J. Vet. Med. B 39, 553-562 (1992) 0 1992 Paul Parey Scientific Publishers, Berlin and Hamburg ISSN 0931-1793
Central Veterinary Institute, Lelystad, The Netherlands
Prevalence of Mycoplasmas in the Respiratory Tracts of Pneumonic Calves E. A. TER LAAK:?,J. H. NOORDERGRAAF and R. P. J. W.
DIELTJES
Address of authors: Central Veterinary Institute, Department of Bacteriology, P. 0. Box 65, 8200 AB Lelystad, The Netherlands
With 8 tables (Received for publication March 29, 1992)
Summary The prevalence of mycoplasmas in the respiratory tracts of 148 pneumonic calves originating from 25 herds in the Netherlands is reported. Four types of culture media were used to isolate mycoplasmas: solid modified EDWARDmedium, 2 types of FRIISmedia, and A7B differential agar medium. Mycoplasmas were isolated both from nasal swab specimens and lung lavage fluids collected from live calves and from nasal mucosa and lung tissue specimens collected post mortem. All of the mycoplasma strains isolated could be identified as either Ureaplasma diversum (isolated from 80 YO of 25 herds), Mycoplasma dispar (92 Yo), M . bovirhinis (88 %), M . bovis (20 Yo), M . bovigenitabm (4 YO), M . arginini (16 "/"), or M. canis (12 YO). Isolation rates of M . dispar and U . diversum were considerably higher from lung lavage fluids than from nasal swab specimens. M . bovis was detected only in fattening herds and not in dairy herds. The respiratory tracts of 75 % of the calves examined contained at least 2 mycoplasrna species. In total, 25 different combinations of mycoplasma species were detected in specimens collected from noses and lungs. The pathogenic species U. diversum and M . dispar had not been isolated before in the Netherlands.
Introduction Respiratory disease in calves is a multifactorial disease to which various species of viruses and bacteria, and also mycoplasmas contribute (24). Not only are mycoplasmas generally specific to one host species, but some bovine mycoplasma species are even specific to an organ, for instance, the respiratory tract. However, little is known about the prevalence of mycoplasma species in the respiratory tracts of pneumonic calves in the Netherlands. The distribution of Mycoplasrna bovis has recently been reviewed (29) and the presence of M . bovirhinis was reported by STRAVER(3). This article reports the prevalence of mycoplasmas in the respiratory tracts of pneumonic calves in the Netherlands. Material and Methods Specimens From 1983 to 1985, specimens from noses and lungs were collected from pneumonic calves younger than six months originating from 25 herds located in the eastern and northern parts of the
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TERLAAK,NOORI)ERGRAAF and DIELTJES
Netherlands. A total of 323 specimens were collected from 69 veal calves and beef cattle from 6 fattening herds and from 79 replacement calves from 19 dairy herds. The number of calves examined in each herd ranged from 1 to 29. Nasal mucosa and lung tissue specimens were collected from autopsied calves. Nasal swab specimens and lung lavage fluids were collected from live calves in the herd; when both types of specimen were collected, the nasal swabs were collected first. Lung lavage fluids were collected as described (7). Lung tissue specimens from autopsied calves were dipped for 6 sec into boiling water, until the color changed from red to brown. The outer side was cut off. The resulting specimen was cut into small pieces and broth was added. The mixture was minced in a Stomacher 80 Lab-Blender (Seward Laboratory, London). The supernatant was immediately stored at -70°C. Mucosal membranes of the nose were scraped o f f and suspended in broth. The supernatant was treated as above. Nasal swab specimens from live calves were collected with a cotton swab that was immediately stored in broth or Hanks’ minimal essential medium. These specimens and lung lavage fluids were also stored at -70°C. The specimens were examined for mycoplasmas after being stored for several months to several years.
Mycoplasma reference strains and antisera Hyperimmune calf antisera against Ureaplasma diversum A417 (serogroup A), D48 (serogroup ARC Institute for Animal Health, B), and T44 (serogroup C ) were obtained from Dr. C . J. HOWARD, Compton, England. Hyperimmune rabbit antisera against M . argznzni G230, M . bovigenitalium PG11, M . bovirhinis PG43, and M .bovts Donetta were obtained from Dr. A. A. POLAK-VOGELZANG, National Institute for Public Health, Bilthoven, the Netherlands. Type strains of the four Mycoplasma species noted above as well as M . dispar and samples of reference antisera against these strains were obtained from D r . E. A. FREUNDTof the former F A O / WHO Collaborating Centre for Animal Mycoplasmas, Arhus, Denmark. M. bovirhinis AF8 was isolated by us and cloned three times according to the method of FREUNDT et al. (9). Hyperimmune antisera against this strain, M . dispar 462/2, and M . bovirhinis PG43 were raised in rabbits by the method of FRIIS(10). M. canis PG14 and hyperimmune rabbit antiserum against it were obtained from Ilr. J. G. TULLY,National Institute of Allergy and Infectious Diseases, Frederick Cancer Research Facility, Frederick, Maryland, the United States.
Mycoplasma media Four types of culture media were used to isolate the mycoplasmas. 1 . Solid modified EDWARD medium (medium B of FREUNDT et al.) was used for less fastidious species o f mycoplasmas (5, 9). 2. Liquid and solid FRIISmedia were used for the isolation of M.dispar (12). Liquid FRIIS medium (‘Medium I’ of FRIISand KROGH)was used in 2 compositions. The NHS25 broth contained 12.5 horse serum and 12.5 ’% porcine serum and was used for primary isolation of M . dispar. The NHS2O broth contained 10%) horse serum and 1 0 % porcine serum and was used to subculture M. dispar strains. SB broth was a selective medium to promote the growth of M. dispar by inhibiting the growth of M . bovirhinis and was used in addition to NHS25 broth for primary isolation of M . dispar (11). The SB broth contained a mixture of hyperimmune rabbit antisera against M. bovirhinis. Antisera were prepared from type strain PG43 and the Dutch field strain AF8, because the growth of certain field strains of M. bovirhinzs was not inhibited by PG43 antiserum alone. Acidification of the liquid media caused by glucose fermenting mycoplasma species was visualized when the phenol red indicator changed from pink to yellow. 3. Liquid FRIISmedium H A U was used to isolate U . diversum and those Mycoplasma species that hydrolyze arginine (‘Medium 11’ of FRIISand KROGH)(12). Liquid FRIISmedium NHU p H 6.0 was used for primary isolation of U . diversum; at p H 6.75 this medium was also used to subculture U . diversum strains (13). The shift of the phenol red indicator to red or purple indicated growth of Mycoplasma species that hydrolyze arginine or U. diversum, which hydrolyzes urea. 4. A7B differential agar medium was used in addition t o FRIISmedia to isolate U . diversum (25). The ureaplasma differential basal agar medium (M52650) and the CVA enrichment (M00170) needed for this medium were obtained from G I B C O Europe Ltd., Paisley, Scotland. Colonies of Ureaplasma grown on A7B agar could be identified when urea was hydrolyzed, causing the accumulation of manganese dioxide and a resulting deep brown stain.
