Significance and Pathogenic Role of Mycoplasma arginini in Cat Diseases R. J. S. Tan, Ek Wang Lim and B. Ishak*
ABSTRACT
it was also isolated from sheep and goats (1), from captive wild cats (7, 8), from Eighty-one strains of Mycoplasma arginini commercial bovine sera (4) and quite comwere isolated from swabs and tissues cultured monly from contaminated tissue cultures from a series of 555 cats. The majority of (10) . In a series of investigations on the inthese strains (84%) were recovered from the cidence and significance of mycoplasmas oropharyngeal regions. M. arginini was experimentally inoculated in cats from 1968 to 1976 M. arginini was into young kittens. No clinical disease was isolated in relatively high frequency from produced. However, there was rapid coloniza- clinically healthy, sick, dead and euthanized tion in the inoculated sites. cats of both sexes ranging from three days old to 17 years of age. Sicknesses included respiratory tract infections (majority of cases), conjunctivitis, tonsillitis, glossitis, urolithiasis and other forms of urogenital tract infections, inappetance, a few cases RESUME of enteritis, dysentery and various forms of food poisoning. Because of this relatively Les auteurs ont isoli 81 souches de Myco- high occurrence in these animals, attempts plasma arginini, a partir de la culture d'ecou- were made to study its pathogenicity in villons et de tissus provenant de 555 chats. cat diseases. La plupart de ces souches (84%) originaient This paper reports on the isolations of du pharynx. M. arginini from cats and the results of a L'inoculation experimentale de ce myco- series of experimental infections of kittens plasme a des chatons ne provoqua pas de with M. arginini. maladie clinique, en depit de sa multiplication rapide aux sites d'injection.
MATERIALS AND METHODS
INTRODUCTION MEDIA
Mycoplasma arginini was first isolated by Barile et al (3) from brain tissues of a scrapie infected sheep and mouse, from joint exudate of an arthritic goat as well as cell cultures established from human, chimpanzee and dog tissues. Subsequently,
*Department of Microbiology, Faculty of Medicine, University of Singapore, College Road, Republic of Singapore. Submitted September 14, 1976.
Volume 41 - July, 1977
The diphasic broth used in the initial isolations consisted of 2 ml of complete mycoplasma agar overlaid with 2 ml of complete mycoplasma broth in one-quarter oz screw capped bottles. Both media were based on those previously described by Tan and Miles (15). Media for strains which utilized arginine for growth consisted of complete mycoplasma broth or agar with 1% L-arginine hydrochloride in place of 1% dextrose. The pH was then adjusted to 6.8-7.0 with 2N
HCl. 349
The strain used for experimental infection was strain MT 293. This strain was originally isolated from the lungs of a very sick cat which was killed in a moribund state.
ISOLATIONS As many swabs as possible were obtained from the eyes, throats, noses, rectum and urogenital tracts of clinically healthy, sick, dead and euthanized cats of both sexes ranging from three days old to 17 years of age. In addition, carcasses of euthanized animals were necropsied and samples from various organs were taken and cultured for mycoplasmas. Blood was obtained aseptically by cardiac puncture. The serum was separated and used for serology. The swabs were inoculated into diphasic broth and incubated at 37°C for two days. Samples were then streaked on mycoplasma agar and incubated in 10% C02 in air at 37°C. These plates were then inspected daily for growth of mycoplasma colonies. Small samples from various organs were minced with sterile scalpel blades and inoculated into diphasic broth and then onto plates as described above. With plates showing no growth, block passes were made onto fresh plates. Specimens were considered negative after the second passage had failed to grow mycoplasmas. Methods used for isolating pathogenic bacteria and viruses in kitten kidney tissue cultures have been described previously (14). MYCOPLASMA STRAIN The original strain of M. arginini was supplied by Dr. W. R. Dowdle. This strain was used to prepare antigens for serology.
