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Isolation of mycoplasmas from milk, lungs and prepuce of cattle
Comp. lmmun. MicrobioL infect. Dis., Vol. 2, pp. 447457 ~) Pergamon Press Ltd., 1980. Printed in Great Britain
0147-9571/79/1201~447 $02.00/0
ISOLATION OF MYCOPLASMAS FROM MILK, LUNGS AND PREPUCE OF CATTLE NATALIA ZALEWSKA-SCHONTHALER Laboratory of Veterinary Hygiene in Warsaw, Lechicka 21, 02-156, Warsaw, Poland
bovis was mainly isolated from 6.25~o of milk samples collected from cows with clinical and sub-clinical mastitis. The main mycoplasma strain isolated from the respiratory tract of calves with symptoms of bronchopneumonia was M. bovirhinis (12.1~o of samples). M. bovigenitalium was most frequently recovered from bull's prepuce (67.1~o of samples). Key words: Mycoplasma sp., Acholeplasma sp., bronchopneumonia, genital tract, mastitis, antibiotics Abstract--M.
ISOLATION DES MYCOPLASMES DU LAIT, DES POUMONS ET DU PREPUCE DU BETAIL R/~sum~-M. boris a surtout &teisole d'echantillons de lait (6,25~) provenant de vaches atteintes de mammites cliniques ou sub-cliniques. Les principales souches de mycoplasma isolees de l'appareil respiratoire de veaux presentant des sympt6mes de bronchopneumonie appartenaient fi l'esp~ce M. bovirhinis (12,1~ des echantillons). Enfin, M. bovigenitalium fut le plus souvent isole de prepuces de taureaux (67,1~o des &:hantillons). Mots-clefs: Mycoplasma sp., Acholeplasma sp., bronchopneumonie, appareil g6nital, mammite, antibiotiques
INTRODUCTION Examinations o f calves with s y m p t o m s o f respiratory tract disease have been carried out in Poland by Truszczyfiski and Pilaszek [1] and N o w a k o w s k i [2, 3]. Their studies have revealed the presence o f M . bovirhinis, M . dispar, M . boris, M . bovQenitafium, A. laidlawii, and A. modicum in those animals. N o w a k o w s k i [3] identified m y c o p l a s m a s in 50~o o f all examined swabs from 1 to 2.5-month-old calves with s y m p t o m s o f the respiratory system disease. G o u r l a y [4] has reported similar results in France. Stipkovits et al. [5] isolated A. axanthum in Hungary. In Italy, Galassi[6, 7] observed 21 outbreaks o f m y c o p l a s m a respiratory tract infection. Infections o f the genital tract o f bulls were reported by Zg6rniak-Nowosielska et al. [8, O] in Poland and by E d w a r d et al. [42], Blom and E r n o [11] in other countries. Zg6rniak-Nowosielska et al. [8, 9] have isolated M . bovigenitalium, A. laidlawii, and A. arginini from semen o f those bulls which did not show clinical symptoms. Similar results were obtained by E r n o [12] in D e n m a r k and J u r m a n o v a [13] in Czechoslovakia. O n o v i r a n et al. [14] have isolated M. bovigenitalium, A. laidlawii, Mycoplasma sp. and strains o f ureaplasma from penial-preputial swabs. M. agalactiae, A. laidlawii a n d A. a x a n t h u m [15-20] have been so far isolated from cows with mastitis symptoms. In Poland, m y c o p l a s m a s were not isolated from milk o f cows suffering from mastitis. 447
448
NATALIA ZALEWSKA-SCHONTHALER
The purpose of this research was to verify the possibility of isolation and classification of mycoplasmas from : cow's milk, washings of bull prepuce, and from the lungs of calves suffering from bronchopneumonia, in central Poland.
MATERIALS AND METHODS
Reference strains of mycoplasmas In this study we used six reference strains: M. bovigenitalium, M. bovis, M. bovirhinis, M. arginini, A. laidlawii and A. granularum supplied by the Veterinary Institute in Pu|awy, Poland, and one Staphylococcus aureus 209 P strain regarded as a standard for antibiotic sensitivity test.
Media On the basis of the preliminary examination of media, the two-phase medium for isolation of mycoplasmas from milk was prepared as described by Wenzl [21]. VF-1 medium prepared as described by Barber and Fabricant [22] was used for isolation and cultivation of the lung tissue and washings of the bull prepuce. The medium was supplemented with 5.5~ of yeast extract and with 0.01~ NAD (nicotinamidadenindinucleotid). The E medium of Roberts and Pojoan [23] was also used. For mycoplasma isolation and identification from milk according to the method proposed by Wenzl [21] milk samples were diluted 1 : 2 in saline solution before inoculation of the medium. To isolate mycoplasmas from lung tissue and from washings of bull's prepuce, 'millipore' membrane filters (650 nm) were employed. Morowitz et al. [24] and Erno and Stipkovits [25] used those filters for experimental investigations of mycoplasma size determination.
Samples 1180 samples of milk including 64 samples taken from cows with clinical and sub-clinical form of mastitis were tested; also one sample of mammary gland and 263 samples of lungs of calves suffering from bronchopneumonia were examined. Seventy-three samples of prepuce washings from bulls without any symptoms of disease were tested. Identification of 87 isolated strains was based on physiological and biochemical tests described by Whittlestone [26], A1-Aubaidi and Fabricant [27] and Erno and Skipkovits [25, 28]. Growth inhibition test (GIT) by Clyde [29] was used for identification of mycoplasma and acholeplasma strains. Sera for growth inhibition test were prepared as described by Morton and Roberts [30] in Pilaszek's modification [31]. In this modification purified mycoplasma antigen was injected into rabbit's foot pat at days 10, 16 and 22 after immunization. This method showed the highest titres of antibodies. Sensitivity of the isolated strains to: streptomycin, oxyterracyclin, chloramphenicol, erythromycin, neomycin and sulphathiasol were determined using the paper disc sensitivity test.
