J. Vet. Med. B 39, 610-616 (1992) 0 1992 Paul Parey Scientific Publishers, Berlin and Hamburg ISSN 0931-1793
Central Veterinary Institute, Lelystad, T h e Netherlands
The Nasal Mycoplasmal Flora of Healthy Calves and Cows E. A. TEK LAAK':., J. H. NOORDERGRAAF and E. BOOMSLUITER Address of authors: Central Veterinary Institute, Department of Bacteriology, P. 0. Box 65, 8200 AB Lelystad, The Netherlands
With 4 tables
(Received for publication March 29, 1992)
Summary The nasal mycoplasmal flora of 270 healthy cows from 27 herds in the Netherlands and 35 healthy calves from 7 of these herds was examined. Various methods for isolating mycoplasmas were compared. The prevalence of the various species was as follows: Ureaplasma diversum in 3 (9%) calves; Mycoplasma dispar in 14 (40 %I) calves; M . bovis in 1 (3 '4)calf; M. bovirhinis in 23 (66 'X,) calves and 16 (6 YO)cows; M. bovoculi in 8 (23 YO)calves and 53 (20 YO) cows; M. ranis in 1 (3 'YO) calf; M . equirhinis in 2 (1 O h ) cows; M . conjunctivae in 2 (1 %) cows; Acholeplasma laidlawii in 1 (3 (Yo) calf and 3 (1 %,) cows; and A . axanthum in 7 (3 %) cows. The noses of healthy calves were less frequently colonized by the pathogenic species U . diversum and contained fewer U . diversum and M . dispar organisms than the noses of pneumonic calves. W e concluded that the mycoplasmal flora of calves and healthy cows was quite different and also that cows play only a minor role in the epidemiology of pathogenic mycoplasma species of calves in the Netherlands.
Introduction The prevalence of mycoplasmas in the respiratory tracts of pneumonic calves has been reported frequently (data reviewed by Ross, 1985) [9]. The prevalence of Ureaplasma diversum and Mycoplasrna dispar has been investigated only occasionally, because these species are fastidious and thus difficult to isolate. Recently, we investigated the prevalence of mycoplasmas, including U . diversum and M.dispar, in the respiratory tracts of pneumonic calves in the Netherlands and the results are reported in an accompanying article [Ill. Studies on the prevalence of mycoplasmas in healthy calves and cows are few. Testing healthy adult cattle at 2 slaughterhouses, POLAK-VOGELZANG and BEUVEKY isolated M.equirhinis, which normally only infects horses, from nasal swab specimens collected from 7 cows (n = 112) and 5 cows (n = 64). It was unknown whether the cows had had contacts with horses [7]. Although these findings were mentioned by GOURLAY and HOWARDin their review [6], no one else has since reported this species in cattle. The aim of the present study was to examine the prevalence of mycoplasmas in healthy calves and adult cattle, paying special attention to mycoplasma species, in particular M . equirhinis, that are not specific to cattle. ::.
Corresponding author.
U.S. Copyright Cluarance Center Code Statement:
0931 - 1793/92/3908-0610$02.50/0
The Nasal Mycoplasmal Flora of Healthy Calves and Cows
61 1
Material and Methods Specimens Nasal swab specimens were collected from cattle during 1987. A total of 270 healthy cows (having given birth to at least one calf) from 27 dairy herds were examined. Ten cows were examined from each herd. A total of 35 healthy calves (under 6 months of age) from 7 of these herds were also examined (5 calves from each herd). A swab specimen was collected from one nostril and was immediately inoculated onto 4 mycoplasma agar media; the swab specimen collected from the other nostril was stored in a tube of NHS25 broth. O n the same day the media were brought to the laboratory to be processed further. Mycoplasma reference strains and antisera The following antisera were obtained from Dr. A. A. POLAK-VOGELZANG, National Institute for Public Health, Bilthoven, the Netherlands: hyperimmune rabbit antisera against Acholeplasma laidlawii PG 8, A. axanthum 5-743, M. bovirbinis PG 43, M. bovis Donetta, M. bovoculi M 165169, and M. equirbinis M 432172. Type strains of the mycoplasma species noted above as well as type strains of M . conjunctivae and