PREPARATION OF ANTIGENS
Antigens used for serology were prepared by first cloning each strain three times on complete mycoplasma agar. On the third cloning, one isolated single colony was picked and inoculated into a 2 ml mycoplasma broth and incubated for 48 hours, after which the entire 2 ml broth culture was inoculated into a 20 ml mycoplasma broth and incubated further for 48-60 hours. Ten ml of this broth culture was then inoculated into a 100 ml volume and incubated for three to seven days (according to the growth rate of each strain) under constant agitation. At the end of the incubation period the bulk broth culture was spun at 3,000 X g in a Sorvall RC2-B centrifuge for ten min. The supernatant was then spun at 23,500 X g for 30 min to deposit the cells. The pellet was resuspended with cold phosphate buffered saline (PBS) redeposited and finally suspended in 1/20 of its original volume with cold PBS (giving a final concentration of 2 X 106 cells per ml). This 20X concentrated antigen was used as inoculum for the experimental infection as well as in direct haemagglutination-inhibition (HAI) and indirect haemagglutination (IHA) tests. For use in comple-
TABLE I. Isolations of M. arginini from Catsa
No. swabs and tissues cultured .......... Total positive swabs and tissues Total number isolatesb ........ . M. arginini ......... Ureaplasmasm. a Othersd .............
Thr.e UGTf Rec.9 Trach.h Lungs Others Total Percent
Eyes
Nose
370
267
555
219
85
11
35
99
85
497
55
11
3
6
37
1579 756
1 11 3 6 916 620 73 e2 i 1 81 68 5 16 16 10 4 97 3 759 68 476 60 -.Untypedp.-e -660 aTotal no. of cats tested -555, which included clinically healthy, sick, dead and euthanized cats bSome swabs yielded more than one mycoplasma type cIsolated from brain of cat which died of ataxia dIncluded - M. felis, M. gateae and A. laidlawii eThroat fUrogenital Tract gRectum hTrachea
350
99 2
103 2 101
47.9 8.8 1.7 82.9 6.6
Can. J. comp. Med.
TABLE II. Summary of Clinical Manifestations after Receiving a 20X Concentrated Inoculum of M. arginini Isolate MT 293
Kitten No.
C/Ta
1...............
2................ 3................ 4................ 5................ 6................ 7................ 8................ 9................
C
C T T C C T T C T T
10 ............... 11 ............... aC - control animals, T - test animals bPyrexia is 39°C and above cWeek old
Age 6 wo"
6 wo 6 wo 6 wo 7 wo 7 wo 7 wo 7wo 8 wo 8 wo 8 wo
Route of Inoculation INd, OIe
IN, OI IN, OI IN, OI
IPf IP IP IP I Pul.g I Pul. I Pul.
Duration of Clinical Signs in Days Postinoculation Pyrexiab Conjunctivitis Anorexia
-
-
2 2- 3 2- 4 2- 3
1 2-3 2- 4 2-3
dIntranasal
"Ocular installation fIntraperitoneal gIntrapulmonary
ment fixation (CF) tests the concentrated antigen was boiled for 35 min in a water bath. Antigens for serological tests were preserved with sodium azide (0.08% final concentration) and stored at -20°C until ready for use. PREPARATION OF SPECIFIC ANTISERA
Homologous antiserum against each representative strain was produced in rabbits. Each rabbit was given a sensitizing dose of a mixture of an equal volume of 20X concentrated antigen and Freund's complete adjuvant. This mixture was administered into the foot-pads of both hind legs (0.1 ml per foot-pad). Two weeks later, a booster dose of 2 ml of a 24-48 hours culture containing approximately 105 colony forming units (c.f.u.) per ml was administered intramuscularly. This was then followed by a weekly booster dose of a similar 2 ml volume intramuscularly. The animal was bled before each booster dose. The antibody titre of the serum was determined by growth inhibition tests (5). When the antibody titre had reached a sufficiently high level to inhibit growth of the homologous mycoplasma by at least 2 mm in width by the disc method of Clyde (5), the animal was bled out by cardiac puncture. To avoid antibodies being formed against pig serum globulins used in the enriched
Volume 41
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July, 1977
media, normal rabbit serum was used in growing cells for antiserum production. IDENTIFICATION OF MYCOPLASMAS
Identification of mycoplasma was performed by the growth inhibition technique of Clyde (5). SEROLOGY
Complement fixation (CF) tests were carried out at 4°C fixation overnight. Complement and antigens were used at four units. A 3 % suspension of sheep erythrocytes was used. Indirect haemagglutination (IHA) tests were carried out in microtitre plates by the method of Herbert (6). EXPERIMENTAL INFECTIONS
Before the experimental infection, all the animals were screened for at least one week. Each experiment was conducted in a different room. The dam and her litter were housed together in a large cage measuring 8 x 5 feet. Both the control and test kittens were housed together and nursed by the same mother. Daily observations were made for clinical signs. Rectal temperatures were taken daily. Swabs were obtained from eyes 351
TABLE III. Isolation of Mycoplasmas, Bacteria and Viruses from Kittens before and after Inoculation of M. arginini Isolate MT 293
Kitten
1............ 2........... 3............ 4............ 5............ 6............ 7............ 8...........