Isolation of mycoplasmas from milk, lungs and prepuce of cattle
449
RESULTS Results of mycoplasma isolation from milk samples from cows with sub-clinical symptoms were shown in Table 1. The data presented in Table 1 showed that all five strains of mycoplasma were isolated from the clinical or sub-clinical form of mastitis. Five mycoplasma strains were isolated together with other bacterial flora, and there was only one case when mycoplasma M 453 strain alone was isolated. Table 2 presented the main serological, biochemical and physiological characteristics of mycoplasma and acholeplasma strains which were isolated from the specimens of the mammary gland secretion and mammary gland tissue of cow's udder. As shown in Table 2, two out of six isolated strains were classified as M. bovis. Both strains of M. bovis showed variable fermentation analogous to the reference strain of M. bovis. Four remaining strains showed physiological reaction characteristic of acholeplasmataceae family. Only one of those strains M 453 was serologically identified as A. laidlawii. Strains M 51 and M 1695 were temporarily classified as A. axanthum, and this should be confirmed by serological investigations. The last of the isolated strains M 2242 was not active biochemically and was not inhibited by the used sera. The strain was designated as A. species. The results o f mycoplasmas and other bacterial isolations from lung samples were presented in Table 3. The results showed that 32 strains of mycoplasma were isolated from mentioned samples. Apart from mycoplasmas, other micro-organisms were isolated in 56.2'~o; they were Pasteurella multocida, Pasteurella haemolytica, Corynebacterium pyogenes and Escherichia coli in lower percentage. Mycoplasmas, as the only microorganisms, were isolated from lungs in 43.8~o but virus infection was not taken into consideration. Table 4 presents the most important characteristics (serological, biochemical and physiological) of the mycoplasma and acholeplasma strains isolated from the lungs of the calves. The presented data revealed that 56.3~o of the isolated strains belonged to M. bovirhinis, 9.4~o to M. boris, 6.2~o to M. bovigenitalium, 9.4~ to A. laidlawii. Physiological reactions of the two remaining strains were typical of the mycoplasmataceae family and therefore they were classified as M. species. Twelve and a half per cent of the strains were not determined because they failed to passage. Isolation of the mycoplasma and acholeplasma strains and other micro-organisms from washings of bull's prepuce were presented in Table 5. In 49 samples from which
Table 1. Isolation of mycoplasmas and other bacteria species from milk of cows with different forms of the m a m m a r y gland infection Strain of mycoplasma No. M M M M M M
2444 37 453* 51 1695 2242
Other bacteria species
The total number of cell elements
Micrococcus Micrococcus Streptococcus agalactiae Micrococcus Corynebacterium pyogenes
* First sample No. M 453 is a portion o f the m a m m a r y gland.
1,200,000 1,300,000 not examined 5 million 5 million 5 million
Form of infection sub-clinical sub-clinical clinical clinical clinical clinical
+ -
-
+ +
-
+
+
.+) ,+) + + + ,+ ,+ + +(6d) +(9d)
-
-
+ (9 d)
-
+ +
+
Biochemical reactions* Sugar fermentation Glucose Xylose Cellobiose
+ , Sensitivity to a n t i s e r u m : , resistance to a n t i s e r u m : d. days: ( + ) , variable results. * N o n e of the m e n t i o n e d strains did lerment m a n n o s e , arginine, urea, sorbitol. ++ K-group as described by Jawad, A I - A u b a i d i and F a b r i c a n t (A. avanthzvn). Reference strains.
M2444 M37 M 453 M51 M 1695 M2242 M. hovi~§ .4. laidlawii§
Strain No,
Serological reactions a n t i s e r u m M. boviM. boviM. hovi.~ ,v,enitalium rhini~ A. laidklwii
+
+
+
+ +
+
-
+
+
+ +
+
-
-
Physiological reactions Growth Growth without in serum 21 C
-
+
+
Sensitivity to digitonin
-
+(14d)
-
+ ( 1 4 d)
+(14d)
Film and spots formation
A ~pe~'ie.~ M . bo vi,~ A. lahtlawii
K{ K¢
M. bovL; M. bo vi,s A. klhlhtwii
Species
Table 2. Classification of the main serological, b i o c h e m i c a l a n d physiological characteristics of m y c o p l a s m a and a c h o l e p l a s m a strains isolated from c o w ' s milk
,-r, :> t--,
,>
7
I s o l a t i o n of m y c o p l a s m a s from milk, lungs a n d prepuce of cattle
451
Table 3. Isolation o f m y c o p l a s m a a n d a c h o l e p l a s m a strains and percentage ratio o f other m i c r o - o r g a n i s m s of calf's lungs N u m b e r of mycoplasm and acholeplasma strains
Species or serogroup
18 3 2 3 2 4
M. bovirhinis M. bovi~ M. bov~,~enilalium A. laidk~wii M..specie~s not classified
Total 32
N u m b e r of strains
E. coil l
P. muhocida
P. haemolvtica
8 1
2 ---
1 1 -1
11
Corvnebacterium pyo~ene~ 2
I
-1
3
3
Per cent of other microorganisms 72.2 33.3 0.0 33.3 50.0 50.0
mycoplasma was isolated, 30.6'~o were bacteria of Campylobacter genus, including 26.5~o of Campylohacter sputorum subspecies sputorum and 4.1~o of Campylobacterfetus subspecies fetus. Table 6 classifies the main serological, physiological and biochemical characteristics of the mycoplasma and acholeplasma strains isolated from the washings of the bull's prepuce. As shown in the table, the strains were identified as M. bov(genitalium in 53.1j°~,, A. laidlawii in 16.3~0, M. bovis in 2°~. Ten and a third per cent of the strains were not classified because it was not possible to passage them. Results of the isolation of mycoplasmas from the respective specimens and their percentage ratio are given in Table 7. Out of 1180 milk specimens and one mammary gland portion six strains (0.5~,i) of mycoplasmas were isolated. Out of 264 specimens of call's lung 32 strains (12.1°~i) of mycoplasmas were isolated. Out of 73 specimens of the washings of bull's prepuce, 49 strains (67.1°o) of Myeoplasma sp. and Acholeplasma sp. were isolated. Results concerning the sensitivity of 47 strains of Mycoplasma sp. and Aeholeplasma sp. and four laboratory strains to antibiotics and sulphathiasol were presented in Table 8. The strains for antibiotic sensitivity test were randomly chosen. All of the strains showed 1001'i, sensitivity to chloramphenicol. All the examined M. bovigenitalium strains were resistant to erythromycin, and from the remaining strains 37.2'~0 of them were sensitive to this antibiotic. Fifty-three per cent of the examined strains were sensitive to neomycin. All of the examined strains were resistant to sulphathiasol. DISCUSSION The use of "millipore' membrane filters with 650-nm mycoplasmas from lung homogenisate and Campylobaeter prepuce as described by Morowitz et al. [24] and Samol and eliminate the overgrowth of other micro-organisms in particularly that of Proteus sp. Isolation of M. bovis and A. infected mammary gland confirmed the studies of Karbe
pores for the isolation of sp. from washings of bull's Korpolifiska [32], allowed to the majority of specimens, laidlawii from milk and from and Mosher [17], Jasper et
( 1)
3
( 1)
2 -( 1)
(2)
3
A. laidlawii
3 --
13 l(-, +)
--
3
3(8d)
Biochemical reactions* fermentation of sugar Glucose Cellobiose
1 1
2
18 3 -3 --
Growth without serum
--
3
--
2
2(14 d) 3(12d)
Film and spots formation
M . bovirhinis M . bovis M . bovi,g e n i t a l i u m A . laidlawii M . ~pecies M . ~'pecie,~
Species or serogroup
3.1 3.1
56.3 9.4 6.2 9.4
Per cent of the strains mycoplasma and acholeplasma
12.5°i; were not tabulated because they failed to
Growth in 21 C
Physiological reactions
* None of the presented strains did ferment mannose, sorbitol, arginine, urea, xylose: d, day. Figure in brackets, doubtful reactions, inhibition zone in m m < 3 m m : ( , +), variable results: four strains passage.
