Zbl. Vet. Med. B, 25, 444-451 (1978) 0 1978 Verlag Paul Parey, Berlin und Hamburg ISSN 0514-7166/ASTM-Coden: ZVRBAZ

Aus dem Institut f u r Mikrobiologie und Tierseuchen der Tierarztlichen Hochschule Hannover

Direktor: Prof . Dr. W . Bisping

Serological and Electrophoretic Characteristics of Mycoplasma hyosynoviae

BY

R. ROSS, H. GREBE and H. KIRCHHOFF

W i t h one figure and 3 tables

(Received for publication June 20, 1977)

Introduction Mycoplasma hyosynoviae has been isolated from joints, lungs and throat

and nasal secretions of swine. The organism has been associated with an acute polyarthritis in 12 to 24 week old swine, especially those with heavy muscling (Ross and DUNCAN, 1970; ROBERTS et al., 1972). Strains of the organism have been shown to differ in pathogenicity and swine have been shown to differ in susceptibility to the arthritis (Ross, 1973). The organism appears to be incapable of acting as a primary factor in swine respiratory disease even though it is occasionally isolated from pneumonic lungs (Go13 et al.? 1975).

Evidence for heterogeneity in biological or antigenic characteristics among different isolates of M . hyosynoviae is very limited. Comparison of strains by growth inhibition (GI) tests revealed a close antigenic relationship among isolates (FRIIS, 1970; GOIS and TAYLOR-ROBINSON, 1972; Ross and KARMON, 1970). Comparisons made with the metabolic-inhibition (MI) test revealed close similarities among strains in one study (ROSS and KARMON, 1970) where- as evidence of heterogeneity was obtained in another study (Go13 and TAYLOR- ROBINSON, 1972). WREGHITT et al. (1974) demonstrated differences in cell proteins of the organism by electrophoresis in polyacrylamide gel. Recogni- tion of heterogeneity is important in the identification of new isolates and in understanding the pathogenic significance of various strains.

Recently, in a survey of arthritic swine joints and lymph nodes sampled at a slaughter plant in Hannover, we isolated 2 strains of M. hyosynoviae (Ross et al., 1977). In the present study, we compare these isolates with the type strains of M . hyosynoviae and 2 additional strains isolated from German

Serologic and Electrophoretic Characteristics of Mycoplasma hyosynoviae 445

swine and provide further evidence of antigenic and electrophoretic differ- ences among strains of the organism.

Material and Methods Mycoplasmas: Mycoplasma hyosynoviae strain S 16, originally isolated

from an arthritic pig in the U. S., was used as the reference strain (Ross and KARMON, 1970). Strain 3053 was isolated from an arthritic joint of a slaughter pig and strain 3222 was isolated from a lymph node of an arthritic slaughter pig (Ross et al., 1977). Strains 932 and 22 were isolated from pharyngeal secretions collected from young boars at the Veterinary School in Hannover. All strains were cloned by selection of single colonies 3 times.

Media: Medium D-TS, consisting of 85 O/o (v/v) PPLO broth (Difco), 15 O/o (v/v) heated turkey serum and 0.5 O/o (w/v) mucin (Difco) was used for routine growth of cultures (ROSS and KARMON, 1970). Solid medium (BHI-TS) consisted of 85 O/o (v/v) fresh beef heart infusion with 0.5 O / o (w/v) mucin, 0.9 O / o agar (agar No. 1, Oxoid) and 15 O / o (v/v) heated turkey serum (Ross and KARMON, 1970). Medium designated D-HS was used for metabolic inhibition tests. It consisted of 70 O/o PPLO broth (v/v), 20 O/o (v/v) unheated horse serum, 10 O/o (v/v) fresh yeast extract, 0.5 O/o (w/v) mucin, 1 O/o (w/v) L arginine HCI, 0.002 O/o phenol red and was adjusted to pH 7.2 (ROSS and KARMON, 1970). An additional medium, designated K N medium, was used for growth of mycoplasmas for electrophoretic techniques. This medium con- sisted of 18.9 g of PPLO broth (Difco), 10 nil of fresh yeast extract, 200 ml of heated horse serum, 2 million IU of penicillin and 900 ml of distilled water.