C/Ta C C T T C C
Preinoculation Throats Eyes
M. gateae M. gateae
Days Postinoculation Recovery of M. arginini from Eyes Throats Lactobacillus spp. 1,2 5,6, 10, 14 1, 3, 7, 10, 14 4-6, 10-14, 20 M. gateaeb
T
T
9..........
10 ........... T 11 ........... T aC- control animals, T - test animals bM. gateae was isolated on days 3 and 14 postinoculation
and throats and cultured for mycoplasmas, pathogenic bacteria and viruses. At the end of the observation week the animals were starved for at least 12 hours (to prevent excessive levels of cholesterols in the blood) and were then anaesthetized with sodium pentobarbitone given intraperitoneally. Blood samples were then obtained by cardiac puncture. Swabs were again obtained and cultured for microorganisms as above. Throat swabs were also obtained from all the dams and cultured for mycoplasmas, pathogenic bacteria and viruses.
Experiment I - A litter of four six week old kittens was used. Two were each given 0.2 ml of a 20X concentrated inoculum of M. arginini into each eye and a 0.5 ml volume intranasally into both nostrils. The remaining two kittens were each given similar volumes of sterile saline.
Experiment II - A second litter of four seven week old kittens was used. Two kittens were each given 0.5 ml of the 20X concentrated inoculum intraperitoneally. The other two kittens were each given similar volumes of sterile saline.
Experiment III - In a third litter of three eight week old kittens, two of them were each given an intrapulmonary inoculum of 0.2 ml of the 20X concentrated culture. The remaining kitten was given a similar volume of sterile saline. The inoculum was injected into the right lung by inserting a 1/4 inch x 26G needle between the third and fourth last ribs, approximately equidistant from the sternum to the posterior edge of the rib cage. The procedure was performed under sodium 352
pentobarbitone anaesthesia. At the end of three weeks all the animals killed and necropsied. Organ samples tested for the presence of M. arginini.
were were
RESULTS ISOLATIONS A total of 81 strains of M. arginini was isolated from swabs and tissues cultured from a series of 555 cats (see Table I). Of these, 68 (84%) were isolated from the oropharyngeal regions. The remaining isolates were recovered from eyes (2.5%), nose (2.5%), urogenital tract (6.2%) and rectum (1.2%). Two strains were isolated from lungs of necropsied animals. One strain was recovered from the brain of an old tom cat which had ataxia and was subsequently euthanized. Other mycoplasmas recovered from cats included M. felis, M. gateae, A. laidlawvii and feline ureaplasmas (Unpublished data ). In addition, there were 60 isolates which could not be identified with the antisera of any of the above species by growth inhibition tests.
EXPERIMENTAL INFECTIONS Clinical - All the control animals (kittens nos. 1, 2, 5, 6 and 9) remained clinically normal throughout the experiment. Two kittens (nos. 3 and 4) were also
Can. J. comp. Med.
TABLE IV. Summary of Serological Tests Performed on Kitten Sera Taken before and after Inoculation of M. arginini Isolate MT 293
C/Te
Kitten 1 ......... 2 ......... 3 ......... 4 ......... 5 ......... 6 ......... 7 ......... 8 ......... 9 ......... 10 ......... 11 .........