M. dispar and samples of reference antisera against these strains were obtained from Dr. E. A. FREUNDT of the former FAO/WHO Collaborating Centre for Animal Mycoplasmas, Wrhus, Denmark. M. bovzrhinis AFX was isolated by us and cloned three times according to the method of FREUNDT et al. [3]. Hyperimmune antisera against this strain, M. conjunctivae H R C 581, M. dispar 462/2, and M. bovirhinis PG 43 were raised in rabbits by the method of FRIIS[4]. M . canis PG 14 and hyperimmune rabbit antiserum directed against this strain were obtained National Institute of Allergy and Infectious Diseases, Frederick Cancer from Dr. J. G. TULLY, Research Facility, Frederick, Maryland, the United States. Mycoplasma media Four types of culture media were used. The first 3 listed here are described in an accompanying article [ 111: 1. Solid modified EDWARD medium for less fastidious mycoplasma species (medium B); 2. Liquid FRIISmedia (NHS25 broth. NHS20 broth, and SB broth) and solid F R I I Smedium for M. dispar; 3. Liquid FRIISmedium N H U p H 6.0 for U . diversum. T o grow colonies, we inoculated liquid media with growth suspected of being U. diversum onto A7B differential agar medium, 4. Solid medium C for Acholeplasma species, which had the same composition as medium B, hut the yeast extract was omitted and the serum content was reduced to 2.5 % (E. A. FREUNDT and H . E R N 0 , personal coinmunication, 1983). Isolation and identification of mycoplasmas One nasal swab specimen collected from each animal was inoculated onto 1 agar plate B, 1 agar plate C, and 2 FRIISagar plates. One FRIISagar plate was incubated in an atmosphere of 5 % CO2 and ' N2. The other 3 agar plates were incubated in a C 0 2 incubator in an atmosphere of 7 "% CO, in 95 % air. The nasal swab specimen that was stored in NHS25 broth was serially diluted tenfold to or 10-6 in NHS25 broth, SB broth, and N H U p H 6.0 broth. All media were incubated at 37°C. Subcultures of the N H U broth medium were made on A7B agar medium. Ureuplasma colonies grown on A7B agar medium were not serotyped, but were regarded to be U . diverrum. Strains suspected of being Acholeplasma species were tested for their resistance to digitonin to distinguish them from Mycoplasma species [2]. Acholeplasma species were identified by the indirect immunofluorescence test and growth inhibition test. Further procedures for isolation and identification of mycoplasmas are described in an accompanying article [11].
Results
All of the mycoplasma strains isolated were identified. Seven mycoplasma species were isolated from the calves. M . dispar and M . bovirhinis were the most prevalent species (Table 1). Six mycoplasma species including M . eguirhznis were isolated from the COWS. M . bovoculi was the most prevalent species; u.diversum and M . dispar were not isolated (Table 2 ) .
612
TERLAAK,NOORDERGRAAF and BOOMSLUITER
Table 1 . Prevalence of mycoplasmas isolated from nasal swab specimens collected from 35 healthy calves originating from 7 herds. Herd no.'>
I/.
diversum 6 7 9 11 13 14 15
No. of calves positive for:. M. M.
M. dispar
M . bovirhinis
bovoculi
A.
laidlawii
2 2
1
5 1
-
1
-
1
5
1
40 %
66 Yo
23 '/o
4 1
M. canis
-
5 5
1 3 4
Total
bovis
3
3 Yo
Five calves were examined from each herd. The herd numbers correspond with those of Table2. M . conjunctivae, M. equrrhinis, and A. axanthum were not isolated.
')
L
Table 2. Prevalence of mycoplasmas isolated from nasal swab specimens collected from 270 healthy cows originating from 27 herds. Herd no.
M. bovirhirris
M. bovoculi
No. of cows positive for:b M. M. conjunctivae equirhinis
A. laidlawii
axanthum
1 %,
3 Yo
A.
1
2 3 4 5 6
1
7
-
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
1 1
Total
1 1
-
2 3
1
2 1 1 2 1
1 9 2 2 3 1 1 3 -
4 8
4
8
6 %,
20 Yo
1 Yo
1 Y"
Ten cows were examined from each herd. U . diversum, M . dispar, M . bovis, and M. canis were not isolated.
I)
613
The Nasal Mycoplasmal Flora of Healthy Calves and Cows Table 3. Endpoints of growth of mycoplasma species in NHS 25 liquid medium.
