrated in the sera

of ten of the 26 animals. However, the IHA test detected antibody titers of >1:160 in It animals and of 1:80 in one of the 26 animals. To determine the specificity of the IHA tests, M. agalactiae subsp. bovis and M. bovigenitalium antigens were reacted with rabbit hyperimmune typing sera produced against 12species of bovine mycoplasmatales. Homolo-gous antisera showed IHA antibody titers of 1:1280 and 1:2560 against M. agalactiae subsp.. bovis and M. bovigenitalium respectively, whereas heterologous antisera showed IHA, antibody titers of _ 1:20. Also eight typespecific bovine antisera were reacted with M. agalactiae subsp. bovis and M. bovigenitalium antigens in homologous and heterologous tests. Homologous reactions showed IHA antibody titers .1:320, whereas heterologous reactions. showed IHA titers of < 1:20. This IHA test promises to be useful for the detection of bovine mycoplasma antibodies in. sera from cattle infected with M. agalactiaesubsp. bovis or M. bovigenitalium. The test issensitive, reproducible and specific and thetechnique is relatively simple and rapid. The antigens were stable for at least seven months.

RASUMe *Animal Pathology Division, Health of Animals Branch, Agriculture Canada, Animal Diseases Research Institute (Western), P.O. Box 640, Lethbridge, Alberta T1J 3Z4 (Cho and Langford) and Veterinary Services Laboratory, Veterinary Services Branch, Ontario Ministry of Agriculture and Food, Box 3612, Guelph, Ontario NiH 6R8 (Ruhnke). Submitted May 21, 1975.

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Les auteurs ont prepare des antigenes sta-bles pour l'epreuve indirecte d'hemagglutination, en utilisant des hematies de mouton traitees a la glutaraldehyde et sensibiliseesaux antigenes Mycoplasma agalactiae var.bovis et Mycoplasma bovigenitalium. L'emploi

Can. J. comp. Me&

de ces antigenes leur permit de deceler des anticorps dans le serum de vaches manifestant des signes de mammite attribuable 'a une infection naturelle par M. agalactiae var. bovis ou M. bovigenitalium. Utilisant 1'etpreuve indirecte d'hemagglutination et celle de l'inhibition de la croissance, ils examinerent, ii differents intervalles, des echantillons preleves sur un total de 200 vaches faisant partie de quatre troupeaux. Ces examens visaient ia diceler la presence de M. agalactiae var. bovis ou des anticorps correspondants. Ils recouvrirent le mycoplasme chez 37 de ces vaches et decelerent des anticorps inhibant la croissance, chez 56 d'entre elles. L'epreuve indirecte d'hemagglutination leur permit de deceler des titres d'anticorps . 1:80 chez 148 vaches dont 76 possedaient un titre . 1:160; par ailleurs, le serum de 116 vaches temoins s'avera depourvu d'anticorps inhibant la croissance et, d'apres l'epreuve indirecte d'hemagglutination, aucun de ces serums ne possedait une concentration d'anticorps superieure 'a 1:40. Ils isolerent M. bovigenitalium du lait de trois des 26 vaches d'un cinquieme troupeau, au cours d'une eruption de mammite. Ils decel'erent des anticorps inhibant la croissance dans le serum de dix de ces 26 vaches. L'epreuve indirecte d'hemagglutination revela cependant une teneur du serum en anticorps . 1:160 chez 13 vaches et de 1:80, chez une autre. Afin de determiner la specificite' de l'epreuye indirecte d'hemagglutination, ils firent reagir les antigenes M. agalactiae var. bovis et M. bovigenitalium avec du serum hyperimmun de lapin, prepare avec 12 especes de mycoplasmes bovins. Les antiserums homologues donnerent, avec l'epreuve indirecte d'hemagglutination, des titres respectifs de 1:1280 et 1:2560 'a l'endroit de M. agalactiae var. bovis et M. bovigenitalium, tandis que les antiserums heterologues revelerent des titres d'anticorps seulement . 1:20. Ils firent aussi reagir huit antiserums bovins specifiques avec M. agalactiae var. bovis et M. bovigenitalium, dans des epreuves indirectes d'hemagglutination homologues et heterologues. Les resultats des premieres revelerent des concentrations d'anticorps ' 1:320, tandis que ceux des secondes ne donnerent que des titres . 1:20. L'epreuve indirecte d'hemagglutination promet de s'averer utile pour deceler des anticorps dans le serum de bovins infectes avec M. agalactiae var. bovis ou M. bovigenitalium. Cette epreuve est sensible, reproductible et specifique; sa technique est aussi relativement simDle et rapide.