C C T T C C T T C T T
Indirect Haemagglutination Tests Bb CC Dd Aa 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1Of 80 0 5 20 160 0 0 0 0 0 10 40 160 160 0 10 40
A 0 0 0 0 0 0 0 0 0 0 0
Complement Fixation Tests C D B 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 10 5 0 40 5 0 0 0 0 40 5 0 40 0 10
aPreinoculation sera bPostinoculation sera taken on day 7 oPostinoculation sera taken on day 14
dPostinoculation sera taken on day 21 eC - control animals, T - test animals Titres in reciprocal dilutions
clinically normal following the inoculation of M. arginini into both eyes and nostrils. The other inoculated animals (nos. 7, 8, 10 and 11) developed slight pyrexia and very mild anorexia following an intraperitoneal or intrapulmonary inoculation as shown in Table II. Isolations - No pathogenic bacteria were isolated from the eyes and throats of all experimental animals before and after inoculation. Neither were viruses isolated from kitten kidney tissue cultures. M. gateae was recovered from the throats of kittens nos. 4 and 6 before inoculation and from throat of kitten no. 5 after inoculation. M. arginini was not isolated from eyes and throats of all animals before inoculation. However, M. arginini was recovered from the eyes of kitten no. 3 on days 1 and 2 after inoculation and from the eyes of kitten no. 4 on days 1, 3, 7, 10 and 14 after inoculation. M. arginini was also recovered from the throat of kitten no. 3 on days 5, 6, 10 and 14 and from the throat of kitten no. 4 on days 4-6, 10-14 and day 20 after inoculation (as shown in Table III). M. arginini was also recovered from the necropsied trachea of kitten no. 4 but not from the other organ samples of all the other animals. M. gateae was isolated from throat swabs of all the dams before inoculation. In addition, M. arginini was recovered from throat swab of the dam in Experiment I at necropsy.
Volume 41
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July, 1977
Serology - Table IV shows the results of indirect haemagglutination (IHA) and complement fixation (CF) tests performed on kitten sera which were obtained at weekly intervals. No antibodies were detected in all preinoculation kitten sera. The postinoculation sera of all control kittens also had no detectable IHA and CF antibodies against M. arginini. Neither were antibodies against M. arginini detected in all postinoculation sera of kittens nos. 3 and 4 following inoculation into the eyes and nostrils. However, low levels of both IHA and CF antibodies were detected in all postinoculation sera of kittens nos. 7, 8, 10 and 11 following intraperitoneal or intrapulmonary inoculation. Necropsy - All the animals were killed and necropsied at day 21 postinoculation. No pathological abnormalities were found in either the control or infected animals.
DISCUSSION A total of 81 strains of M. arginini was isolated from swabs and tissues cultured from a series of 555 cats (see Table I). The majority of these strains (84%) were recovered from the oropharyngeal regions of cats. This suggests that M. arginini
353
has an affinity for the mucous membrane of the oropharyngeal regions. The recovery of M. arginini from the lungs of two necropsied sick cats suggests its possible involvement in cat pneumonia. Its recovery from the brain of a cat with ataxia also suggests that M. arginini could be involved in certain disease syndromes of the central nervous system. Unfortunately, no histopathological studies were available for direct correlation with isolation studies. Similar organisms were recovered from brain tissues of a scrapie infected sheep and mouse (3) and from a lioness (7). However, these authors could not establish an etiological role for M. arginini isolated from brains of animals. The isolation of 'M. felis from cats in New Zealand has been reported and characterized (13, 16). Its pathogenic role in young kittens has been described previously (11). The isolations of A. laidlanvii and feline ureaplasmas have been reported elsewhere (12, 15, 18). Their significance in the cat hosts is the subject of another paper (Unpublished data). Results of experimental infections of M. arginini in kittens suggest that our strain MT 293 is not pathogenic for cats. There was no clinical manifestation in two kittens (nos. 3 and 4) following inoculation into the eyes and nostrils. However, kittens nos. 7, 8, 10 and 11 developed a slight pyrexia and mild anorexia. From the authors' own experience, pyrexia and anorexia are not unexpected in animals given a large dose of mycoplasma antigens. In this case, it could probably be due to the pyrogenic properties of M. arginini. The case with which M. arginini could be recovered from the eyes and throats of kittens following intraocular and intranasal inoculations of the 20X concentrated antigens clearly demonstrates the predilection of the organisms for mucous membranes of the eyes and nasopharynx. Its persistence in these sites suggests rapid colonization without any ill effects on the host. Like A. laidlawii, M. arginini also has a wide distribution in nature (3, 4, 9, 10). It was first reported in sick cats (including Siamese and Burmese pedigrees) by Tan and Miles (17). Since then, it has not been reported by workers in other countries, although Heyward et al (7) isolated a strain from the lung and brain of a lioness which died of encephalitis. That isolate was originally named M. leonis (7).
354
Subsequently, it was found to be synonymous with M. arginini by Tully et al (19). The relatively high occurrence of M. arginini in cats in this study suggests that M. arginini might be a common inhabitant in this animal host, although it has only been recorded in New Zealand and also here in Singapore.