Prevalence of Mycoplasmas in the Respiratory Tracts
555
Cultural procedures Specimens were inoculated onto modified EDWARDagar, FRIIS agar, and A7B agar that were incubated in a C 0 2 incubator in an atmosphere of 7 % C O z in air. In addition, the specimens were serially diluted tenfold to in NHS25 broth, SB broth, H A U broth, and NHU p H 6.0 broth. The broth media NHS25, NHS20, and SB were incubated in rolling drums in which the tubes rotated once per min. The broth media H A U and NHU were incubated without rotation. All media were incubated at 37°C. A7B agar was inspected daily for 6 days; the other media were inspected daily for 14 days. A rapid acidification of NHS25 broth after 1 to 2 days of incubation indicated the growth of M . bovirhinis. Typical mycoplasma colonies then developed on agar plates inoculated with these broth cultures. However, a slow acidification of NHS25 broth after 3 t o 5 days of incubation indicated the growth of M . dispar. These tubes were stored at -70°C. The contents of the tubes with the highest dilution indicating growth were serially diluted tenfold up to 10-6 in NHS20 broth. Again, a slow acidification of NHS2O broth indicated the growth of M . dispar. Acidifying tubes were stored at -70°C. The contents of the tubes with the highest dilution indicating growth were inoculated onto FRIISagar. Growth of centerless colonies indicated M . dispar. A rapid alkalinization of the H A U and N H U broth media, often after only 1 to 2 days of incubation, indicated the growth of U . diversum o r Mycoplasma species that hydrolyze arginine. These tubes were stored at -70°C. The contents of the tube with the highest dilution indicating growth was plated onto A7B agar and FRIISagar and serially diluted tenfold to in NHU p H 6.75 broth. These tubes were stored at -70°C. The contents of the tube with the highest dilution indicating growth was then plated onto A7B agar. Deep brown colonies were recognized as Ureaplasma colonies.
Identification of rnycoplasmas Typical mycoplasma colonies (colonies having a fried egg appearance) grown o n agar media were subcultured or were identified by the indirect immunofluorescence test (23) o r indirect immunoperoxidase test (22). A conjugate of horse antirabbit immunoglobulins and fluorescein isothiocyanate (Red Cross Laboratory, Amsterdam) was used in the immunofluorescence test and a conjugate of goat antirabbit immunoglobulins (prepared by Dr. R. H. MELOEN,Central Veterinary Institute, Lelystad) and horseradish peroxidase in the immunoperoxidase test; in this test 4-chloro-1-naphtol was used as chromogen. The growth inbibation test was used for some strains (2). Cultures suspected of being M . dispar were identified as described earlier (27). Nineteen primary cultures of Ureuplasmct were purified three times by conventional filtration cloning techniques in which a membrane filter with 450-nm pores was used (Milley-HV; Millipore Corp., Bedford, Mass., USA)-(30). After the first filtration step, the culture was serially diluted tenfold in 6 tubes with N H U p H 6.0 broth and the contents of each tube were transferred to 8 wells of a microtiter plate that was sealed with adhesive tape and incubated at 37°C for 7 days. The contents of one of the wells containing the highest dilution of viable culture was serially diluted tenfold in 4 tubes of NHU p H 6.0 broth. The tube containing the highest viable dilution was used for the next filtration step. Colonies of purified cultures were grown o n A7B agar. The Ureaplasma species and serogroups were identified by the indirect immunofluorescence test (18). For some of the strains the biochemical characteristics were determined. Fermentation of glucose was determined in BG broth (containing 0.4% glucose) (9); pH was adjusted to 7.6. Arginine hydrolysis was determined by BA broth ( p H 7.0) and phosphatase was determined as described earlier (9). The production of film and spots was determined on modified EDWARDagar medium o r egg yolk agar medium (8).
Results Mycoplasmas were isolated from all herds examined. Eleven Ureaplasma strains were identified as U . diversum. The remaining 8 strains did not develop colonies on A7B agar after being purified. The following serogroups were identified: serogroup A, 9 strains; and serogroup B, 2 strains. Because the various species of Ureaplasma are host-specific and only small amounts of hyperimmune antisera against the 3 serogroups of U . diversum were available, it was assumed that the remaining Ureaplasma strains belonged to the species U. diversum. U.diversum was isolated from 80 % of the 25 herds. All of the other mycoplasma strains isolated could be identified as either M . dispar (isolated from 92 % of the herds), M . bovirhinis (88 Yo), M . bovis (20 %), M . bovigenitaliurn (4 YO),M . arginini (16 %), or M . canis (12 %) (Table 1).The pathogenic species U . diversum and M . dispur had not been isolated before in the Netherlands.
TERLAAK,NOORDERCRAAF and DIELTJES
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Table 1. Prevalence of mycoplasmas isolated from the respiratory tracts of 148 calves originating from 25 herds Type of herdl No. of calves tested per herd
M. diversum dispar I/.
No. of calves positive for: M. M. M.bowiM. bovirhinis bowis genitalium arginini
M. canis
Fattening 23 29 4 1 10 2
19 16 1 1 1 2
9 21 1
6 3 9 6 4 1 9 3 6 2 2 1 2
3 3 4 1 3 6 2 6 1 2
6 I 6 3 4 1 9 3 6 2 2 1 2 1 4 4 5 2 4
13 24 1
1 1
14
5
5
-
-
1
1 3 2
-
2
Dairy
~~~~
Total
1
-
4 6 5 4 5
2 5 4 2
1
3 9 5 4 1 9
3 6 1 2
~
84 (57%)
98 (66 "A))
106 (72 O h )
25 (17 "/o)
4 (3 Yo)
12 (8%)
3 (2%))
No mycoplasmas were isolated from 46 (14 Y o ) of the 321 specimens; 1 species was isolated from 77 (24 Yo) specimens; 2 species were isolated from 102 (32 Yo) specimens; 3 species were isolated from 81 (25 %) specimens; and 4 species were isolated from 15 (5 Yo) specimens (Table 2). When 3 species were isolated, the most common combination was U . diversum, M . dispar, and M . bovirhinis. This combination was detected in 41 calves (28 Yo). The combination of M . dispar and M . bovirhinis was detected in 27 calves (18 %); and the combination of U. diverswn and M . bovirhinis was detected in 9 calves (6 YO).Two calves contained as many as 5 mycoplasma species in their respiratory tracts. U. diversum, M . bovirhinis, M . bovis, and M . arginini were isolated from the nose of an autopsied calf, while U. diversum, M . dispar, M . bowirbinis, and M . bovzs were isolated from its lung tissue. M.bovirhinis and M . arginini were isolated from a nasal swab specimen collected from a live calf, while U. diversum, M . dispay, and M . canis were isolated from lung lavage fluid. The respiratory tracts of 75 % of all calves examined contained at least 2 mycoplasma species. Mycoplasmas were isolated from both the nose and lungs of live and dead calves (Table 3). Isolation rates of U. diversum and M . dispar were considerably higher from lung lavage fluids than from nasal swab specimens when these 2 specimens were compared as
557
Prevalence of Mycoplasmas in the Respiratory Tracts Table 2. Combinations of mycoplasma species isolated from respiratory tract specimens’
No. of
Mycoplasma species isolated
specimens
13 22 12 43 1 1 6 2 2 57 3 1 5 5 1 1 4 1 1 1 1 5
U. diversum
M.
+ + +
-
+ + +
-
i
dispar -
M. bowirhinis bowis
M.bowigenitalium
-
M.
M.
arginini
canis
-
-
-
-
-
-
-
+
-
-
-
-
t
M.