1
2 3 1
18
18 3
M . bovi.s
M . bovi,~enitalium
Serological reaction antiserum
M. bovirhini.s
Strain No.
Sensitivity to digitonin
Table 4. Classification of the main serological, biochemical and physiological characteristics of mycoplasma and acholeplasma strains from the calf's lungs
©: Z
6~
N
>.
Z
t,3
Isolation of mycoplasmas from milk. lungs and prepuce of cattle
453
Table 5. Isolation ol mycoplasma and acholeplasma strains and percentage ratio of other micro-organisms recovered from the washings of bull's prepuce Isolated mycoplasma and acholeplasma strains Number Per cent 16 1 8 14
53. I 2.(I 16.3 28.6
Total 49
100.0
Species or serogroup Mycoplasma Acholeplasma M.bov(~enimlium M. bovi~
Campvh~bacter Campyh~bacter /etu.~ .~pulorum ~. /em~ ~. hubulu~
2
A. laidlawii not classified
2
8
Per cent o1" other microorganisms
3 2
20.4 0.0 6.1 4.1
13
30.6
al. [16] and, Gourlay and Scott [10]. On the basis of their biochemical characteristics the two strains isolated from milk were classified as A. axanthum in our study. Our results correspond with observations of Whenert et al. [20] who have also isolated A. axanthum from milk. It is, however, necessary to confirm these results by serological tests. Isolation of Mycoplasma sp. and Acholeplasma sp. in 6.25~, of milk samples obtained from cows with clinical and sub-clinical mastitis suggests that like in West Germany or Japan there is mycoplasma infection of bovine mammary gland in Poland, as well. We suspect that mycoplasmas can be one of the etiological agents in these infections. It should be pointed out that the isolation ofA. laidlawii from the mammary gland was associated with leukemic changes in the mammary tissue. Mycoplasmas were isolated together with other micro-organisms from milk by Pan and Ogata [19]; this is in agreement with the results of our research. Two strains of mycoplasmas were isolated from milk samples in which the number of cell elements was higher than 1,200,000 per ml. In both cases bacteria of Micrococcus sp. were isolated from the milk samples. Three strains were isolated from milk of cattle suffering from the clinical form of mastitis but other bacteria like Streptococcus agalactiae, Micrococcus sp. and Col3,nebacterium pyogenes were also isolated from these samples.
Strains M. boris isolated from the milk and the reference strain which were subjected to fermentation reacted variably to glucose; this is confirmed by observations of Edward and Moore [33], Leach [18] and other authors. The majority of authors suggested that M. boris does not ferment glucose. Edward and Moore [33] showed that the lowering of the pH was caused by horse serum or yeast extract added to culture medium rather than by glucose fermentation by M. bovis. In our studies the level of glucose in culture medium was not determined. As suggested by Edward and Moore [33] this may explain the positive fermentation observed in our studies. Results of mycoplasma isolation from the mammary gland tissue of cows udder in Poland (6.25~ of the clinical cases of mastitis) differ from the data reported by Pan and Ogata [19] in Japan. The authors isolated mycoplasmas from 78~o of all examined milk samples. A1-Aubaidi and Fabricant [27] isolated mycoplasmas from milk samples in 40~o. Results of isolation of mycoplasmas from 12.1~ of cases of pneumonia are generally in accordance with the results obtained by Galassi [6, 7], Nowakowski [3] and also by Pilaszek and Truszczyfiski [34]. As in the studies of Shimizu et al. [35], Mengik and Jurmanova [36] and Gossger [37], M. bovirhinis was isolated in the highest percentage. 16
-
8
---
2(10
8
d)
"
8
Biochemical reactions* fermentation of sugar Glucose Cellobiose
7
1
26
-"
8
Growth without serum 26 l(12d)
Film and spots fc,rma!ion
examined specimens 1180 264 73
Organ
Mammar~ gland Lungs Prepuce
2 3 1
M. bovi.~
2 26
M. hovi~,,enilalium
18
M. hovirhini~
I 3 8
3 6 14
A. laidlawii Unclassified
Isolated specimens
0.5 12.1 67.1
Per cent
Table 7. Isolation of mycoplasma and acholeplasma strains from the cattle of central Poland Number of
Total
M. bovivenitalium M. hovi.~ A. iaMlawii M. .~pe{qe~ M. v~ecie~
Species or serogroup
89.7
53.1 2.0 16.3 14.3 4.0
Per cent of the strains mycoplasma and acholeplasma
10.3". were not tabulated because they tailed to passage.
."
8
--
Growth in 21 C
Physiological reactions
* None of the presented strains did ferment mannose, sorbitol, arginine, urea, xylose: d, days: five strains
_"~
7
8
26 --
1
1
Serological reactions antiserum M. M. hoviA. hovirhini~ M. hovi.~ .~enitalium h&llawii
26
Strain No.
Sensitivity to digitonin
Table 6. Classification of the main serological, biochemical and physiological characteristics of mycoplasma and acholeplasma strains isolated from the washings of bull's prepuce
3
~=.
,>
> t...>~ > N ~r"
4~
96.6 100
15 11
51
Total
11.8
3.4 0
0 27.8
ms
Streptomycin
3.9
0 0
14.3 0
ns
70.6
100 72.8
100 34.4
s
27.4
0 18.1
0 66.6
ms
2.0
0 9.1
0 0
ns
100
100 100 0
0 0
0 0
ms
0
0 0
0 0
ns
Chloramphenicol
100 100
s
37.2
0 100
17.7
0 0
85.7 16.7
ms
0 44.4
ms
45.1
100 0
Erythromycin
14.3 38.9
s
Per cent of strains with different sensitivity Oxytetracycline
0
0 0
0 0
s
s, sensitivity > 30 mm: ms, medium sensitive 27 24 mm: ns, non-sensitive ~< 23 mm. The examined strains were resistant to sulphathiasol.