ACKNOWLEDGMENTS We are greatly indebted to Drs. J. Tully and W. R. Dowdle of the National Institutes of Health, Bethesda, Maryland, U.S.A., for providing us with the strains M. felis, M. gateae and M. arginini. REFERENCES 1. AL-AUBAIDI, J. M., W. D. TAYLOR, G. R. RIJBASH and A. H. DARDIN. Identification and characterization of M. arginini from Bighorn sheep (Ovis canadensis) and goats. Am. J. vet. Res. 33: 87-90. 1972. 2. BARILE, M. F., R. T. SCHIMKE and D. B. RIGGS. Presence of arginine dihydrolase pathway in mycoplasmas. J. Bact. 91: 189-192. 1966. 3. BARILE, M. F., R. A. DELGUIDICE, T. R. CARSKI, C. J. GIBBS and J. A. MORRIS. Isolation and characterization of Mycoplasma arginini: spec. nov. Proc. Soc. exp. Biol. Med. 129: 489-494. 1968. 4. BARILE, M. F. and J. KERN. Isolation of M. arginini from commercial bovine sera and its implication in contaminated cell cultures. Proc. Soc. exp. Biol. Med. 138: 432-437. 1971. 5. CLYDE, W. A. Jr. Mycoplasma species identification based upon growth inhibition by specific antisera. J. Immun. 92: 958-965. 1964. 6. HERBERT, W. J. Indirect haemagglutination. In Handbook of Experimental Immunology. D. M. Weir, Ed. pp. 720-744. Oxford and Edinburgh: Blackwell Scientific Publications. 1967. 7. HEYWARD, J. T., M. Z. SABRY and W. R. DOWDLE. Characterization of mycoplasma species of feline origin. Am. J. vet. Res. 30: 615-622. 1969. 8. HILL, A. The isolation of Mycoplasma arginini from captive wild cats. Vet. Rec. 91: 224-225. 1972. 9. LEACH, R. H. The occurrence of Mycoplasma arginini in several animal hosts. Vet. Rec. 87: 319-320. 1970. 10. STEWART, S. M. and D. YOUNG. Isolation of Mycoplasma arginini from tissue cultures. Lancet 1: 347. 1971. 11. TAN, R. J. S. Susceptibility of kittens to Mycoplasma felis infection. Jap. J. exp. Med. 44: 235-240. 1974. 12. TAN, R. J. S. and J. G. MARKHAM. Feline Tstrain mycoplasmas. Jap. J. exp. Med. 41: 247-248. 1971. 13. TAN, R. J. S. and J. G. MARKHAM. Isolation of mycoplasma from cats with conjunctivitis. N. Z. vet. J. 19: 28. 1971. 14. TAN, R. J. S. and J. A. R. MILES. Further studies on feline respiratory virus diseases. I. Vaccination experiments. N. Z. vet. J. 19: 15-18. 1971. 15. TAN, R. J. S. and J. A. R. MILES. Mycoplasma isolations from clinically normal cats. Br. vet. J. 128: 87-90. 1972. 16. TAN, R. J. S. and J. A. R. MILES. Characterization of mycoplasmas isolated from cats with conjunctivitis. N.Z. vet. J. 21: 27-32. 1973. 17. TAN, R. J. S. and J. A. R. MILES. Incidence and significance of mycoplasmas in sick cats. Res. vet. sci. 16: 27-34. 1974. 18. TAN, R. J. S. and J. A. R. MILES. Possible role of feline T-strain mycoplasmas in cat abortion. Aust. vet. J. 50: 142-145. 1974. 19. TULLY, J. G., R. A. DELGUIDICE and M. F. BARILE. Synonymy of Mycoplasma arginini and Mycoplasma leonis. Int. J. syst. Bact. 22: 47-49. 1972.
Can. J. comp. Med.