Antisera: Cells for preparation of antigen were grown for 24 hr in D-TS broth, harvested by centrifugation and washed 3 times with 0.25 M NaCl. Soluble antigen of each strain was prepared by extraction of the mycoplasmal cells with 1 O/o sodium desoxycholate (DOC) (Ross and KARMON, 1970).

Antisera were produced by injection of rabbits intramuscularly with aqueous solutions of DOC extracts of the mycoplasmas with equal volumes of complete Freund adjuvant. The immunization regimen consisted of 3 injec- tions of 5 mg of antigen at 4 to 5 day intervals followed by a 4th injection of 5 mg of antigen 21 to 28 days after the third injection. Blood was collected 7 to 10 days following the last injection of antigen.

Serological Tests: Growth inhibition was done as described by CLYDE (1964) except that sterile filter-paper discs 10 mm in diameter charged with 0.04 ml of antiserum were used. BHI-TS plates were inoculated with 0.05 ml of lo-* or dilutions of 24 hour D-TS cultures and streaked with a bent glass rod.

Metabolic inhibition was done in microtiter plates according to the pro- cedures described by PURCELL et al. (1966) for use with arginine-utilizing mycoplasmas. Cultures were passaged 3 to 4 times in D-HS medium prior to use in the MI test. Seed cultures consisted of 24 hour growth in D-HS medium diluted 10-1 and lo-* in fresh D-HS. Fluid for serum dilutions consisted of 1 part unheated guinea pig serum and 9 parts D-HS medium. The final con- centration of guinea pig serum was calculated to be about 6 O/o following dilution of sera and addition of seed culture. Test sera were heated at 56 OC prior to use. Sera were not diluted prior to dropping in the first microtiter well. Plates were incubated at 37 OC until a pH shift of approximately 0.5 units occurred in wells containing culture with no antiserum. Titers were ex- pressed as the highest serum dilution with no evidence of color change and reflected the serum concentrations prior to addition of seed culture and growth medium.

446 Ross, G R E B E and K I R C H H O F F

Direct immunofluorescence was done according to the epifluorescence procedure (DEL GIUDICE et al., 1967). Conjugates were prepared from 3 times ammonium sulfate-precipitated globulins from hyperimmune rabbit sera at 1 : 70 fluorescein isothiocyanate to protein ratios. Colonies were grown on BHI-TS agar for 2 to 3 days. A Zeiss microscope equipped with an inverted fluorescence light system was used. Gradings of fluorescence were 1 + = weak, 2 + = moderate and 3 + = strong.

Electrophoresis of cell proteins: Electrophoretic patterns of mycoplasmal cell proteins were obtained using the techniques described by RAZIN and ROT- TEM (1967) and RAZIN (1968) and as modified for horizontal flat gel poly- acrylamide electrophoresis by BODEN and KIRCHHOFF (1977). In this system, gels are not prepared in the electrophoresis chamber, rather they are polymeriz- ed in the vertical position and then transferred to the electrophoresis chamber for electrophoresis in the horizontal position. The apparatus used in this study was an LKB Multiphor 21 17.

Polyacrylamide gel was prepared from the following stock solutions:

Stock Solution I 60 g acrylamide (Serva, Heidelberg) 1.6 g N, N’-methylenebisacrylamide 144.14 g urea 280 ml acetic acid 600 ml distilled water

Stock Solution I I 3 g ammonium persulfate 120 g urea 200 ml distilled water

Gel solution was prepared immediately prior to use by mixing 60 ml of stock solution I, 20 ml of stock solution I1 and 0.4 ml of N N N N - t e t r a - methylethylenediamine. The gel solution was poured into a chamber formed by two glass plates, a separator gasket (LKB) and clamps (LKB). Polymeriza- tion was carried out at room temperature overnight. Following removal of one of the glass plates with a thin spatula, the gel was placed exposed side upwards on the cooling plate of the electrophoresis apparatus.