84.3
87.5 72.2
s
7 18
strain
strains
M. bovL~ M. bovirhinis M. bov~eenitalium A. laidlawii
of
acholeplasma
Mycoplasma and N u m be r
Table 8. Sensitivity to antibiotics of mycoplasma and acholeplasma strains isolated from cattle
53.0
0 81.8
71.4 61.2
ms
Neomycin
47.0
100 18.2
28.6 38.8
ns
t~
g
o
a"
(3
,-,.,
7
456
NATALIA ZALEWSKA-SCH()NTHALER
G a l a s s i [6] i s o l a t e d - - f r o m l- to 2 - w e e k - o l d c a l v e s - - M , bovis in 58.5'~/o o f cases, M . bovigenitalium in 7.4~,q, a n d M . arginini in 4.2~o o f cases. Stipkovits et al. [5] isolated A. a x a n t h u m from lungs, G e o r g e et al. [38] M . dispar. Oghiso Y o i c h o et al. [39], however, received quite different data. T h e y isolated T - m y c o p l a s m a from lungs o f calves suffering from p n e u m o n i a in 9 0 ~ o f cases, a n d M . bovirhinis a n d M . bovigenitalium in lower percentage. Similarly to o t h e r a u t h o r s [4, 40] while e x a m i n i n g by the g r o w t h inhibition test we o b s e r v e d few c r o s s - r e a c t i o n s between them. The c r o s s - r e a c t i o n was o b s e r v e d between M . bovirhinis antigen a n d M . bovigenitalium sera, but also between M . boris a n d M . bovigenitalium sera. M y c o p l a s m a s were recovered in 67.1% from bull's p r e p u c e washings. Similarly, high p e r c e n t a g e - - 4 7 . 3 ~ o o f m y c o p l a s m a isolation from bull's semen in P o l a n d - - w a s r e p o r t e d by Z g 6 r n i a k - N o w o s i e l s k a a n d B r a n n y [8]. T h e a u t h o r s isolated m a i n l y M . bovigenitalium a n d A. laidlawii a n d M . bovis to a lesser extent. This is in a g r e e m e n t with o u r results. L a n g f o r d [41] a n d O n o v i r a n et al. [14] isolated m y c o p l a s m a s from p e n i a l - p r e p u t i a l washings in 65.9~o, f r o m semen in 34.9~o. The a u t h o r s n o t e d higher frequency o f m y c o p l a s m a isolation from the p e n i a l - p r e p u t i a l washings t h a n from bull's semen. This is also in a c c o r d a n c e with o u r a n d o t h e r a u t h o r s ' r e s u l t s . However, studies c a r r i e d out so far, d o n o t solve the m a i n p r o b l e m , i.e. the role o f m y c o p l a s m a s in the genital tract o f ill bulls. This p r o b l e m is still open for discussion. The results o f tests for m y c o p l a s m a sensitivity to antibiotics a n d s u l p h a t h i a s o l r e p o r t e d by N o w a k o w s k i [3] were not c o m p a r a b l e with o u r findings. O u r tests in vitro s h o w e d 100~o sensitivity to c h l o r a m p h e n i c o l a n d 8 4 . 3 ~ sensitivity to s t r e p t o m y c i n , while in N o w a k o w s k i ' s [3] studies the s a m e p e r c e n t a g e o f m y c o p l a s m a strains were sensitive to streptomycin and chloramphenicol.
REFERENCES 1. Truszczyflski, M. and Pilaszek, J., Ocena metod izolacji i identyfikacji gatunkow Mycoplasmatales wystgpujocych u byd|a, Medycyna wet. 32, 217 221 (1976). 2. Nowakowski, J., Wyosobnienie mykoplazm od cielRt z odoskrzelowym zapaleniem ptuc, Medycyna wet. 27, 403 404 (1971). 3. Nowakowski, J., Charakterystyka krajowych szczepow mykoplazm wyosobnionych od bydta. Doctoral thesis, Agriculture Academy, Lublin, Poland (1975). 4. Gourlay, R. N., Significance of mycoplasmas infections in cattle, J. Am. vet. med. A.~s. 163, 905 909 (1973). 5. Stipkovits, L., Schimmel, D. and Varga, L., Occurrence of Acholeplasma axanthum in cattle, Arch. exp. Vet. Med. 28, 559 562 (1974). 6. Galassi, D., Mycoplasma in beef cattle herd. VIII International Conference on Diseases of Cattle, Abstr. Milano 9(133, 17 18 (1974). 7. Galassi, D., Mycoplasmosis in Italian fattening calves rearing, VIII International Meetinq, on Diseases ~[ Cattle, pp. 42 51. T.E.P.-Tipogr. Edit. Piacentino Gallart C Milano (1975). 8. Zgorniak-Nowosielska, I. and Branny, J., Mykoptasmy w nasieniu buhajow. I. Izolacja i niekt6re wla~ciwosci mykoplazm wyst~puj~cych w nasieniu buhajow, Medycyna wet. 1, 51 53 (1970). 9. Zgorniak-Nowosielska, I., Branny, J. and Pastuszak, J., Some characteristics of bovine mycoplasmas, In Vitro 2 (1), 168 174 Pardubice (1973). 10. Gourlay, R. N. and Scott, E. J., Isolation of Mycoplasma agalactiae var. boris and infectious bovine rhinitracheitis virus from an outbreak of mastitis in France, Vet. Rec. 95, 534-535 (19743. 11. Blom, E. and Erno,, H., Infection of the genital organs of bulls, Acta vet. stand. 8, 186 188 (19673. 12. Erno,. H., Bovine mycoplasmas: identification of 100 strains isolated from semen of bulls, Acta vet. stand. 16, 321 323 (1975). 13. Jurmanova, K., Mycoplasmas isolated from the genital tract and semen of bulls, In Vitro 2 (1), 70 75 Pardubice (1973).