-
+
-
+
-
+
-
-
-
+
+
+ +
-
+ + + + + + + + +
-
+ + + + -
+ + + + + + +
-
-
+ + + +
4
3 3
= A total of 321 specimens was examined; no mycoplasmas were isolated from 46 specimens; one mycoplasma species was isolated from 77 specimens; + = mycoplasma species isolated; - = mycoplasma species not isolated.
Table3. Prevalence of mycoplasmas in noses and lungs of live and dead calves Calves/ Type of specimen Live calves nose lungs Dead calves nose lungs Total
No. of
Yo Specimens positive for: M . bovi-
M. diversum dispar
M.bovi- M. rhinis bowis
69 103
38 51
57 63
81 80
4 11
7 4
6 7
2
34 115
26 58
44 34
38 30
24 28
-
3 3
3 3
321
48
49
58
17
3
5
2
specimens U .
M.
M.
genitalium arginini canis
TERLAAK,NOORDBRGRAAF and DIELTJES
558
Table+. Isolation of mycoplasmas from 60 live calves from which both a nasal swab specimen and lung lavage fluid were collected Type of specimen
%, Specimens positive for: M.
M. di,uersum dispur
M. M. bovirhinis bovis
37 65
87 93
U.
M.
M. cunis
boviarginini genitulium
~
Nasal swab Lung lavage fluid
53 75
5 5
7 3
8 7
-
3
Table 5. Efficacy ot three culture media for the isolation of U. diversum from 236 respiratory tract specimensA
No. of qpeciniens
Growth of specimens in medium HAU NHU
A713 55 4 6 25 4 23 119
I Only specimens t h x were inoculated into the three media are included; A7B = agar medium (25); H A U = liquid medium according to FRIISand KROCH (12); according to SHEPARDand COMBS N H U = liquid medium p H 6.0 according to FRIIS et al. (13); + = U.dsversum isolated; - = I/. dsversum not isolated.
Table 6. Growth promoting capacity of two liquid media for the isolation of U . dzversum from 26 nose specimens and 98 lung specimen9 Type of specimen/ Medium Nose HAU
NHU
% Specimen5 with no growth
% Specimens with endpoints of growth at a dilution of: lo-' 10-2 lo-' 10-4 10-5 lo-"
46 K
4 31
12 27
15 27
15 4
8 4
-
21 6
3 3
6 15
15 17
16 22
21 15
16 20
-
Lungs
HAU NHU
Abbreviations are defined in the footnotes of Table 5; 2 Ureuplasmu diversum was isolated from each specimen in at least one medium.
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559
Table 7. Efficacy of two media for the isolation of M . dzspar from 276 respiratory tract specimens. No. of specimens
Growth of specimens in medium: SB NHS
54 62 18 142 ~
~
~~
~
Only specimens that were inoculated into both media are included; NHS = 'medium I' according to FRIISand KROGH (12); SB = selective medium according to FRIIS(11); + = M,dzspar isolated; - = M . dzspar not isolated.
paired specimens from each calf, but M . bovirhinis was isolated as frequently from the nose as from the lungs (Table 4). When the media used t o isolate U.diversurn were compared, the organism was isolated from 28 YO of the specimens inoculated onto A7B agar, from 37 % inoculated into H A U broth, and from 4 6 % inoculated into NHU broth. In total, U.diversum was isolated from 5 0 % of the specimens grown on these 3 media. When U.diversum was isolated from A7B agar, it was also isolated from one or both broth media (Table 5). Both H A U broth and NHU broth contained the same number of U.diversum organisms. Lung specimens contained approximately 100 times more U . diversum organisms than nose specimens (Table 6). M . dispar was isolated from 26 % of the specimens inoculated into NHS broth and from 42 % of the specimens inoculated into SB broth. In total, M . dispur was isolated from 49 % of the specimens grown on these 2 media (Table 7). Both NHS broth and SB broth contained the same number of M . dispur organisms. Lung specimens contained approximately 100 times more M . dispur organisms than nose specimens (Table 8).
Discussion
To study the prevalence of mycoplasmas in the respiratory tracts of pneumonic calves, we used four types of media to isolate these organisms from 323 specimens collected over a 3-year period. Mycoplasmas were isolated from most of the pneumonic calves and 7 species of mycoplasmas were isolated. T w o pathogenic species, U. diversum and M . dispar,
Table 8. Growth promoting capacity of two media for the isolation of M . dzspar from 43 nose specimens and 91 lung specimens. Type of % Specimens specimen/ with no test Medium resultb Nose NHS SB
% Specimens with endpoints of growth at a dilution of: lo-' 10-2 10-3 10-4 10-5 10-6
-
53 9
7 26
19 26
14 7
5 7
2
26
43 15
2 11
3 12
7 22
13 15
18 15
14 9
-
Lungs
NHS SB ~~
~~
Abbreviations are defined in the footnotes of Table 7; a Mycoplasrna dzspar was isolated from each specimen in at least one medium; b Tubes contained growth of M . bovzrhznrs or M . bows, or contained no cultures, or were not tested.
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TERLAAK,NOOKDERGKAAF and DIELTJES
that had not been isolated before in the Netherlands, were prevalent. The numbers of organisms of the most prevalent species sometimes exceeded 106 organisms per gram specimen (U. diversum, Table 6; M. dispar, Table 8; results for M. bovirbinis and M . bovis are not shown). Although it is well-known that mycoplasmal inhibitors (e. g., lysolecithin) are released in tissue suspensions (26), we concluded that these substances apparently do not harm the viability of mycoplasmas when the tissue suspensions are stored at -70 "C, even when stored for years. Although the lung lavage technique was described by GOURLAY (14) in 1983, it has seldom been used to study the mycoplasmal flora of calf lungs. DEVRIESE et al. (4) and BINDERet al. (1) isolated mycoplasmas from lung lavage fluids collected from calves at a clinic. Because the lavage tube is passed through the nostril of the calf, it has been thought that lung lavage fluid might be contaminated with organisms from the nose. However, we often isolated mycoplasma species from the nose that were not isolated from lung lavage fluids. Thus, we conclude that reliable specimens of the lung flora can be collected from live calves in the field by this method. U . diversum in particular can be isolated more frequently from the lungs than from the nose (Tables 3 and 4). The value of the lung lavage technique for isolating viruses and pulmonary cells as well has already been reported (19). Because M . dispar is fastidious and slow-growing, it is easily overgrown in culture by M . bowirbinis, which also catabolizes glucose, but which grows faster and is easily cultured. Because the latter is often present in specimens that contain M.dispar, FRIIS(11) devised a selective medium to suppress the growth of M. bovirbinis and to support the growth of M . dispar. We prepared this medium and were able