ABSTRACT
it was also isolated from sheep and goats (1), from captive wild cats (7, 8), from Eighty-one strains of Mycoplasma arginini commercial bovine sera (4) and quite comwere isolated from swabs and tissues cultured monly from contaminated tissue cultures from a series of 555 cats. The majority of (10) . In a series of investigations on the inthese strains (84%) were recovered from the cidence and significance of mycoplasmas oropharyngeal regions. M. arginini was experimentally inoculated in cats from 1968 to 1976 M. arginini was into young kittens. No clinical disease was isolated in relatively high frequency from produced. However, there was rapid coloniza- clinically healthy, sick, dead and euthanized tion in the inoculated sites. cats of both sexes ranging from three days old to 17 years of age. Sicknesses included respiratory tract infections (majority of cases), conjunctivitis, tonsillitis, glossitis, urolithiasis and other forms of urogenital tract infections, inappetance, a few cases RESUME of enteritis, dysentery and various forms of food poisoning. Because of this relatively Les auteurs ont isoli 81 souches de Myco- high occurrence in these animals, attempts plasma arginini, a partir de la culture d'ecou- were made to study its pathogenicity in villons et de tissus provenant de 555 chats. cat diseases. La plupart de ces souches (84%) originaient This paper reports on the isolations of du pharynx. M. arginini from cats and the results of a L'inoculation experimentale de ce myco- series of experimental infections of kittens plasme a des chatons ne provoqua pas de with M. arginini. maladie clinique, en depit de sa multiplication rapide aux sites d'injection.
MATERIALS AND METHODS
INTRODUCTION MEDIA
Mycoplasma arginini was first isolated by Barile et al (3) from brain tissues of a scrapie infected sheep and mouse, from joint exudate of an arthritic goat as well as cell cultures established from human, chimpanzee and dog tissues. Subsequently,
*Department of Microbiology, Faculty of Medicine, University of Singapore, College Road, Republic of Singapore. Submitted September 14, 1976.
Volume 41 - July, 1977
The diphasic broth used in the initial isolations consisted of 2 ml of complete mycoplasma agar overlaid with 2 ml of complete mycoplasma broth in one-quarter oz screw capped bottles. Both media were based on those previously described by Tan and Miles (15). Media for strains which utilized arginine for growth consisted of complete mycoplasma broth or agar with 1% L-arginine hydrochloride in place of 1% dextrose. The pH was then adjusted to 6.8-7.0 with 2N
HCl. 349
The strain used for experimental infection was strain MT 293. This strain was originally isolated from the lungs of a very sick cat which was killed in a moribund state.
ISOLATIONS As many swabs as possible were obtained from the eyes, throats, noses, rectum and urogenital tracts of clinically healthy, sick, dead and euthanized cats of both sexes ranging from three days old to 17 years of age. In addition, carcasses of euthanized animals were necropsied and samples from various organs were taken and cultured for mycoplasmas. Blood was obtained aseptically by cardiac puncture. The serum was separated and used for serology. The swabs were inoculated into diphasic broth and incubated at 37°C for two days. Samples were then streaked on mycoplasma agar and incubated in 10% C02 in air at 37°C. These plates were then inspected daily for growth of mycoplasma colonies. Small samples from various organs were minced with sterile scalpel blades and inoculated into diphasic broth and then onto plates as described above. With plates showing no growth, block passes were made onto fresh plates. Specimens were considered negative after the second passage had failed to grow mycoplasmas. Methods used for isolating pathogenic bacteria and viruses in kitten kidney tissue cultures have been described previously (14). MYCOPLASMA STRAIN The original strain of M. arginini was supplied by Dr. W. R. Dowdle. This strain was used to prepare antigens for serology.
PREPARATION OF ANTIGENS
Antigens used for serology were prepared by first cloning each strain three times on complete mycoplasma agar. On the third cloning, one isolated single colony was picked and inoculated into a 2 ml mycoplasma broth and incubated for 48 hours, after which the entire 2 ml broth culture was inoculated into a 20 ml mycoplasma broth and incubated further for 48-60 hours. Ten ml of this broth culture was then inoculated into a 100 ml volume and incubated for three to seven days (according to the growth rate of each strain) under constant agitation. At the end of the incubation period the bulk broth culture was spun at 3,000 X g in a Sorvall RC2-B centrifuge for ten min. The supernatant was then spun at 23,500 X g for 30 min to deposit the cells. The pellet was resuspended with cold phosphate buffered saline (PBS) redeposited and finally suspended in 1/20 of its original volume with cold PBS (giving a final concentration of 2 X 106 cells per ml). This 20X concentrated antigen was used as inoculum for the experimental infection as well as in direct haemagglutination-inhibition (HAI) and indirect haemagglutination (IHA) tests. For use in comple-
TABLE I. Isolations of M. arginini from Catsa
No. swabs and tissues cultured .......... Total positive swabs and tissues Total number isolatesb ........ . M. arginini ......... Ureaplasmasm. a Othersd .............