Isolation of mycoplasmas from milk, lungs and prepuce of cattle
457
14. Onoviran, O., Truscott, R. B., Fish, N. A., Barker, C. A. V. and Ruhnke, L., The recovery of mycoplasmas from the genital tracts of bulls in artificial breeding units in Ontario, Can. J. comp. Med. 39, 474-475 (1975). 15. Cottew, C. S., Mycoplasmas isolated from cattle in Australia, Aus.t. vet. J. 46, 378 381 (1970). 16. Jasper, D. E., Al-Aubaidi, J. M. and Fabricant, J., Epidemiologic observations on mycoplasma mastitis, Cornell Vet. 64, 407 415 (1974). 17. Karbe, E. and Mosher, A. H., Pathogenese der Mycoplasmen Mastitis beim Rind, Zentbl. VetMed. 15, 817-827 (1968). 18. Leach, R. H., Further studies on classification of bovine strains of Mycoplasmatales with proposals for new species Acholeplasma modicum and Mycoplasma alcalescens, J. gen. Microbiol. 75, 135-153 (1973). 19. Pan, J. and Ogata, M., New sero-types of Mycoplasma laidlawii isolated from mastitis milk and urogenital tracts of cattle, II. Typing ofM. laidlawii by biological and serological methods, Jap. J. vet. Sci. 31, 313 323 (1969). 20. Whenert, C., Teichmann, G., Hanke, H., Schimmel, D. and Latzsch, C., Course of Mycoplasma infection of the udder, Mh. Vet.-Med. 32, 55-59 (1977). 21. Wenzl, J., Zum Vorkommen von Mycoplasmen in Kuhmilch, Inaug.-Dissertation zur Erlangung der veterin/irmedizinischen Doktorwfirde der Tier~irztlichen Fakult~it der Ludwig-Maximilian-Universit~it, Miinchen (1971). 22. Barber, T. and Fabricant, J., Primary isolation of mycoplasma organisms (PPLO) from mammalian sources, J. Bact. 83, 1268 1273 (1968). 23. Roberts, D. H. and Pojoan, C., Identification of Mycoplasma hyorhinis, Br. vet. J. 127, 582-586 (1971). 24. Morowitz, H. J., Tourtellotte, M. E. and Pollack, H.E., Use of porous cellulose ester membranes in the primary isolation and size determination of pleuropneumonia-like organisms, J. Bact. 85, 134-135 (1963). 25. Erno, H. and Stipkovits, L., Bovine mycoplasmas: cultural and biochemical studies. I, Acta vet. stand. 14, 43~449 (1973). 26. Whittlestone, P., Isolation methods from Microbiologist, Shapton, D. A. and Gould, G. W. (Eds), pp. 51-61. Academic Press, London and New York (1969). 27. AI-Aubaidi, J. N. and Fabricant, J., Technique for isolation of mycoplasma from cattle, Cornell Vet. 58, 555 571 (1968). 28. Erno, H. and Stipkovits, L., Bovine mycoplasmas: cultural and biochemical studies II, Acta vet. scand. 14, 450 463 (1973). 29. Clyde, W. A., Mycoplasma species identification based upon growth inhibition by specific antisera, J. Immun. 92, 958 965 (1964). 30. Morton, H. E. and Roberts, R. J., Production of anti-Mycoplasma CPPLO) antibodies in rabbits, Proc. Soc. exp. Biol. Med. 125, 538 543 (1957). 31. Pilaszek, J., Personal communication. 32. Sam61, S. and Korpolifiska, M., Zastosowanie filtr6w membranowych w rutynowej diagnostyce mgtwika plodowego, Medycyna wet. 30, 246-249 (1974). 33. Edward, D. G. and Moore, W. B., Determination of utilisation of glucose by mycoplasmas, In Vitro 2(1), 148-155, Pardubice (1973). 34. Pilaszek, J. and Truszczyfiski, M., Metody laboratoryjne w przyzyciowej diagnostyce mykoplazmozy u ciel~t, MedytTna wet. 32, 152 156 (1976). 35. Shimizu, T., Nosaka, D. and Nakamura, N., An enzootic of calf pneumonia associated with Mycoplasma bovirhinis, Jap. J. vet. Sci. 35, 535-537 (1973). 36. Mengik, J. and Jurmanova, K., Occurrence ofMycoplasma bovirhinis in connection with pneumonia of calves. In Vitro 2(1), 56-62 Pardubice (1973). 37. Gossger, K. W., Untersuchung fiber das Vorkommen von Mycoplasmen bei respiratorischen K/ilbererkrankungen unter besonderer Beriiksichtigung der enzootischen Pneumonie der Mastk~ilber. Inaug. Dissertation Tier/irztlischen Fakult~it der Ludwig-Maximilian-Universit/it Mfinchen (1974). 38. George, T. D., Horsfall, N. and Sullivan, N. D., A subclinical pneumonia of calves associated with Mycoplasma di~par, Aust. vet. J. 49, 580-586 (1973). 39. Oghiso, Yoichi, Yamamoto, Koshi, Goto, Naoaki, Takahashi, Reiji and Fuiwara, Kosaku, Pathological studies on bovine pleuropneumonia in special reference to isolation of mycoplasmas, Jap. J. vet. Sci. 38, 15-24 (1976). 40. Erno, H. and Jurmanova, K., Serological studies of bovine mycoplasmas, In Vitro 2(1), 15%167 Pardubice (1973). 41. Langford, E. V., Mycoplasma infections in the bovine genital tract, Can. vet. J. 15, 300-301 (1974). 42. Edward, D. G., Hancock, J. L. and Hignett, S. L., Isolation of pleuropneumonia-like organisms from the bovine genital tract, Vet. Rec. 59, 329-330 (1947). 43. Truscott, R. B., Onoviran, O., Ruhnke, H. D. and Fish, N. A., In vitro antimicrobial sensitivity of mycoplasmas isolated from the bovine genital tract, Can. J. comp. Med. 39, 416-420 (1975).
0147-9571/79/1201~447 $02.00/0
ISOLATION OF MYCOPLASMAS FROM MILK, LUNGS AND PREPUCE OF CATTLE NATALIA ZALEWSKA-SCHONTHALER Laboratory of Veterinary Hygiene in Warsaw, Lechicka 21, 02-156, Warsaw, Poland
bovis was mainly isolated from 6.25~o of milk samples collected from cows with clinical and sub-clinical mastitis. The main mycoplasma strain isolated from the respiratory tract of calves with symptoms of bronchopneumonia was M. bovirhinis (12.1~o of samples). M. bovigenitalium was most frequently recovered from bull's prepuce (67.1~o of samples). Key words: Mycoplasma sp., Acholeplasma sp., bronchopneumonia, genital tract, mastitis, antibiotics Abstract--M.
ISOLATION DES MYCOPLASMES DU LAIT, DES POUMONS ET DU PREPUCE DU BETAIL R/~sum~-M. boris a surtout &teisole d'echantillons de lait (6,25~) provenant de vaches atteintes de mammites cliniques ou sub-cliniques. Les principales souches de mycoplasma isolees de l'appareil respiratoire de veaux presentant des sympt6mes de bronchopneumonie appartenaient fi l'esp~ce M. bovirhinis (12,1~ des echantillons). Enfin, M. bovigenitalium fut le plus souvent isole de prepuces de taureaux (67,1~o des &:hantillons). Mots-clefs: Mycoplasma sp., Acholeplasma sp., bronchopneumonie, appareil g6nital, mammite, antibiotiques
INTRODUCTION Examinations o f calves with s y m p t o m s o f respiratory tract disease have been carried out in Poland by Truszczyfiski and Pilaszek [1] and N o w a k o w s k i [2, 3]. Their studies have revealed the presence o f M . bovirhinis, M . dispar, M . boris, M . bovQenitafium, A. laidlawii, and A. modicum in those animals. N o w a k o w s k i [3] identified m y c o p l a s m a s in 50~o o f all examined swabs from 1 to 2.5-month-old calves with s y m p t o m s o f the respiratory system disease. G o u r l a y [4] has reported similar results in France. Stipkovits et al. [5] isolated A. axanthum in Hungary. In Italy, Galassi[6, 7] observed 21 outbreaks o f m y c o p l a s m a respiratory tract infection. Infections o f the genital tract o f bulls were reported by Zg6rniak-Nowosielska et al. [8, O] in Poland and by E d w a r d et al. [42], Blom and E r n o [11] in other countries. Zg6rniak-Nowosielska et al. [8, 9] have isolated M . bovigenitalium, A. laidlawii, and A. arginini from semen o f those bulls which did not show clinical symptoms. Similar results were obtained by E r n o [12] in D e n m a r k and J u r m a n o v a [13] in Czechoslovakia. O n o v i r a n et al. [14] have isolated M. bovigenitalium, A. laidlawii, Mycoplasma sp. and strains o f ureaplasma from penial-preputial swabs. M. agalactiae, A. laidlawii a n d A. a x a n t h u m [15-20] have been so far isolated from cows with mastitis symptoms. In Poland, m y c o p l a s m a s were not isolated from milk o f cows suffering from mastitis. 447
448
NATALIA ZALEWSKA-SCHONTHALER
The purpose of this research was to verify the possibility of isolation and classification of mycoplasmas from : cow's milk, washings of bull prepuce, and from the lungs of calves suffering from bronchopneumonia, in central Poland.