to suppress the growth of M.bovirbinis in 62 specimens (Table 7). The presence of M . bowis also may hamper the isolation of M . dispar, because it grows faster than M . dispar and causes a slight acidification in media with o r without glucose, although it does not catabolize glucose (15). Indeed, when specimens contained quantities of M. bovis, both NHS and SB broth media were overgrown by this organism. As a result, we were not able to isolate M.dispar from these specimens. This could be the reason why M . dispar was less prevalent in fattening calves (46 Oh)than in dairy calves (84 Yo). M.bovzs was prevalent only in fattening calves. In addition, M.bovirbinis was less prevalent in fattening calves (57 Yo) than in dairy calves (85 Yo) (Tables 1 and 3). These findings indicate that a higher prevalence of M.bovis is associated with a lower isolation rate of M . dispar and M. bovirbinis. Whether M. dispar and M. bovirbinis are less prevalent when M . bovis is prevalent, however, is not clear. Therefore, we recommend that hyperimmune antiserum against M . bovis should be included with hyperimmune antiserum against M . bovirbinis in the selective medium used to isolate M.dispar, when M.bovis is prevalent. Because in Denmark, where the selective broth for M. dispar was developed, M . bovis still occurs rarely (6), it was not necessary to include M . bowis antiserum in SB broth. Although SB broth enabled us to isolate M . dispar from specimens containing M . bovirinis (Table 7), it slightly inhibited the growth of M. dispar. Whether this phenomenon was caused by the hyperimmune M. bovirhinis antisera or by other medium components is not clear. Despite this finding, however, we conclude that SB broth is a valuable medium for isolating M . dispar. When we used 2 liquid media to isolate U . diversum, the use of a third medium (A7B agar) did not increase the total number of isolates. We conclude from this that the A7B agar medium can be omitted for the primary isolation of U. diversum. It was, however, useful to confirm the growth of U.diwersum in the liquid media. When nose specimens were inoculated onto the agar medium alone, I/. diversum was isolated from only 12 Yo of the 65 nose specimens; whereas when 2 liquid media were added to the agar medium, U . divevsum was isolated from 2 7 % of the specimens. Because lung specimens contained more U. diversum organisms, it was concluded that A7B agar supported the growth of U . diversum only when it was present in quantity. Of the 117 U. diversum isolates, 75 % were obtained from H A U broth and 93 Yo from NHU broth, leading us to conclude that N H U broth was superior for this purpose (Table5). Moreover, because of its low pH, NHU
Prevalence of Mycoplasmas in the Respiratory Tracts
561
broth inhibited true bacteria and acidifying Mycoplusma species better than H A U broth. H A U broth, however, is useful for isolating mycoplasmas that hydrolyze arginine, because these organisms generally do not grow in N H U broth. Eighty-two percent of the U . divevsum strains (n = 11) examined with serogroupspecific antisera belonged to serogroup A. These findings are in contrast to those reported by HOWARD and GOURLAY (IS), who found that 81 % of the strains (n = 54) isolated from the respiratory tracts of cattle from 4 countries belonged to serogroup B but only 15 % to serogroup A. Seven strains isolated from calves from 3 herds were identified as M . canis. Since the present study was ended, this species has been isolated from 17 other herds. This finding is notable, because M . cunis has never been reported in cattle before. There were indications that M . canis was transmitted by dogs that licked milk from the noses of the calves after feeding. The identification and characterization of M . canis strains is described elsewhere (28). Although Acholephsma species could have been detected by the methods used, none of these species were isolated. Only a few studies on the mycoplasmal flora of the respiratory tract of pneumonic calves have included methods for isolating U. divevsum and M . dispar (12, 17,21). Another study used in addition to these direct methods also indirect methods to detect the presence of these organisms (20). These studies were conducted in countries other than the Netherlands where U. diversum and M.dispar were also found to be prevalent. M . bovivhinis was often found to be less prevalent, however, than we found it to be. Reliable information on the prevalence of mycoplasmas in a particular herd can be obtained when both lung lavage fluids and nasal swab specimens are collected from 5 pneumonic calves. Although the mycoplasmas isolated from upper respiratory tract specimens may not necessarily be the ones involved in the pneumonic lesions (16), most mycoplasma species in a herd can be detected, even when only nasal swab specimens are tested.
Acknowledgements We thank G. M. ZIMMERfor collecting the specimens, E. A. FREUNDT and H. E K N(the ~ former F A O / W H O Collaborating Centre for Animal Mycoplasmas, Arhus, Denmark) for general advice in culturing mycoplasmas, and N . F. FRIIS(State Veterinary Serum Laboratory, Copenhagen, Denmark) for advice in culturing bovine mycoplasmas.
References 1. BINDER,A., G. AMTSBERG,S. DOSE, W. FISCHER, H. SCHOLZ, and H. KIRCHHOFF, 1990: Isolation of mycoplasmas and bacteria from cattle with respiratory diseases. J. Vet. Med. B 37, 430-435. (In German) 2. CLYDE,JK., W. A,, 1983: Growth inhibition tests. In: S. RUIN, and J. G. TULLY,eds. Methods in Mycoplasmology I. New York: Academic Press Inc., 405-410. 3. COTTEW,G. S., and R. H. LEACH,1969: Mycoplasmas of cattle, sheep, and goats. In: L. HAYPLICK, ed. The Mycoplasmutales and the L-phase of bacteria. New York: Appleton-CenturyCrofts, Meredith Corporation, 546. and H . THOONE,1987: Bacteriological findings in bovine 4. DEVKIESB, L., J. NUYTTEN,P. DEPREZ, respiratory infections. Vlaams Diergeneeskd. Tijdschr. 56, 438-446. (In Dutch) 5. EUWARD,D . G . ff., 1947: A selective medium for pleuropneumonia-like organisms. J. Gen. Microbiol., 1, 238-243. MADSEN,N . F . FKIIS,A MEYLING,and P.AHRENS,1991: A field 6. FEBNSTKA, A,, E. BISGAARD study of Mycoplasma bovis infection in cattle. J. Vet. Med. B, 38, 195-202. U., P. J. Q U I N Nand , J. HANNAN, 1983: Bronchopulmonary lavage in the calf: a new 7. FOGAKTHY, technique. Ir. Vet. J., 37, 35-38. E. A,, 1983: Film and spot production. In: S. RAZIN, and J. G . TULLY, eds. Methods in 8. FREUNDT, Mycoplasmology I. N e w York: Academic Press Inc., 373-374.