Volume 40

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January, 1976

INTRODUCTION

Reliable serological tests for detecting antibodies in serum from cattle naturally exposed to bovine mycoplasmatales are not well established with the exception of Mycoplasma mycoides subsp. mycoides infection in which the complement-fixation test is the most reliable method (8). Employing hyperimmune rabbit sera against bovine mycoplasmatales, antibodies have been detected by means of metabolic inhibition (7), immunodiffusion, growth precipitation and growth inhibition tests (6) and by counterimmunoelectrophoresis (2). However, these techniques have some limitation for detecting and quantifying bovine mycoplasma antibodies since they are either time consuming, laborious to perform or qualitative rather than quantitative. The indirect hemagglutination (IHA) test has been shown to be a sensitive serological test for the detection of antibodies against various mycoplasma species in man and animals (10, 11,12, 15). In the present investigation, a procedure was developed for preparing stable antigens by sensitizing glutaraldehyde-fixed sheep erythrocytes with the two mycoplasma antigens. The sensitized cells were used in the IHA test for the detection of mycoplasma antibodies in cows affected with mastitis caused by either M. agalactiae subsp. bovis or M. bovigenitalium. IHA test results were compared with growth inhibition and mycoplasma isolation and the specificity of the IHA test was studied.

MATERIALS AND METHODS TYPE STRAINS M. agalactiae subsp. bovis Donetta strain and M. bovigenitalium PG11 obtained from the National Collection of Type Cultures, Great Britain', were used. 'L. R. Hill, NCTC, Central Public Health Laboratory, Colindale Avenue, Colindale, London, England NW9 5HT.

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iMEDIA Horse serum broth (HSB) of the type described by Hayflick (9) and consisting of Difco PPLO broth supplement with a boiled yeast extract (10% V/V of a 25% aqueous extract) and 20% unheated horse serum as described by Langford and Leach (14) was modified by the addition of 0.024 gm of DNA calf thymus origin2 per 100 ml of medium. Horse serum agar (HSA) was prepared in a similar manner substituting Difco PPLO agar for Difco PPLO broth3. PREPARATION OF ANTIGEN

Organisms were grown in 1,000 ml of HSB for two to three days and concentrated by centrifugation at 14,000xg for one hour at 5°C. The pellet was washed twice with 0.85% saline at the same speed and resuspended in 0.85% saline at a protein concentration of 10 mg/ml determined by a standard biuret method. Sodium azide at a final concentration of 0.01% was added as a preservative and the antigen was stored at 5°C until required. BOVINE SERA AND MILK SAMPLES

Sera and milk samples were collected at intervals as recorded in Table I. Cows in four herds (200 cows of herds A, B, C in Ontario and D in Alberta) had a mastitis from which M. agalactiae subsp. bovis was isolated. From herd A, 51 animals, 163 blood samples and 145 milk samples were collected at five different time intervals. From herds B, C and D, a total of 149 animals, 183 blood samples and 181 milk samples were collected at two different times. Samples of milk and blood were collected on the same day from these four herds. Herd E (26 cows, Alberta) had a mastitis from which M. bovigenitalium was isolated from three cows of this herd. Starting two weeks after the collection of milk samples, a total of 41 blood samples were collected on three visits at intervals of about three months. By the owner's observations, animals 1 to 14 showed clinical symptoms of mastitis and animals 15 to 2Sigma Products, St. Louis, MissourL 3Difco Laboratories, Detroit, Michigan.