MATERIALS AND METHODS
Reference strains of mycoplasmas In this study we used six reference strains: M. bovigenitalium, M. bovis, M. bovirhinis, M. arginini, A. laidlawii and A. granularum supplied by the Veterinary Institute in Pu|awy, Poland, and one Staphylococcus aureus 209 P strain regarded as a standard for antibiotic sensitivity test.
Media On the basis of the preliminary examination of media, the two-phase medium for isolation of mycoplasmas from milk was prepared as described by Wenzl [21]. VF-1 medium prepared as described by Barber and Fabricant [22] was used for isolation and cultivation of the lung tissue and washings of the bull prepuce. The medium was supplemented with 5.5~ of yeast extract and with 0.01~ NAD (nicotinamidadenindinucleotid). The E medium of Roberts and Pojoan [23] was also used. For mycoplasma isolation and identification from milk according to the method proposed by Wenzl [21] milk samples were diluted 1 : 2 in saline solution before inoculation of the medium. To isolate mycoplasmas from lung tissue and from washings of bull's prepuce, 'millipore' membrane filters (650 nm) were employed. Morowitz et al. [24] and Erno and Stipkovits [25] used those filters for experimental investigations of mycoplasma size determination.
Samples 1180 samples of milk including 64 samples taken from cows with clinical and sub-clinical form of mastitis were tested; also one sample of mammary gland and 263 samples of lungs of calves suffering from bronchopneumonia were examined. Seventy-three samples of prepuce washings from bulls without any symptoms of disease were tested. Identification of 87 isolated strains was based on physiological and biochemical tests described by Whittlestone [26], A1-Aubaidi and Fabricant [27] and Erno and Skipkovits [25, 28]. Growth inhibition test (GIT) by Clyde [29] was used for identification of mycoplasma and acholeplasma strains. Sera for growth inhibition test were prepared as described by Morton and Roberts [30] in Pilaszek's modification [31]. In this modification purified mycoplasma antigen was injected into rabbit's foot pat at days 10, 16 and 22 after immunization. This method showed the highest titres of antibodies. Sensitivity of the isolated strains to: streptomycin, oxyterracyclin, chloramphenicol, erythromycin, neomycin and sulphathiasol were determined using the paper disc sensitivity test.
Isolation of mycoplasmas from milk, lungs and prepuce of cattle
449
RESULTS Results of mycoplasma isolation from milk samples from cows with sub-clinical symptoms were shown in Table 1. The data presented in Table 1 showed that all five strains of mycoplasma were isolated from the clinical or sub-clinical form of mastitis. Five mycoplasma strains were isolated together with other bacterial flora, and there was only one case when mycoplasma M 453 strain alone was isolated. Table 2 presented the main serological, biochemical and physiological characteristics of mycoplasma and acholeplasma strains which were isolated from the specimens of the mammary gland secretion and mammary gland tissue of cow's udder. As shown in Table 2, two out of six isolated strains were classified as M. bovis. Both strains of M. bovis showed variable fermentation analogous to the reference strain of M. bovis. Four remaining strains showed physiological reaction characteristic of acholeplasmataceae family. Only one of those strains M 453 was serologically identified as A. laidlawii. Strains M 51 and M 1695 were temporarily classified as A. axanthum, and this should be confirmed by serological investigations. The last of the isolated strains M 2242 was not active biochemically and was not inhibited by the used sera. The strain was designated as A. species. The results o f mycoplasmas and other bacterial isolations from lung samples were presented in Table 3. The results showed that 32 strains of mycoplasma were isolated from mentioned samples. Apart from mycoplasmas, other micro-organisms were isolated in 56.2'~o; they were Pasteurella multocida, Pasteurella haemolytica, Corynebacterium pyogenes and Escherichia coli in lower percentage. Mycoplasmas, as the only microorganisms, were isolated from lungs in 43.8~o but virus infection was not taken into consideration. Table 4 presents the most important characteristics (serological, biochemical and physiological) of the mycoplasma and acholeplasma strains isolated from the lungs of the calves. The presented data revealed that 56.3~o of the isolated strains belonged to M. bovirhinis, 9.4~o to M. boris, 6.2~o to M. bovigenitalium, 9.4~ to A. laidlawii. Physiological reactions of the two remaining strains were typical of the mycoplasmataceae family and therefore they were classified as M. species. Twelve and a half per cent of the strains were not determined because they failed to passage. Isolation of the mycoplasma and acholeplasma strains and other micro-organisms from washings of bull's prepuce were presented in Table 5. In 49 samples from which
Table 1. Isolation of mycoplasmas and other bacteria species from milk of cows with different forms of the m a m m a r y gland infection Strain of mycoplasma No. M M M M M M
2444 37 453* 51 1695 2242
Other bacteria species
The total number of cell elements
Micrococcus Micrococcus Streptococcus agalactiae Micrococcus Corynebacterium pyogenes
* First sample No. M 453 is a portion o f the m a m m a r y gland.
1,200,000 1,300,000 not examined 5 million 5 million 5 million
Form of infection sub-clinical sub-clinical clinical clinical clinical clinical
+ -
-
+ +
-
+
+
.+) ,+) + + + ,+ ,+ + +(6d) +(9d)
-
-
+ (9 d)
-
+ +
+
Biochemical reactions* Sugar fermentation Glucose Xylose Cellobiose
+ , Sensitivity to a n t i s e r u m : , resistance to a n t i s e r u m : d. days: ( + ) , variable results. * N o n e of the m e n t i o n e d strains did lerment m a n n o s e , arginine, urea, sorbitol. ++ K-group as described by Jawad, A I - A u b a i d i and F a b r i c a n t (A. avanthzvn). Reference strains.