Thr.e UGTf Rec.9 Trach.h Lungs Others Total Percent
Eyes
Nose
370
267
555
219
85
11
35
99
85
497
55
11
3
6
37
1579 756
1 11 3 6 916 620 73 e2 i 1 81 68 5 16 16 10 4 97 3 759 68 476 60 -.Untypedp.-e -660 aTotal no. of cats tested -555, which included clinically healthy, sick, dead and euthanized cats bSome swabs yielded more than one mycoplasma type cIsolated from brain of cat which died of ataxia dIncluded - M. felis, M. gateae and A. laidlawii eThroat fUrogenital Tract gRectum hTrachea
350
99 2
103 2 101
47.9 8.8 1.7 82.9 6.6
Can. J. comp. Med.
TABLE II. Summary of Clinical Manifestations after Receiving a 20X Concentrated Inoculum of M. arginini Isolate MT 293
Kitten No.
C/Ta
1...............
2................ 3................ 4................ 5................ 6................ 7................ 8................ 9................
C
C T T C C T T C T T
10 ............... 11 ............... aC - control animals, T - test animals bPyrexia is 39°C and above cWeek old
Age 6 wo"
6 wo 6 wo 6 wo 7 wo 7 wo 7 wo 7wo 8 wo 8 wo 8 wo
Route of Inoculation INd, OIe
IN, OI IN, OI IN, OI
IPf IP IP IP I Pul.g I Pul. I Pul.
Duration of Clinical Signs in Days Postinoculation Pyrexiab Conjunctivitis Anorexia
-
-
2 2- 3 2- 4 2- 3
1 2-3 2- 4 2-3
dIntranasal
"Ocular installation fIntraperitoneal gIntrapulmonary
ment fixation (CF) tests the concentrated antigen was boiled for 35 min in a water bath. Antigens for serological tests were preserved with sodium azide (0.08% final concentration) and stored at -20°C until ready for use. PREPARATION OF SPECIFIC ANTISERA
Homologous antiserum against each representative strain was produced in rabbits. Each rabbit was given a sensitizing dose of a mixture of an equal volume of 20X concentrated antigen and Freund's complete adjuvant. This mixture was administered into the foot-pads of both hind legs (0.1 ml per foot-pad). Two weeks later, a booster dose of 2 ml of a 24-48 hours culture containing approximately 105 colony forming units (c.f.u.) per ml was administered intramuscularly. This was then followed by a weekly booster dose of a similar 2 ml volume intramuscularly. The animal was bled before each booster dose. The antibody titre of the serum was determined by growth inhibition tests (5). When the antibody titre had reached a sufficiently high level to inhibit growth of the homologous mycoplasma by at least 2 mm in width by the disc method of Clyde (5), the animal was bled out by cardiac puncture. To avoid antibodies being formed against pig serum globulins used in the enriched
Volume 41
-
July, 1977
media, normal rabbit serum was used in growing cells for antiserum production. IDENTIFICATION OF MYCOPLASMAS
Identification of mycoplasma was performed by the growth inhibition technique of Clyde (5). SEROLOGY
Complement fixation (CF) tests were carried out at 4°C fixation overnight. Complement and antigens were used at four units. A 3 % suspension of sheep erythrocytes was used. Indirect haemagglutination (IHA) tests were carried out in microtitre plates by the method of Herbert (6). EXPERIMENTAL INFECTIONS
Before the experimental infection, all the animals were screened for at least one week. Each experiment was conducted in a different room. The dam and her litter were housed together in a large cage measuring 8 x 5 feet. Both the control and test kittens were housed together and nursed by the same mother. Daily observations were made for clinical signs. Rectal temperatures were taken daily. Swabs were obtained from eyes 351
TABLE III. Isolation of Mycoplasmas, Bacteria and Viruses from Kittens before and after Inoculation of M. arginini Isolate MT 293
Kitten
1............ 2........... 3............ 4............ 5............ 6............ 7............ 8...........
C/Ta C C T T C C
Preinoculation Throats Eyes
M. gateae M. gateae
Days Postinoculation Recovery of M. arginini from Eyes Throats Lactobacillus spp. 1,2 5,6, 10, 14 1, 3, 7, 10, 14 4-6, 10-14, 20 M. gateaeb
T
T
9..........