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26 were apparently normal. Normal sera were collected from 116 cattle in the herd of the Animal Diseases Research Institute (Western) at Lethbridge and served as control sera. HYPERIMMUNE RABBIT TYPING SERA AGAINST BOVINE MYCOPLASMATALES

Rabbit hyperimmune sera against 12 reference strains of bovine mycoplasmatales were prepared essentially as described elsewhere (2). The mycoplasmatales organisms were incorporated in incomplete Freund adjuvant and inoculated into foot pads and subcutaneously. ISOLATION OF MYCOPLASMAS At Lethbridge, mycoplasmas were isolated by inoculating 0.2 ml of milk into HSB with inhibitors (13). After incubation at 37°C for three days the cultures were transferred to HSA and incubated an additional one to three days at 37°C. At Guelph, quarter milk samples were plated directly on solid medium as described by Davies (4). Cloning, if necessary, was by filtration as described in a report of the Subcommittee on Mycoplasmatales (18).

IDENTIFICATION OF MYCOPLASMA This was by growth precipitation (6), growth inhibition based on Clyde's method (3) or modified as follows: wells 5 mm in diameter, 10 mm apart were cut in agar approximately 4-5 mm deep which had been previously inoculated with thte test organism. Test serum in an amount of 0.05 ml was placed in a well and the zone and type of inhibition was read 48 and 72 hours later. Direct immunofluorescence using incident light as described by Del Giudice (5) was also used. PREPARATION OF GLUTARALDEHYDE-FIxED SHEEP ERYTHROCYTES The glutaraldehyde-fixed sheep erythrocytes were prepared as described by Bing et al (1) with the following modification by Dr. R. Weinbach (personal communication, Suny Downstate Medical Center, Brooklyn, N.Y.). Sheep erythrocytes, col-

Can. J. comp. Med.

lected in Alsever's solution, were washed four times with phosphate buffered glucose (PBG), pH 7.2 (76 ml of 0.15M disodium phosphate, 24 ml of 0.15M monopotassium phosphate, 100 ml of 5.4% glucose in distilled water). An equal volume of 0.2% glutaraldehyde4 dissolved in PBG was mixed with 20%1 :160, 40 sera reacted at 1 :80 and 64 sera had titers .1:40. On an individual animal basis during the 445 day period of examination M. agalactiae subsp. bovis was isolated on one or more occasions from 14 of 51 animals, and growth inhibiting antibodies were detected in sera from 16 of the animals. With the IHA tests, antibody titers . 1:160 were detected from 23 of 51 animals on one or more occasions and 18 animals showed their highest IHA antibody titers of 1:80 on one or more occasions. Ten animals had IHA antibody titers 0.5 mm < 1.0 mm (suspicious) -Reciprocal of highest dilution causing complete agglutination of treated cells fBlanks: samples were not collected cNumber of animals tested for isolation, growth inhibition and indirect hemagglutination -

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TABLE III. Summary and Correlation Among Isolation, Growth Inhibition and Indirect Hemagglutination Test in Bovine Mastitis Caused by M. agalactiae subsp. boris

Total Animals Tested A .51 B .26 C .48 D .75

Herd

TOTAL.200 Correlation with: Isolation of M. agalactiae subsp. bovis. Growth Inhibition...

IHA

Isolation of M. agalactiae subsp. bovis Isol.. 14 7 9 7 37

Isol N P

37 31 1 5 33 4 0

S N >160

.-

80

1.0 mm (P: positive) 'Growth dGrowth inhibiting zone > 0.5 mm < 1.0 mm (S: suspicious) eReciprocal of highest dilution causing complete agglutination of sensitized cells

IHA Titers

.160' 23 11 20 22 76

80 18 5

13 36 72

.40 10 10 15 17 52

33 43 43 8 25 76

4 68 4 1 67

0 52 0 0 52

72 52

0

0

116

TABLE IV. Demonstration of Antibody from Cattle Showing Mastitis Caused by M. bovigenitalium Using Growth Inhibition (GI) and Indirect Hemagglutination (IHA) Tests

Animal No. la .+ 2 .3. .+ 4 5 .....................-

Dec. 17/1973 IHA GI

160b 160 5120

160 20

........................

6....1280 7 .................... 8 .....................9. .+ 10 .....................

11......................

+ + + +

160