Mycoplasmal cells were grown in D-TS medium as well as in KN medium. The cells were harvested by centrifugation at 13.000 x g for 30 min, washed twice in 0.25 M NaCl and dissolved by adding 1 to 2 ml of phenol-acetic acid-water (2 : 1 : 0.5, w/v/v), The insoluble material was remov- ed by centrifugation at 30,000 x g for 15 min. The amount of protein in the supernates was determined by the biuret method (Merckotest, Merck, Darmstadt) and diluted to a final concentration of 1.5 mg/ml with phenol- acetic acid-water.

Wells were made in the anodal end of the gel using a home-made punch and charged with 80 to 120 j t l of protein solution. Electrophoresis was carried out at room temperature, using tap water for cooling, for seven hours at a constant current of 50 ma. The gels were stained for 30 min at 60 O C with Coomassie brilliant blue (Serva, Heidelberg). Dye was prepared as a stock 1 percent aqueous solution, then diluted 1 : 20 with trichloroacetic acid imme- diately prior to use. The gels were stained as well as destained on the glass plates. Destainiiig was done with a mixture of methanol (20 O / o ) , acetic acid (2 O/o) and glycerol (0.1 O/o). Stained gels were evaluated by recording the pherograms with a Zeiss MQ 3 photometer a t a wave-length of 546 nm.

Strain

S 16 3053

22 932

3222

Antiserum

S 16 3053 22 932 3222 - L.8= 1.3 1.5 2.3 0 4.0 - 4.5 2.0 5.0 0 2.7 1 .o - 4.3 3.8 3.8 4.7 2.3 5.0 - L.3 0 5.3 4.5 7.5 4.0 7.5 - -

Strain

S 16 3053

22 932

3222

Antiserum

S 16 3053 22 932 3222

- 16’ ( 4 d L 8 4 8 4 4 16 8 8 L - 32 16 8 4 4 4 - 32 4

64 - 8 4 4 0

Ross, GREBE and KIRCHHOFF 448

difference in titer among 10 strains of English origin by the use of the MI test. The greater difference among strains obtained with a given antiserum in our comparison (Table 3 ) , as great as 32-fold, provides even more evidence of antigenic heterogeneity of the species. Evidence of close similarities between isolates from widely separated areas (England, U.S. and Denmark) has also been obtained (GOIS and TAYLOR-ROBINSON. 1972).

Antibody sensitivity of strain 3222, as determined with G I and MI tests, appeared to be greater than that of the other 4 strains. Seemingly, this sensitivity related to antibodies to the type-specific antigen common to strains 3222 and 22 as well as to antigens from the other strains, ie strain S 16.

Differences in sensitivity of mycoplasmas to anti- body in MI tests were related to degree of adap- tation to medium by FORSHAW and FALLON (1972). They demonstrated that freshly isolated strains of M . pulmonis were inhibited by higher dilutions of antibody to that organism than were established strains. Although strain 3222 was exa- mined at the 26th to 28th passage level in MI tests, a level similar to that of the other 4 strains of M . hyosynowiae, it had the greatest tendency to become non-viable in broth or on agar medium of the 5 strains examined.

Fig. 1. Pherograms of electrophoretic patterns of the cell proteins of M. hyosynoviae strains 3053, 22, 932, and

3222 and of the type strain S 16

Antigenic heterogeneity of strains is well known among mycoplasma species. The extent of heterogeneity in M . hyorhinis causes difficulties in the