No. of specimens with endpoints Mycoplasma species/ Source of specimen
U. diversurn calved M . dispar calves calves. M . bovirhinis calves cows M . bovoculr calves cows A . axanthurn cows A . laidlawzi calves cows
of growth at a dilution of: 10-2 10-3
Central Veterinary Institute, Lelystad, T h e Netherlands
The Nasal Mycoplasmal Flora of Healthy Calves and Cows E. A. TEK LAAK':., J. H. NOORDERGRAAF and E. BOOMSLUITER Address of authors: Central Veterinary Institute, Department of Bacteriology, P. 0. Box 65, 8200 AB Lelystad, The Netherlands
With 4 tables
(Received for publication March 29, 1992)
Summary The nasal mycoplasmal flora of 270 healthy cows from 27 herds in the Netherlands and 35 healthy calves from 7 of these herds was examined. Various methods for isolating mycoplasmas were compared. The prevalence of the various species was as follows: Ureaplasma diversum in 3 (9%) calves; Mycoplasma dispar in 14 (40 %I) calves; M . bovis in 1 (3 '4)calf; M. bovirhinis in 23 (66 'X,) calves and 16 (6 YO)cows; M. bovoculi in 8 (23 YO)calves and 53 (20 YO) cows; M. ranis in 1 (3 'YO) calf; M . equirhinis in 2 (1 O h ) cows; M . conjunctivae in 2 (1 %) cows; Acholeplasma laidlawii in 1 (3 (Yo) calf and 3 (1 %,) cows; and A . axanthum in 7 (3 %) cows. The noses of healthy calves were less frequently colonized by the pathogenic species U . diversum and contained fewer U . diversum and M . dispar organisms than the noses of pneumonic calves. W e concluded that the mycoplasmal flora of calves and healthy cows was quite different and also that cows play only a minor role in the epidemiology of pathogenic mycoplasma species of calves in the Netherlands.
Introduction The prevalence of mycoplasmas in the respiratory tracts of pneumonic calves has been reported frequently (data reviewed by Ross, 1985) [9]. The prevalence of Ureaplasma diversum and Mycoplasrna dispar has been investigated only occasionally, because these species are fastidious and thus difficult to isolate. Recently, we investigated the prevalence of mycoplasmas, including U . diversum and M.dispar, in the respiratory tracts of pneumonic calves in the Netherlands and the results are reported in an accompanying article [Ill. Studies on the prevalence of mycoplasmas in healthy calves and cows are few. Testing healthy adult cattle at 2 slaughterhouses, POLAK-VOGELZANG and BEUVEKY isolated M.equirhinis, which normally only infects horses, from nasal swab specimens collected from 7 cows (n = 112) and 5 cows (n = 64). It was unknown whether the cows had had contacts with horses [7]. Although these findings were mentioned by GOURLAY and HOWARDin their review [6], no one else has since reported this species in cattle. The aim of the present study was to examine the prevalence of mycoplasmas in healthy calves and adult cattle, paying special attention to mycoplasma species, in particular M . equirhinis, that are not specific to cattle. ::.
Corresponding author.
U.S. Copyright Cluarance Center Code Statement:
0931 - 1793/92/3908-0610$02.50/0
The Nasal Mycoplasmal Flora of Healthy Calves and Cows
61 1
Material and Methods Specimens Nasal swab specimens were collected from cattle during 1987. A total of 270 healthy cows (having given birth to at least one calf) from 27 dairy herds were examined. Ten cows were examined from each herd. A total of 35 healthy calves (under 6 months of age) from 7 of these herds were also examined (5 calves from each herd). A swab specimen was collected from one nostril and was immediately inoculated onto 4 mycoplasma agar media; the swab specimen collected from the other nostril was stored in a tube of NHS25 broth. O n the same day the media were brought to the laboratory to be processed further. Mycoplasma reference strains and antisera The following antisera were obtained from Dr. A. A. POLAK-VOGELZANG, National Institute for Public Health, Bilthoven, the Netherlands: hyperimmune rabbit antisera against Acholeplasma laidlawii PG 8, A. axanthum 5-743, M. bovirbinis PG 43, M. bovis Donetta, M. bovoculi M 165169, and M. equirbinis M 432172. Type strains of the mycoplasma species noted above as well as type strains of M . conjunctivae and M. dispar and samples of reference antisera against these