M2444 M37 M 453 M51 M 1695 M2242 M. hovi~§ .4. laidlawii§
Strain No,
Serological reactions a n t i s e r u m M. boviM. boviM. hovi.~ ,v,enitalium rhini~ A. laidklwii
+
+
+
+ +
+
-
+
+
+ +
+
-
-
Physiological reactions Growth Growth without in serum 21 C
-
+
+
Sensitivity to digitonin
-
+(14d)
-
+ ( 1 4 d)
+(14d)
Film and spots formation
A ~pe~'ie.~ M . bo vi,~ A. lahtlawii
K{ K¢
M. bovL; M. bo vi,s A. klhlhtwii
Species
Table 2. Classification of the main serological, b i o c h e m i c a l a n d physiological characteristics of m y c o p l a s m a and a c h o l e p l a s m a strains isolated from c o w ' s milk
,-r, :> t--,
,>
7
I s o l a t i o n of m y c o p l a s m a s from milk, lungs a n d prepuce of cattle
451
Table 3. Isolation o f m y c o p l a s m a a n d a c h o l e p l a s m a strains and percentage ratio o f other m i c r o - o r g a n i s m s of calf's lungs N u m b e r of mycoplasm and acholeplasma strains
Species or serogroup
18 3 2 3 2 4
M. bovirhinis M. bovi~ M. bov~,~enilalium A. laidk~wii M..specie~s not classified
Total 32
N u m b e r of strains
E. coil l
P. muhocida
P. haemolvtica
8 1
2 ---
1 1 -1
11
Corvnebacterium pyo~ene~ 2
I
-1
3
3
Per cent of other microorganisms 72.2 33.3 0.0 33.3 50.0 50.0
mycoplasma was isolated, 30.6'~o were bacteria of Campylobacter genus, including 26.5~o of Campylohacter sputorum subspecies sputorum and 4.1~o of Campylobacterfetus subspecies fetus. Table 6 classifies the main serological, physiological and biochemical characteristics of the mycoplasma and acholeplasma strains isolated from the washings of the bull's prepuce. As shown in the table, the strains were identified as M. bov(genitalium in 53.1j°~,, A. laidlawii in 16.3~0, M. bovis in 2°~. Ten and a third per cent of the strains were not classified because it was not possible to passage them. Results of the isolation of mycoplasmas from the respective specimens and their percentage ratio are given in Table 7. Out of 1180 milk specimens and one mammary gland portion six strains (0.5~,i) of mycoplasmas were isolated. Out of 264 specimens of call's lung 32 strains (12.1°~i) of mycoplasmas were isolated. Out of 73 specimens of the washings of bull's prepuce, 49 strains (67.1°o) of Myeoplasma sp. and Acholeplasma sp. were isolated. Results concerning the sensitivity of 47 strains of Mycoplasma sp. and Aeholeplasma sp. and four laboratory strains to antibiotics and sulphathiasol were presented in Table 8. The strains for antibiotic sensitivity test were randomly chosen. All of the strains showed 1001'i, sensitivity to chloramphenicol. All the examined M. bovigenitalium strains were resistant to erythromycin, and from the remaining strains 37.2'~0 of them were sensitive to this antibiotic. Fifty-three per cent of the examined strains were sensitive to neomycin. All of the examined strains were resistant to sulphathiasol. DISCUSSION The use of "millipore' membrane filters with 650-nm mycoplasmas from lung homogenisate and Campylobaeter prepuce as described by Morowitz et al. [24] and Samol and eliminate the overgrowth of other micro-organisms in particularly that of Proteus sp. Isolation of M. bovis and A. infected mammary gland confirmed the studies of Karbe
pores for the isolation of sp. from washings of bull's Korpolifiska [32], allowed to the majority of specimens, laidlawii from milk and from and Mosher [17], Jasper et
( 1)
3
( 1)
2 -( 1)
(2)
3
A. laidlawii
3 --
13 l(-, +)
--
3
3(8d)
Biochemical reactions* fermentation of sugar Glucose Cellobiose
1 1
2
18 3 -3 --
Growth without serum
--
3
--
2
2(14 d) 3(12d)
Film and spots formation
M . bovirhinis M . bovis M . bovi,g e n i t a l i u m A . laidlawii M . ~pecies M . ~'pecie,~
Species or serogroup
3.1 3.1
56.3 9.4 6.2 9.4
Per cent of the strains mycoplasma and acholeplasma
12.5°i; were not tabulated because they failed to
Growth in 21 C
Physiological reactions
* None of the presented strains did ferment mannose, sorbitol, arginine, urea, xylose: d, day. Figure in brackets, doubtful reactions, inhibition zone in m m < 3 m m : ( , +), variable results: four strains passage.
1
2 3 1
18
18 3
M . bovi.s
M . bovi,~enitalium
Serological reaction antiserum
M. bovirhini.s
Strain No.
Sensitivity to digitonin
Table 4. Classification of the main serological, biochemical and physiological characteristics of mycoplasma and acholeplasma strains from the calf's lungs
©: Z
6~
N
>.
Z
t,3
Isolation of mycoplasmas from milk. lungs and prepuce of cattle
453
Table 5. Isolation ol mycoplasma and acholeplasma strains and percentage ratio of other micro-organisms recovered from the washings of bull's prepuce Isolated mycoplasma and acholeplasma strains Number Per cent 16 1 8 14
53. I 2.(I 16.3 28.6
Total 49
100.0
Species or serogroup Mycoplasma Acholeplasma M.bov(~enimlium M. bovi~
Campvh~bacter Campyh~bacter /etu.~ .~pulorum ~. /em~ ~. hubulu~
2
A. laidlawii not classified
2
8
Per cent o1" other microorganisms
3 2
20.4 0.0 6.1 4.1
13
30.6
al. [16] and, Gourlay and Scott [10]. On the basis of their biochemical characteristics the two strains isolated from milk were classified as A. axanthum in our study. Our results correspond with observations of Whenert et al. [20] who have also isolated A. axanthum from milk. It is, however, necessary to confirm these results by serological tests. Isolation of Mycoplasma sp. and Acholeplasma sp. in 6.25~, of milk samples obtained from cows with clinical and sub-clinical mastitis suggests that like in West Germany or Japan there is mycoplasma infection of bovine mammary gland in Poland, as well. We suspect that mycoplasmas can be one of the etiological agents in these infections. It should be pointed out that the isolation ofA. laidlawii from the mammary gland was associated with leukemic changes in the mammary tissue. Mycoplasmas were isolated together with other micro-organisms from milk by Pan and Ogata [19]; this is in agreement with the results of our research. Two strains of mycoplasmas were isolated from milk samples in which the number of cell elements was higher than 1,200,000 per ml. In both cases bacteria of Micrococcus sp. were isolated from the milk samples. Three strains were isolated from milk of cattle suffering from the clinical form of mastitis but other bacteria like Streptococcus agalactiae, Micrococcus sp. and Col3,nebacterium pyogenes were also isolated from these samples.