Serologic and Electrophoretic Characteristics of Mycoplasma hyosynoviae 449

Strain

S 16 3053 22 932 3222

Antiserum

S 16 3053 22 932 3222 - 8,192' 16 256 64 32

2,048 8 32,768 - 1,024 32,768 128 32 16,384 32,768 4 2

11,048,576

16 2,048 256 32,766 4 -

- 131,072 256 > 1,048,576 2,048

Summary Five strains of Mycoplasma hyosynoviae isolated from joints, a lymph

node, and pharyngeal secretions of swine were compared by means of serological and electrophoresis techniques. Comparison by growth inhibition, immunofluorescence and metabolic inhibition techniques revealed varying degrees of antigenic relatedness and type-specificity among the strains. Evidence of type-specificity was most pronounced with the metabolic inhibition technique. Immunofluorescence appeared to be the most reliable for identification of all strains. Patterns obtained by electrophoresis of cell proteins from the five strains had the same number of bands and band distribution, but band intensity as reflected by the height of the peaks obtained in pherograms varied considerably.

Acknowledgements This work was supported by Sonderforschungsbereich 54 (Rheumatoide

Krankheiten des Tieres) der Deutschen Forschungsgemeinschaft. Dr. ROSS was

450 Koss, GREHE and KIRCHHOFF

the reci ient of a Senior American Scientist Award from the Alexander von

Iowa, U.S.A. Humbo P d t Foundation and was on leave from Iowa State University, Ames,

Zusammenfassung Serologische und elektrophoretische Eigenschaften

von Mycoplasma hyos y noviae Funf Mycoplasma hyosynoviae-Stamme, die aus Gelenken, Lymphknoten

und Pharynx von Schweinen isoliert wurden, werden serologisch und elektro- phoretisch untersucht. Der Vergleich im Wachstumshemmtest, Stofiwechsel- hemmtest und Epiimmunfluoreszenztest zeigt unterschiedliche Grade antigener Verwandtschaft und Typspezifitat. Die Typspezifitat kam am starksten im Stoff wechselhemmtest zum Ausdruck. Fur die Bestimmung der Stamme erwies sich der Immunfluoreszenztest am geeignetsten. In der Elektrophorese zeigten die funf M . hyosynoviae-Stamme die gleiche Anzahl von Banden, die sich auch im gleichen Abstand voneinander befanden. Die Starke der Banden, bzw. die Hohe der peaks im Pherogramm war jedoch unterschiedlich.

Resume Proprii t is sirologiques et Clectrophoritiques

de Mycoplasma hyosynoviae Cinq souches de Mycoplasma hyosynoviae isolkes 2~ partir d’articulations,

de ganglions et du pharynx de porcs ont k t k examinkes skrologiquement et klectrophorktiquement. La comparaison entre le test d’inhibition de croissance, le test d’inhibition mktabolique et le test d’kpiimmunofluorescence a montrk diffkrents degrks de parent6 antigknique et de spkcificitk de type. La spkcifi- citk de type est apparue le mieux dans le test d’inhibition mktabolique. Le test d’immuiiofluorescence fut le plus adkquat dans la dktermination des souches. L’klectrophorbe a montrk le meme nombre de bandes pour les cinq souches de Mycoplasma hyosynoviae, avec une distance kgale les skparant. L’intensitk des bandes ainsi que la hauteur des pointes dans le phkrogramme furent toute- fois diffkrentes.

Resumen Propiedades serol6gicas y electroforiticas de Mycoplasma hyosynoviae Se examinaron serol6gica y electroforkticamente cinco estirpes de Myco-

plasma hyosynoviae, aisladas en articulaciones, ganglios linfiticos y faringes de cerdos. La comparacibn en las pruebas de inhibici6n del crecimiento, de in- hibici6n del metabolismo y de epiinmunofluorescencia muestra grados diver- sos de parentesco antigknico y especificidad de tipo. La especificidad de tip0 se expresaba de forma mis intensa en la prueba de inhibicih del metabolismo. Para la asignacibn de las estirpes result6 ser la prueba de inmunofluorescencia la mis adecuada. En la electroforesis mostraban las cinco estirpes M . hyosyno- viae la misma cantidad de bandas, las cuales se hallaban tambikn a la misma distancia entre si. Sin embargo, eran diferentes la intensidad de las bandas, resp. la altura de las cimas en el ferograma.

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