strains were obtained from Dr. E. A. FREUNDT of the former FAO/WHO Collaborating Centre for Animal Mycoplasmas, Wrhus, Denmark. M. bovzrhinis AFX was isolated by us and cloned three times according to the method of FREUNDT et al. [3]. Hyperimmune antisera against this strain, M. conjunctivae H R C 581, M. dispar 462/2, and M. bovirhinis PG 43 were raised in rabbits by the method of FRIIS[4]. M . canis PG 14 and hyperimmune rabbit antiserum directed against this strain were obtained National Institute of Allergy and Infectious Diseases, Frederick Cancer from Dr. J. G. TULLY, Research Facility, Frederick, Maryland, the United States. Mycoplasma media Four types of culture media were used. The first 3 listed here are described in an accompanying article [ 111: 1. Solid modified EDWARD medium for less fastidious mycoplasma species (medium B); 2. Liquid FRIISmedia (NHS25 broth. NHS20 broth, and SB broth) and solid F R I I Smedium for M. dispar; 3. Liquid FRIISmedium N H U p H 6.0 for U . diversum. T o grow colonies, we inoculated liquid media with growth suspected of being U. diversum onto A7B differential agar medium, 4. Solid medium C for Acholeplasma species, which had the same composition as medium B, hut the yeast extract was omitted and the serum content was reduced to 2.5 % (E. A. FREUNDT and H . E R N 0 , personal coinmunication, 1983). Isolation and identification of mycoplasmas One nasal swab specimen collected from each animal was inoculated onto 1 agar plate B, 1 agar plate C, and 2 FRIISagar plates. One FRIISagar plate was incubated in an atmosphere of 5 % CO2 and ' N2. The other 3 agar plates were incubated in a C 0 2 incubator in an atmosphere of 7 "% CO, in 95 % air. The nasal swab specimen that was stored in NHS25 broth was serially diluted tenfold to or 10-6 in NHS25 broth, SB broth, and N H U p H 6.0 broth. All media were incubated at 37°C. Subcultures of the N H U broth medium were made on A7B agar medium. Ureuplasma colonies grown on A7B agar medium were not serotyped, but were regarded to be U . diverrum. Strains suspected of being Acholeplasma species were tested for their resistance to digitonin to distinguish them from Mycoplasma species [2]. Acholeplasma species were identified by the indirect immunofluorescence test and growth inhibition test. Further procedures for isolation and identification of mycoplasmas are described in an accompanying article [11].
Results
All of the mycoplasma strains isolated were identified. Seven mycoplasma species were isolated from the calves. M . dispar and M . bovirhinis were the most prevalent species (Table 1). Six mycoplasma species including M . eguirhznis were isolated from the COWS. M . bovoculi was the most prevalent species; u.diversum and M . dispar were not isolated (Table 2 ) .
612
TERLAAK,NOORDERGRAAF and BOOMSLUITER
Table 1 . Prevalence of mycoplasmas isolated from nasal swab specimens collected from 35 healthy calves originating from 7 herds. Herd no.'>
I/.
diversum 6 7 9 11 13 14 15
No. of calves positive for:. M. M.
M. dispar
M . bovirhinis
bovoculi
A.
laidlawii
2 2
1
5 1
-
1
-
1
5
1
40 %
66 Yo
23 '/o
4 1
M. canis
-
5 5
1 3 4
Total
bovis
3
3 Yo
Five calves were examined from each herd. The herd numbers correspond with those of Table2. M . conjunctivae, M. equrrhinis, and A. axanthum were not isolated.
')
L
Table 2. Prevalence of mycoplasmas isolated from nasal swab specimens collected from 270 healthy cows originating from 27 herds. Herd no.
M. bovirhirris
M. bovoculi
No. of cows positive for:b M. M. conjunctivae equirhinis
A. laidlawii
axanthum
1 %,
3 Yo
A.
1
2 3 4 5 6
1
7
-
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
1 1
Total
1 1
-
2 3
1
2 1 1 2 1
1 9 2 2 3 1 1 3 -
4 8
4
8
6 %,
20 Yo
1 Yo
1 Y"
Ten cows were examined from each herd. U . diversum, M . dispar, M . bovis, and M. canis were not isolated.
I)
613
The Nasal Mycoplasmal Flora of Healthy Calves and Cows Table 3. Endpoints of growth of mycoplasma species in NHS 25 liquid medium.
No. of specimens with endpoints Mycoplasma species/ Source of specimen
U. diversurn calved M . dispar calves calves. M . bovirhinis calves cows M . bovoculr calves cows A . axanthurn cows A . laidlawzi calves cows
of growth at a dilution of: 10-2 10-3