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9. FREUNIIT,E. A., H . ERN^, and R. M. LEMCKE,1979: Identification of mycoplasmas. In: T. BERG A N , and J. R. NORRIS,eds. Methods in Microbiology 13. London: Academic Press Inc., 377-434. 10. Ficiis, N . F., 1977: Mycoplusmu suipneumoniae and Mycoplusmu flocculare in the growth precipitation test. Acta Vet. Scand., 18, 168-175. 11. FKIIS,N. F., 1979: Selective isolation of slowly growing acidifying mycoplasmas from swine and cattle. Acta Vet. Scand., 20, 607-609. 12. FRIIS,N . F., and H. V. KROGH,1983: Isolation of mycoplasmas from Danish cattle. Nord. Vet. Med., 35, 74-81. and B. BLOCH,1980: Ureaplusmu isolated from the respiratory tract 13. FRIIS,N . F., K. B. PEDERSEN, of mink. Acta Vet. Scand., 21, 134-136. 14. GOURLAY, R. N., 1983: Lavage techniques for recovery of animal mycoplasmas. In: J. G. TULLY, and S.RAZIN,eds. Methods in mycoplasmology 11. New York: Academic Press Inc., 149-151. 15. GOURLAY, R. N., and C. J. HOWARD,1979: Bovine mycoplasmas. In: J. G. TULLY,and R.F. WHrrcoMB, eds. The mycoplasmas 11. New York: Academic Press Inc., 49-102. 16. GOURLAY, R. N., and C. J. HOWARD,1982: Respiratory mycoplasmosis. In: C . E. CORNELIUS, and C . F. SIMPSON,eds. Advances in veterinary science and comparative medicine. D. L. DUNWORTH, ed. Vol. 26. The respiratory system. New York: Academic Press. Inc., 289-332. and J.E. COOPER,1970: Studies of the microbiology and 17. GOUKLAY, R.N., A. MACKENZIE, pathology of pneumonic lungs of calves. J. Comp. Path., 80, 575-584. 18. HOWARD,C . J., and R. N . GOURLAY,1981: Identification of ureaplasmas from cattle using antisera prepared in gnotobiotic calves. J. Gen. Microbiol., 126, 365-369. T. G., G. M. ZIMMER, I?. J. STRAVEK, and P. W. DE LEEUW,1986: Diagnosis of bovine 19. KIMMAN, respiratory syncytial virus infections improved by virus detection in lung lavage samples. Am. J.Vet. Res., 47, 143-147. 20. KNUDTSON, W. U., D. E. REED, and G. DANIELS,1986: Identification of Mycoplasmutales in pneumonic calf lungs. Vet. Microbiol., 11, 79-91. O . A . , E.E. OSE,and T. MATSUOKA, 1979: The incidence of Mycoplusmu dispar, 21. MUENSTER, Ureaplusma and conventional Mycoplusmu in the pneumonic calf lung. Can. J. Comp. Med., 43, 392-398. 22. POLAK-VOGBLZANG, A. A., R. HAGENAAKS, and J. NAGEL, 1978: Evaluation of an indirect inimunoperoxidase test for identification of Acboleplusrna and Mycoplusmu. J. Gen. Microbiol., 106,241 -249. S., and F. T. BLACK,1972: Direct and indirect immunofluorcscence of unfixed and 23. ROSENI)AL, fixed mycoplasina colonies. Acta Path. Microbiol. Scand. Section B, 80, 615-622. eds. Handbuch der 24. Ross, R. F., 1985: Mycoplasmas of cattle. In: H. BLOBEI.and TH. SCHLIESSER, bakteriellen Infektionen bei Tieren V. Stuttgart: Gustav Fischer Verlag, 3 14-344. 25. SHEPARD, M. C., and R. S. COMBS,1979: Enhancement of Ureuplasmu urealyticum growth o n a differential agar medium (A7B) by a polyamine, putrescine. J. Clin. Microbiol., 10, 931 -933. 26. TAYLOR-ROBINSON, D., and T. A. CHEN,1983: Growth inhibitory factors in animal and plant eds. Methods in mycoplasmology 1. New York: Academic tissues. In: S.RAZIN,and J. G. TULLY, Press Iuc., 109- 114. 1987: An improved method for the identification of 27. TERLAAK,E. A,, and J. H. NOORDERGKAAF, Mycoplusmu dispar. Vet. Microbiol., 14, 25-31. D. L. ROSE, P. CARLE,J.M. BovE, and 28. TERLAAK,E.A., J.G. TULLY,J . H . NOORDERGRAAF, M. A. SMITS:Recognition of Mycoplusmu cunis as part of the mycoplasmal flora of the bovine respiratory tract. Vet. Microbiol., in press. and G. M. ZIMMER:Increased prevalence of Mycoplasmu bowis 29. TBRLAAK,E. A,, G. H. WENTINK, in the Netherlands. Vet. Quart., 4, 100-104. 30. TUI.I.Y,J. G., 1983: Cloning and filtration techniques for mycoplasmas. In: S. KAZIN,and J. G. TULLY, eds. Methods in mycoplasmology I. New York: Academic Press Inc., 173-177.
Central Veterinary Institute, Lelystad, The Netherlands
Prevalence of Mycoplasmas in the Respiratory Tracts of Pneumonic Calves E. A. TER LAAK:?,J. H. NOORDERGRAAF and R. P. J. W.
DIELTJES
Address of authors: Central Veterinary Institute, Department of Bacteriology, P. 0. Box 65, 8200 AB Lelystad, The Netherlands
With 8 tables (Received for publication March 29, 1992)
Summary The prevalence of mycoplasmas in the respiratory tracts of 148 pneumonic calves originating from 25 herds in the Netherlands is reported. Four types of culture media were used to isolate mycoplasmas: solid modified EDWARDmedium, 2 types of FRIISmedia, and A7B differential agar medium. Mycoplasmas were isolated both from nasal swab specimens and lung lavage fluids collected from live calves and from nasal mucosa and lung tissue specimens collected post mortem. All of the mycoplasma strains isolated could be identified as either Ureaplasma diversum (isolated from 80 YO of 25 herds), Mycoplasma dispar (92 Yo), M . bovirhinis (88 %), M . bovis (20 Yo), M . bovigenitabm (4 YO), M . arginini (16 "/"), or M. canis (12 YO). Isolation rates of M . dispar and U . diversum were considerably higher from lung lavage fluids than from nasal swab specimens. M . bovis was detected only in fattening herds and not in dairy herds. The respiratory tracts of 75 % of the calves examined contained at least 2 mycoplasrna species. In total, 25 different combinations of mycoplasma species were detected in specimens collected from noses and lungs. The pathogenic species U. diversum and M . dispar had not been isolated before in the Netherlands.
Introduction Respiratory disease in calves is a multifactorial disease to which various species of viruses and bacteria, and also mycoplasmas contribute (24). Not only are mycoplasmas generally specific to one host species, but some bovine mycoplasma species are even specific to an organ, for instance, the respiratory tract. However, little is known about the prevalence of mycoplasma species in the respiratory tracts of pneumonic calves in the Netherlands. The distribution of Mycoplasrna bovis has recently been reviewed (29) and the presence of M . bovirhinis was reported by STRAVER(3). This article reports the prevalence of mycoplasmas in the respiratory tracts of pneumonic calves in the Netherlands. Material and Methods Specimens From 1983 to 1985, specimens from noses and lungs were collected from pneumonic calves younger than six months originating from 25 herds located in the eastern and northern parts of the
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TERLAAK,NOORI)ERGRAAF and DIELTJES
Netherlands. A total of 323 specimens were collected from 69 veal calves and beef cattle from 6 fattening herds and from 79 replacement calves from 19 dairy herds. The number of calves examined in each herd ranged from 1 to 29. Nasal mucosa and lung tissue specimens were collected from autopsied calves. Nasal swab specimens and lung lavage fluids were collected from live calves in the herd; when both types of specimen were collected, the nasal swabs were collected first. Lung lavage fluids were collected as described (7). Lung tissue specimens from autopsied calves were dipped for 6 sec into boiling water, until the color changed from red to brown. The outer side was cut off. The resulting specimen was cut into small pieces and broth was added. The mixture was minced in a Stomacher 80 Lab-Blender (Seward Laboratory, London). The supernatant was immediately stored at -70°C. Mucosal membranes of the nose were scraped o f f and suspended in broth. The supernatant was treated as above. Nasal swab specimens from live calves were collected with a cotton swab that was immediately stored in broth or Hanks’ minimal essential medium. These specimens and lung lavage fluids were also stored at -70°C. The specimens were examined for mycoplasmas after being stored for several months to several years.
