Mycoplasmas f rom donkeys and horses in the Sudan

fermentation Les du groupe que Mycoplasme


Introduction
Mycoplasmas and acholeplasmas have been isolated from genital and respiratory tracts of horses by several workers (9), but until now there were no reports on the isolation of these organisms from donkeys. They are useful animals in the Sudan since they are used for transport purposes in many parts of the country.
Isolation of mycoplasmas from donkeys and horses has not been previously attempted in the Sudan. The present work was, therefore, carried out to investigate the prevalence of mycoplasmas in these two animal species and to find out whether or not donkeys and horses harbour Mycoplasma mycoides subsp. mycoides (M. mycoides) and Mycoplasma mycoides subsp. capri (M. cap@, the causal agents of contagious bovine and caprine pleuropneumonia. These two diseases are among the most important for cattle and goats in the country.

Samples
Three hundred and ninety eight samples consisting of nasal swabs were collected from 300 donkeys and 69 horses in Khartoum and Wad Medani areas and 29 uterine washings from mares in Khartoum. Swabs were collected with sterile absorbent cotton wool swabs and uterine washings were sent to the laboratory for bacteriological examination. Swabs collected from animals in Wad Medani area were sent to Khartoum in a transport medium. All samples were kept on ice during transport.
Culture media PPLO and Brucella media (Difco) were used as solid and liquid media for isolation and propagation of cultures. The media were supplemented with 20 % horse serum, 0.02 % DNA, 500 IU/ml penicillin and 10 % vlv of a 25 % aqueous solution of yeast extract (Oxoid). PPLO broth was also used as transport and basa1 medium for biochemical tests.

Culture methods
Swabs without transport medium were streaked directly on the solid medium, then immersed in the liquid medium and stirred by an electrical stirrer for half ,a minute. Serial tenfold dilutions of the suspension were made up to 10" in the liqtiid medium and after two days of incubation, 0.2 ml of each dilution was used to inoculate liquid and .s&id media (H. KIR-CHHOFF, persona1 communication, 1988). Swabs in the transport medium were stirred and, similarly treated.
Swabs from the Khartoum area were examined within two hours after collection and those from Wad Medaqi after 36 hours. Uterine washings, were centrifuged at 3000 rpm for 10 minutes and thel deposit was used to inoculate liquid and solid media. Inoculated media were incubated aerobically in a humid container at 37 "C.

Identification of isolates
Cloned cultures were identified by the following tests : reversion and filtrability (5), sterol requirement for growth (4) sensitivity to digitonin (6).

Biological and biochemical tests
The tests used were : liquefaction of coagulated serum, glucose fermentation, arginine hydrolysis, phosphatase activity (7), haemolysis of red blood cells (l), tetrazolium reduction (10) and production of films and spots (3).

Isolation of mycoplasmas
Seventeen isolates (4.27 %) were recovered from 398 samples. Twelve isolates (4 %) were obtained from 300 donkey nasal swabs, three isolates (4.3 %) were obtained from 69 horse nasal swabs and two isolates (6.89 %) were recovered from 29 uterine washings. All isolates showed typical fried-egg colony type which appeared on microscopic examination after 24 to 48 hours of incubation. Both PPLO and Brucella media were suitable for isolation, but growth was better and the colonies were larger on PPLO agar.
Nine isolates, i.e. six from donkey nasal swabs, one from horse nasal swab and two from uterine washings, were lost during storage at -20 "C. These isolates were subjected to the reversion test only and showed no change in colony appearance. They were, therefore, assumed to be either mycoplasma or acholeplasma.

Biological and biochemical properties of isolates
The remaining eight isolates showed no change in colonial morphology when grown in inhibitor-free media ; they required serum for growth, were sensitive to digitonin and could be filtered through a 450 nm millipore filter. Accordingly, the organisms were identified as mycoplasmas.
Only four isolates hydrolysed arginine and the other four neither fermented glucose nor hydrolysed arginine. All isolates gave negative results in the other tests.
On the basis of glucose fermentation and arginine hydrolysis, the isolates were divided into two groups. The first group included the four isolates hydrolysing arginine of which three were from donkeys nasal swabs and the fourth from horse nasal swab. The second group included the remaining four strains which neither fermented glucose nor hydrolysed arginine. Three of these strains were from donkey nasal swabs and the fourth was from horse nasal swab.

Serological tests
All isolates did not react with any of the antisera used in the two tests.

Discussion
The present work is the first study on equine mycoplasmas in the Sudan. The isolation rate from donkey and horse nasal swabs and mare uterine washings were 4, 4.3 and 6.89 %, respectively.
The isolates possessed cultural and biochemical properties which justified their identification as mycoplasmas.
None of the isolates reacted with any of the antisera used in the tests. It should be important, therefore, to test the isolates with antisera prepared against other mycoplasma species. It should also be noticed that mycoplasmas other than those common to horses were isolated from the respiratory tract of these animals such as M. felis (9). Surprisingly no acholeplasma was isolated during this study although they were reported elsewhere (9). However, nine isolates were lost before complete identification. Some of these might have been acholeplasmas.
An important fact revealed by the serological tests was that none of the isolates reacted with antisera to M. mycoides and M. capri which indicates that horses and donkeys do not carry these cattle and goats important pathogens.