Strains M. boris isolated from the milk and the reference strain which were subjected to fermentation reacted variably to glucose; this is confirmed by observations of Edward and Moore [33], Leach [18] and other authors. The majority of authors suggested that M. boris does not ferment glucose. Edward and Moore [33] showed that the lowering of the pH was caused by horse serum or yeast extract added to culture medium rather than by glucose fermentation by M. bovis. In our studies the level of glucose in culture medium was not determined. As suggested by Edward and Moore [33] this may explain the positive fermentation observed in our studies. Results of mycoplasma isolation from the mammary gland tissue of cows udder in Poland (6.25~ of the clinical cases of mastitis) differ from the data reported by Pan and Ogata [19] in Japan. The authors isolated mycoplasmas from 78~o of all examined milk samples. A1-Aubaidi and Fabricant [27] isolated mycoplasmas from milk samples in 40~o. Results of isolation of mycoplasmas from 12.1~ of cases of pneumonia are generally in accordance with the results obtained by Galassi [6, 7], Nowakowski [3] and also by Pilaszek and Truszczyfiski [34]. As in the studies of Shimizu et al. [35], Mengik and Jurmanova [36] and Gossger [37], M. bovirhinis was isolated in the highest percentage. 16
-
8
---
2(10
8
d)
"
8
Biochemical reactions* fermentation of sugar Glucose Cellobiose
7
1
26
-"
8
Growth without serum 26 l(12d)
Film and spots fc,rma!ion
examined specimens 1180 264 73
Organ
Mammar~ gland Lungs Prepuce
2 3 1
M. bovi.~
2 26
M. hovi~,,enilalium
18
M. hovirhini~
I 3 8
3 6 14
A. laidlawii Unclassified
Isolated specimens
0.5 12.1 67.1
Per cent
Table 7. Isolation of mycoplasma and acholeplasma strains from the cattle of central Poland Number of
Total
M. bovivenitalium M. hovi.~ A. iaMlawii M. .~pe{qe~ M. v~ecie~
Species or serogroup
89.7
53.1 2.0 16.3 14.3 4.0
Per cent of the strains mycoplasma and acholeplasma
10.3". were not tabulated because they tailed to passage.
."
8
--
Growth in 21 C
Physiological reactions
* None of the presented strains did ferment mannose, sorbitol, arginine, urea, xylose: d, days: five strains
_"~
7
8
26 --
1
1
Serological reactions antiserum M. M. hoviA. hovirhini~ M. hovi.~ .~enitalium h&llawii
26
Strain No.
Sensitivity to digitonin
Table 6. Classification of the main serological, biochemical and physiological characteristics of mycoplasma and acholeplasma strains isolated from the washings of bull's prepuce
3
~=.
,>
> t...>~ > N ~r"
4~
96.6 100
15 11
51
Total
11.8
3.4 0
0 27.8
ms
Streptomycin
3.9
0 0
14.3 0
ns
70.6
100 72.8
100 34.4
s
27.4
0 18.1
0 66.6
ms
2.0
0 9.1
0 0
ns
100
100 100 0
0 0
0 0
ms
0
0 0
0 0
ns
Chloramphenicol
100 100
s
37.2
0 100
17.7
0 0
85.7 16.7
ms
0 44.4
ms
45.1
100 0
Erythromycin
14.3 38.9
s
Per cent of strains with different sensitivity Oxytetracycline
0
0 0
0 0
s
s, sensitivity > 30 mm: ms, medium sensitive 27 24 mm: ns, non-sensitive ~< 23 mm. The examined strains were resistant to sulphathiasol.
84.3
87.5 72.2
s
7 18
strain
strains
M. bovL~ M. bovirhinis M. bov~eenitalium A. laidlawii
of
acholeplasma
Mycoplasma and N u m be r
Table 8. Sensitivity to antibiotics of mycoplasma and acholeplasma strains isolated from cattle
53.0
0 81.8
71.4 61.2
ms
Neomycin
47.0
100 18.2
28.6 38.8
ns
t~
g
o
a"
(3
,-,.,
7
456
NATALIA ZALEWSKA-SCH()NTHALER
G a l a s s i [6] i s o l a t e d - - f r o m l- to 2 - w e e k - o l d c a l v e s - - M , bovis in 58.5'~/o o f cases, M . bovigenitalium in 7.4~,q, a n d M . arginini in 4.2~o o f cases. Stipkovits et al. [5] isolated A. a x a n t h u m from lungs, G e o r g e et al. [38] M . dispar. Oghiso Y o i c h o et al. [39], however, received quite different data. T h e y isolated T - m y c o p l a s m a from lungs o f calves suffering from p n e u m o n i a in 9 0 ~ o f cases, a n d M . bovirhinis a n d M . bovigenitalium in lower percentage. Similarly to o t h e r a u t h o r s [4, 40] while e x a m i n i n g by the g r o w t h inhibition test we o b s e r v e d few c r o s s - r e a c t i o n s between them. The c r o s s - r e a c t i o n was o b s e r v e d between M . bovirhinis antigen a n d M . bovigenitalium sera, but also between M . boris a n d M . bovigenitalium sera. M y c o p l a s m a s were recovered in 67.1% from bull's p r e p u c e washings. Similarly, high p e r c e n t a g e - - 4 7 . 3 ~ o o f m y c o p l a s m a isolation from bull's semen in P o l a n d - - w a s r e p o r t e d by Z g 6 r n i a k - N o w o s i e l s k a a n d B r a n n y [8]. T h e a u t h o r s isolated m a i n l y M . bovigenitalium a n d A. laidlawii a n d M . bovis to a lesser extent. This is in a g r e e m e n t with o u r results. L a n g f o r d [41] a n d O n o v i r a n et al. [14] isolated m y c o p l a s m a s from p e n i a l - p r e p u t i a l washings in 65.9~o, f r o m semen in 34.9~o. The a u t h o r s n o t e d higher frequency o f m y c o p l a s m a isolation from the p e n i a l - p r e p u t i a l washings t h a n from bull's semen. This is also in a c c o r d a n c e with o u r a n d o t h e r a u t h o r s ' r e s u l t s . However, studies c a r r i e d out so far, d o n o t solve the m a i n p r o b l e m , i.e. the role o f m y c o p l a s m a s in the genital tract o f ill bulls. This p r o b l e m is still open for discussion. The results o f tests for m y c o p l a s m a sensitivity to antibiotics a n d s u l p h a t h i a s o l r e p o r t e d by N o w a k o w s k i [3] were not c o m p a r a b l e with o u r findings. O u r tests in vitro s h o w e d 100~o sensitivity to c h l o r a m p h e n i c o l a n d 8 4 . 3 ~ sensitivity to s t r e p t o m y c i n , while in N o w a k o w s k i ' s [3] studies the s a m e p e r c e n t a g e o f m y c o p l a s m a strains were sensitive to streptomycin and chloramphenicol.
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