Mycoplasma reference strains and antisera Hyperimmune calf antisera against Ureaplasma diversum A417 (serogroup A), D48 (serogroup ARC Institute for Animal Health, B), and T44 (serogroup C ) were obtained from Dr. C . J. HOWARD, Compton, England. Hyperimmune rabbit antisera against M . argznzni G230, M . bovigenitalium PG11, M . bovirhinis PG43, and M .bovts Donetta were obtained from Dr. A. A. POLAK-VOGELZANG, National Institute for Public Health, Bilthoven, the Netherlands. Type strains of the four Mycoplasma species noted above as well as M . dispar and samples of reference antisera against these strains were obtained from D r . E. A. FREUNDTof the former F A O / WHO Collaborating Centre for Animal Mycoplasmas, Arhus, Denmark. M. bovirhinis AF8 was isolated by us and cloned three times according to the method of FREUNDT et al. (9). Hyperimmune antisera against this strain, M . dispar 462/2, and M . bovirhinis PG43 were raised in rabbits by the method of FRIIS(10). M. canis PG14 and hyperimmune rabbit antiserum against it were obtained from Ilr. J. G. TULLY,National Institute of Allergy and Infectious Diseases, Frederick Cancer Research Facility, Frederick, Maryland, the United States.
Mycoplasma media Four types of culture media were used to isolate the mycoplasmas. 1 . Solid modified EDWARD medium (medium B of FREUNDT et al.) was used for less fastidious species o f mycoplasmas (5, 9). 2. Liquid and solid FRIISmedia were used for the isolation of M.dispar (12). Liquid FRIIS medium (‘Medium I’ of FRIISand KROGH)was used in 2 compositions. The NHS25 broth contained 12.5 horse serum and 12.5 ’% porcine serum and was used for primary isolation of M . dispar. The NHS2O broth contained 10%) horse serum and 1 0 % porcine serum and was used to subculture M. dispar strains. SB broth was a selective medium to promote the growth of M. dispar by inhibiting the growth of M . bovirhinis and was used in addition to NHS25 broth for primary isolation of M . dispar (11). The SB broth contained a mixture of hyperimmune rabbit antisera against M. bovirhinis. Antisera were prepared from type strain PG43 and the Dutch field strain AF8, because the growth of certain field strains of M. bovirhinzs was not inhibited by PG43 antiserum alone. Acidification of the liquid media caused by glucose fermenting mycoplasma species was visualized when the phenol red indicator changed from pink to yellow. 3. Liquid FRIISmedium H A U was used to isolate U . diversum and those Mycoplasma species that hydrolyze arginine (‘Medium 11’ of FRIISand KROGH)(12). Liquid FRIISmedium NHU p H 6.0 was used for primary isolation of U . diversum; at p H 6.75 this medium was also used to subculture U . diversum strains (13). The shift of the phenol red indicator to red or purple indicated growth of Mycoplasma species that hydrolyze arginine or U. diversum, which hydrolyzes urea. 4. A7B differential agar medium was used in addition t o FRIISmedia to isolate U . diversum (25). The ureaplasma differential basal agar medium (M52650) and the CVA enrichment (M00170) needed for this medium were obtained from G I B C O Europe Ltd., Paisley, Scotland. Colonies of Ureaplasma grown on A7B agar could be identified when urea was hydrolyzed, causing the accumulation of manganese dioxide and a resulting deep brown stain.
Prevalence of Mycoplasmas in the Respiratory Tracts
555
Cultural procedures Specimens were inoculated onto modified EDWARDagar, FRIIS agar, and A7B agar that were incubated in a C 0 2 incubator in an atmosphere of 7 % C O z in air. In addition, the specimens were serially diluted tenfold to in NHS25 broth, SB broth, H A U broth, and NHU p H 6.0 broth. The broth media NHS25, NHS20, and SB were incubated in rolling drums in which the tubes rotated once per min. The broth media H A U and NHU were incubated without rotation. All media were incubated at 37°C. A7B agar was inspected daily for 6 days; the other media were inspected daily for 14 days. A rapid acidification of NHS25 broth after 1 to 2 days of incubation indicated the growth of M . bovirhinis. Typical mycoplasma colonies then developed on agar plates inoculated with these broth cultures. However, a slow acidification of NHS25 broth after 3 t o 5 days of incubation indicated the growth of M . dispar. These tubes were stored at -70°C. The contents of the tubes with the highest dilution indicating growth were serially diluted tenfold up to 10-6 in NHS20 broth. Again, a slow acidification of NHS2O broth indicated the growth of M . dispar. Acidifying tubes were stored at -70°C. The contents of the tubes with the highest dilution indicating growth were inoculated onto FRIISagar. Growth of centerless colonies indicated M . dispar. A rapid alkalinization of the H A U and N H U broth media, often after only 1 to 2 days of incubation, indicated the growth of U . diversum o r Mycoplasma species that hydrolyze arginine. These tubes were stored at -70°C. The contents of the tube with the highest dilution indicating growth was plated onto A7B agar and FRIISagar and serially diluted tenfold to in NHU p H 6.75 broth. These tubes were stored at -70°C. The contents of the tube with the highest dilution indicating growth was then plated onto A7B agar. Deep brown colonies were recognized as Ureaplasma colonies.
Identification of rnycoplasmas Typical mycoplasma colonies (colonies having a fried egg appearance) grown o n agar media were subcultured or were identified by the indirect immunofluorescence test (23) o r indirect immunoperoxidase test (22). A conjugate of horse antirabbit immunoglobulins and fluorescein isothiocyanate (Red Cross Laboratory, Amsterdam) was used in the immunofluorescence test and a conjugate of goat antirabbit immunoglobulins (prepared by Dr. R. H. MELOEN,Central Veterinary Institute, Lelystad) and horseradish peroxidase in the immunoperoxidase test; in this test 4-chloro-1-naphtol was used as chromogen. The growth inbibation test was used for some strains (2). Cultures suspected of being M . dispar were identified as described earlier (27). Nineteen primary cultures of Ureuplasmct were purified three times by conventional filtration cloning techniques in which a membrane filter with 450-nm pores was used (Milley-HV; Millipore Corp., Bedford, Mass., USA)-(30). After the first filtration step, the culture was serially diluted tenfold in 6 tubes with N H U p H 6.0 broth and the contents of each tube were transferred to 8 wells of a microtiter plate that was sealed with adhesive tape and incubated at 37°C for 7 days. The contents of one of the wells containing the highest dilution of viable culture was serially diluted tenfold in 4 tubes of NHU p H 6.0 broth. The tube containing the highest viable dilution was used for the next filtration step. Colonies of purified cultures were grown o n A7B agar. The Ureaplasma species and serogroups were identified by the indirect immunofluorescence test (18). For some of the strains the biochemical characteristics were determined. Fermentation of glucose was determined in BG broth (containing 0.4% glucose) (9); pH was adjusted to 7.6. Arginine hydrolysis was determined by BA broth ( p H 7.0) and phosphatase was determined as described earlier (9). The production of film and spots was determined on modified EDWARDagar medium o r egg yolk agar medium (8).
Results Mycoplasmas were isolated from all herds examined. Eleven Ureaplasma strains were identified as U . diversum. The remaining 8 strains did not develop colonies on A7B agar after being purified. The following serogroups were identified: serogroup A, 9 strains; and serogroup B, 2 strains. Because the various species of Ureaplasma are host-specific and only small amounts of hyperimmune antisera against the 3 serogroups of U . diversum were available, it was assumed that the remaining Ureaplasma strains belonged to the species U. diversum. U.diversum was isolated from 80 % of the 25 herds. All of the other mycoplasma strains isolated could be identified as either M . dispar (isolated from 92 % of the herds), M . bovirhinis (88 Yo), M . bovis (20 %), M . bovigenitaliurn (4 YO),M . arginini (16 %), or M . canis (12 %) (Table 1).The pathogenic species U . diversum and M . dispur had not been isolated before in the Netherlands.
TERLAAK,NOORDERCRAAF and DIELTJES
556
Table 1. Prevalence of mycoplasmas isolated from the respiratory tracts of 148 calves originating from 25 herds Type of herdl No. of calves tested per herd
M. diversum dispar I/.
No. of calves positive for: M. M. M.bowiM. bovirhinis bowis genitalium arginini
M. canis
Fattening 23 29 4 1 10 2
19 16 1 1 1 2
9 21 1
6 3 9 6 4 1 9 3 6 2 2 1 2
3 3 4 1 3 6 2 6 1 2
6 I 6 3 4 1 9 3 6 2 2 1 2 1 4 4 5 2 4
13 24 1
1 1
14
5
5
-
-
1
1 3 2
-
2
Dairy
~~~~
Total
1
-
4 6 5 4 5
2 5 4 2
1
3 9 5 4 1 9
3 6 1 2
~
84 (57%)
98 (66 "A))
106 (72 O h )
25 (17 "/o)
4 (3 Yo)
12 (8%)
3 (2%))
No mycoplasmas were isolated from 46 (14 Y o ) of the 321 specimens; 1 species was isolated from 77 (24 Yo) specimens; 2 species were isolated from 102 (32 Yo) specimens; 3 species were isolated from 81 (25 %) specimens; and 4 species were isolated from 15 (5 Yo) specimens (Table 2). When 3 species were isolated, the most common combination was U . diversum, M . dispar, and M . bovirhinis. This combination was detected in 41 calves (28 Yo). The combination of M . dispar and M . bovirhinis was detected in 27 calves (18 %); and the combination of U. diverswn and M . bovirhinis was detected in 9 calves (6 YO).Two calves contained as many as 5 mycoplasma species in their respiratory tracts. U. diversum, M . bovirhinis, M . bovis, and M . arginini were isolated from the nose of an autopsied calf, while U. diversum, M . dispar, M . bowirbinis, and M . bovzs were isolated from its lung tissue. M.bovirhinis and M . arginini were isolated from a nasal swab specimen collected from a live calf, while U. diversum, M . dispay, and M . canis were isolated from lung lavage fluid. The respiratory tracts of 75 % of all calves examined contained at least 2 mycoplasma species. Mycoplasmas were isolated from both the nose and lungs of live and dead calves (Table 3). Isolation rates of U. diversum and M . dispar were considerably higher from lung lavage fluids than from nasal swab specimens when these 2 specimens were compared as
557
Prevalence of Mycoplasmas in the Respiratory Tracts Table 2. Combinations of mycoplasma species isolated from respiratory tract specimens’
No. of
Mycoplasma species isolated
specimens
13 22 12 43 1 1 6 2 2 57 3 1 5 5 1 1 4 1 1 1 1 5
U. diversum
M.
+ + +
-
+ + +
-
i
dispar -
M. bowirhinis bowis
M.bowigenitalium
-
M.
M.
arginini
canis
-
-
-
-
-
-
-
+
-
-
-
-
t
M.
-
+
-
+
-
+
-
-
-
+
+
+ +
-
+ + + + + + + + +
-
+ + + + -
+ + + + + + +
-
-
+ + + +
4
3 3
= A total of 321 specimens was examined; no mycoplasmas were isolated from 46 specimens; one mycoplasma species was isolated from 77 specimens; + = mycoplasma species isolated; - = mycoplasma species not isolated.
Table3. Prevalence of mycoplasmas in noses and lungs of live and dead calves Calves/ Type of specimen Live calves nose lungs Dead calves nose lungs Total
No. of
Yo Specimens positive for: M . bovi-
M. diversum dispar
M.bovi- M. rhinis bowis
69 103
38 51
57 63
81 80
4 11
7 4
6 7
2
34 115
26 58
44 34
38 30
24 28
-
3 3
3 3
321
48
49
58
17
3
5
2
specimens U .
M.
M.
genitalium arginini canis
TERLAAK,NOORDBRGRAAF and DIELTJES
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Table+. Isolation of mycoplasmas from 60 live calves from which both a nasal swab specimen and lung lavage fluid were collected Type of specimen
%, Specimens positive for: M.
M. di,uersum dispur
M. M. bovirhinis bovis
37 65
87 93
U.
M.
M. cunis
boviarginini genitulium
~
Nasal swab Lung lavage fluid
53 75
5 5
7 3
8 7
-
3
Table 5. Efficacy ot three culture media for the isolation of U. diversum from 236 respiratory tract specimensA
No. of qpeciniens
Growth of specimens in medium HAU NHU
A713 55 4 6 25 4 23 119
I Only specimens t h x were inoculated into the three media are included; A7B = agar medium (25); H A U = liquid medium according to FRIISand KROCH (12); according to SHEPARDand COMBS N H U = liquid medium p H 6.0 according to FRIIS et al. (13); + = U.dsversum isolated; - = I/. dsversum not isolated.
Table 6. Growth promoting capacity of two liquid media for the isolation of U . dzversum from 26 nose specimens and 98 lung specimen9 Type of specimen/ Medium Nose HAU
NHU
% Specimen5 with no growth
% Specimens with endpoints of growth at a dilution of: lo-' 10-2 lo-' 10-4 10-5 lo-"
46 K
4 31
12 27
15 27
15 4
8 4
-
21 6
3 3
6 15
15 17
16 22
21 15
16 20
-
Lungs
HAU NHU
Abbreviations are defined in the footnotes of Table 5; 2 Ureuplasmu diversum was isolated from each specimen in at least one medium.
Prevalence of Mycoplasmas in the Respiratory Tracts
559
Table 7. Efficacy of two media for the isolation of M . dzspar from 276 respiratory tract specimens. No. of specimens
Growth of specimens in medium: SB NHS
54 62 18 142 ~
~
~~
~
Only specimens that were inoculated into both media are included; NHS = 'medium I' according to FRIISand KROGH (12); SB = selective medium according to FRIIS(11); + = M,dzspar isolated; - = M . dzspar not isolated.
paired specimens from each calf, but M . bovirhinis was isolated as frequently from the nose as from the lungs (Table 4). When the media used t o isolate U.diversurn were compared, the organism was isolated from 28 YO of the specimens inoculated onto A7B agar, from 37 % inoculated into H A U broth, and from 4 6 % inoculated into NHU broth. In total, U.diversum was isolated from 5 0 % of the specimens grown on these 3 media. When U.diversum was isolated from A7B agar, it was also isolated from one or both broth media (Table 5). Both H A U broth and NHU broth contained the same number of U.diversum organisms. Lung specimens contained approximately 100 times more U . diversum organisms than nose specimens (Table 6). M . dispar was isolated from 26 % of the specimens inoculated into NHS broth and from 42 % of the specimens inoculated into SB broth. In total, M . dispur was isolated from 49 % of the specimens grown on these 2 media (Table 7). Both NHS broth and SB broth contained the same number of M . dispur organisms. Lung specimens contained approximately 100 times more M . dispur organisms than nose specimens (Table 8).
Discussion
To study the prevalence of mycoplasmas in the respiratory tracts of pneumonic calves, we used four types of media to isolate these organisms from 323 specimens collected over a 3-year period. Mycoplasmas were isolated from most of the pneumonic calves and 7 species of mycoplasmas were isolated. T w o pathogenic species, U. diversum and M . dispar,
Table 8. Growth promoting capacity of two media for the isolation of M . dzspar from 43 nose specimens and 91 lung specimens. Type of % Specimens specimen/ with no test Medium resultb Nose NHS SB
% Specimens with endpoints of growth at a dilution of: lo-' 10-2 10-3 10-4 10-5 10-6
-
53 9
7 26
19 26
14 7
5 7
2
26
43 15
2 11
3 12
7 22
13 15
18 15
14 9
-
Lungs
NHS SB ~~
~~
Abbreviations are defined in the footnotes of Table 7; a Mycoplasrna dzspar was isolated from each specimen in at least one medium; b Tubes contained growth of M . bovzrhznrs or M . bows, or contained no cultures, or were not tested.
560
TERLAAK,NOOKDERGKAAF and DIELTJES
that had not been isolated before in the Netherlands, were prevalent. The numbers of organisms of the most prevalent species sometimes exceeded 106 organisms per gram specimen (U. diversum, Table 6; M. dispar, Table 8; results for M. bovirbinis and M . bovis are not shown). Although it is well-known that mycoplasmal inhibitors (e. g., lysolecithin) are released in tissue suspensions (26), we concluded that these substances apparently do not harm the viability of mycoplasmas when the tissue suspensions are stored at -70 "C, even when stored for years. Although the lung lavage technique was described by GOURLAY (14) in 1983, it has seldom been used to study the mycoplasmal flora of calf lungs. DEVRIESE et al. (4) and BINDERet al. (1) isolated mycoplasmas from lung lavage fluids collected from calves at a clinic. Because the lavage tube is passed through the nostril of the calf, it has been thought that lung lavage fluid might be contaminated with organisms from the nose. However, we often isolated mycoplasma species from the nose that were not isolated from lung lavage fluids. Thus, we conclude that reliable specimens of the lung flora can be collected from live calves in the field by this method. U . diversum in particular can be isolated more frequently from the lungs than from the nose (Tables 3 and 4). The value of the lung lavage technique for isolating viruses and pulmonary cells as well has already been reported (19). Because M . dispar is fastidious and slow-growing, it is easily overgrown in culture by M . bowirbinis, which also catabolizes glucose, but which grows faster and is easily cultured. Because the latter is often present in specimens that contain M.dispar, FRIIS(11) devised a selective medium to suppress the growth of M. bovirbinis and to support the growth of M . dispar. We prepared this medium and were able to suppress the growth of M.bovirbinis in 62 specimens (Table 7). The presence of M . bowis also may hamper the isolation of M . dispar, because it grows faster than M . dispar and causes a slight acidification in media with o r without glucose, although it does not catabolize glucose (15). Indeed, when specimens contained quantities of M. bovis, both NHS and SB broth media were overgrown by this organism. As a result, we were not able to isolate M.dispar from these specimens. This could be the reason why M . dispar was less prevalent in fattening calves (46 Oh)than in dairy calves (84 Yo). M.bovzs was prevalent only in fattening calves. In addition, M.bovirbinis was less prevalent in fattening calves (57 Yo) than in dairy calves (85 Yo) (Tables 1 and 3). These findings indicate that a higher prevalence of M.bovis is associated with a lower isolation rate of M . dispar and M. bovirbinis. Whether M. dispar and M. bovirbinis are less prevalent when M . bovis is prevalent, however, is not clear. Therefore, we recommend that hyperimmune antiserum against M . bovis should be included with hyperimmune antiserum against M . bovirbinis in the selective medium used to isolate M.dispar, when M.bovis is prevalent. Because in Denmark, where the selective broth for M. dispar was developed, M . bovis still occurs rarely (6), it was not necessary to include M . bowis antiserum in SB broth. Although SB broth enabled us to isolate M . dispar from specimens containing M . bovirinis (Table 7), it slightly inhibited the growth of M. dispar. Whether this phenomenon was caused by the hyperimmune M. bovirhinis antisera or by other medium components is not clear. Despite this finding, however, we conclude that SB broth is a valuable medium for isolating M . dispar. When we used 2 liquid media to isolate U . diversum, the use of a third medium (A7B agar) did not increase the total number of isolates. We conclude from this that the A7B agar medium can be omitted for the primary isolation of U. diversum. It was, however, useful to confirm the growth of U.diwersum in the liquid media. When nose specimens were inoculated onto the agar medium alone, I/. diversum was isolated from only 12 Yo of the 65 nose specimens; whereas when 2 liquid media were added to the agar medium, U . divevsum was isolated from 2 7 % of the specimens. Because lung specimens contained more U. diversum organisms, it was concluded that A7B agar supported the growth of U . diversum only when it was present in quantity. Of the 117 U. diversum isolates, 75 % were obtained from H A U broth and 93 Yo from NHU broth, leading us to conclude that N H U broth was superior for this purpose (Table5). Moreover, because of its low pH, NHU
Prevalence of Mycoplasmas in the Respiratory Tracts
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broth inhibited true bacteria and acidifying Mycoplusma species better than H A U broth. H A U broth, however, is useful for isolating mycoplasmas that hydrolyze arginine, because these organisms generally do not grow in N H U broth. Eighty-two percent of the U . divevsum strains (n = 11) examined with serogroupspecific antisera belonged to serogroup A. These findings are in contrast to those reported by HOWARD and GOURLAY (IS), who found that 81 % of the strains (n = 54) isolated from the respiratory tracts of cattle from 4 countries belonged to serogroup B but only 15 % to serogroup A. Seven strains isolated from calves from 3 herds were identified as M . canis. Since the present study was ended, this species has been isolated from 17 other herds. This finding is notable, because M . cunis has never been reported in cattle before. There were indications that M . canis was transmitted by dogs that licked milk from the noses of the calves after feeding. The identification and characterization of M . canis strains is described elsewhere (28). Although Acholephsma species could have been detected by the methods used, none of these species were isolated. Only a few studies on the mycoplasmal flora of the respiratory tract of pneumonic calves have included methods for isolating U. divevsum and M . dispar (12, 17,21). Another study used in addition to these direct methods also indirect methods to detect the presence of these organisms (20). These studies were conducted in countries other than the Netherlands where U. diversum and M.dispar were also found to be prevalent. M . bovivhinis was often found to be less prevalent, however, than we found it to be. Reliable information on the prevalence of mycoplasmas in a particular herd can be obtained when both lung lavage fluids and nasal swab specimens are collected from 5 pneumonic calves. Although the mycoplasmas isolated from upper respiratory tract specimens may not necessarily be the ones involved in the pneumonic lesions (16), most mycoplasma species in a herd can be detected, even when only nasal swab specimens are tested.
Acknowledgements We thank G. M. ZIMMERfor collecting the specimens, E. A. FREUNDT and H. E K N(the ~ former F A O / W H O Collaborating Centre for Animal Mycoplasmas, Arhus, Denmark) for general advice in culturing mycoplasmas, and N . F. FRIIS(State Veterinary Serum Laboratory, Copenhagen, Denmark) for advice in culturing bovine mycoplasmas.
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