10 ........... T 11 ........... T aC- control animals, T - test animals bM. gateae was isolated on days 3 and 14 postinoculation
and throats and cultured for mycoplasmas, pathogenic bacteria and viruses. At the end of the observation week the animals were starved for at least 12 hours (to prevent excessive levels of cholesterols in the blood) and were then anaesthetized with sodium pentobarbitone given intraperitoneally. Blood samples were then obtained by cardiac puncture. Swabs were again obtained and cultured for microorganisms as above. Throat swabs were also obtained from all the dams and cultured for mycoplasmas, pathogenic bacteria and viruses.
Experiment I - A litter of four six week old kittens was used. Two were each given 0.2 ml of a 20X concentrated inoculum of M. arginini into each eye and a 0.5 ml volume intranasally into both nostrils. The remaining two kittens were each given similar volumes of sterile saline.
Experiment II - A second litter of four seven week old kittens was used. Two kittens were each given 0.5 ml of the 20X concentrated inoculum intraperitoneally. The other two kittens were each given similar volumes of sterile saline.
Experiment III - In a third litter of three eight week old kittens, two of them were each given an intrapulmonary inoculum of 0.2 ml of the 20X concentrated culture. The remaining kitten was given a similar volume of sterile saline. The inoculum was injected into the right lung by inserting a 1/4 inch x 26G needle between the third and fourth last ribs, approximately equidistant from the sternum to the posterior edge of the rib cage. The procedure was performed under sodium 352
pentobarbitone anaesthesia. At the end of three weeks all the animals killed and necropsied. Organ samples tested for the presence of M. arginini.
were were
RESULTS ISOLATIONS A total of 81 strains of M. arginini was isolated from swabs and tissues cultured from a series of 555 cats (see Table I). Of these, 68 (84%) were isolated from the oropharyngeal regions. The remaining isolates were recovered from eyes (2.5%), nose (2.5%), urogenital tract (6.2%) and rectum (1.2%). Two strains were isolated from lungs of necropsied animals. One strain was recovered from the brain of an old tom cat which had ataxia and was subsequently euthanized. Other mycoplasmas recovered from cats included M. felis, M. gateae, A. laidlawvii and feline ureaplasmas (Unpublished data ). In addition, there were 60 isolates which could not be identified with the antisera of any of the above species by growth inhibition tests.
EXPERIMENTAL INFECTIONS Clinical - All the control animals (kittens nos. 1, 2, 5, 6 and 9) remained clinically normal throughout the experiment. Two kittens (nos. 3 and 4) were also
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TABLE IV. Summary of Serological Tests Performed on Kitten Sera Taken before and after Inoculation of M. arginini Isolate MT 293
C/Te
Kitten 1 ......... 2 ......... 3 ......... 4 ......... 5 ......... 6 ......... 7 ......... 8 ......... 9 ......... 10 ......... 11 .........
C C T T C C T T C T T
Indirect Haemagglutination Tests Bb CC Dd Aa 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1Of 80 0 5 20 160 0 0 0 0 0 10 40 160 160 0 10 40
A 0 0 0 0 0 0 0 0 0 0 0
Complement Fixation Tests C D B 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 10 5 0 40 5 0 0 0 0 40 5 0 40 0 10
aPreinoculation sera bPostinoculation sera taken on day 7 oPostinoculation sera taken on day 14
dPostinoculation sera taken on day 21 eC - control animals, T - test animals Titres in reciprocal dilutions
clinically normal following the inoculation of M. arginini into both eyes and nostrils. The other inoculated animals (nos. 7, 8, 10 and 11) developed slight pyrexia and very mild anorexia following an intraperitoneal or intrapulmonary inoculation as shown in Table II. Isolations - No pathogenic bacteria were isolated from the eyes and throats of all experimental animals before and after inoculation. Neither were viruses isolated from kitten kidney tissue cultures. M. gateae was recovered from the throats of kittens nos. 4 and 6 before inoculation and from throat of kitten no. 5 after inoculation. M. arginini was not isolated from eyes and throats of all animals before inoculation. However, M. arginini was recovered from the eyes of kitten no. 3 on days 1 and 2 after inoculation and from the eyes of kitten no. 4 on days 1, 3, 7, 10 and 14 after inoculation. M. arginini was also recovered from the throat of kitten no. 3 on days 5, 6, 10 and 14 and from the throat of kitten no. 4 on days 4-6, 10-14 and day 20 after inoculation (as shown in Table III). M. arginini was also recovered from the necropsied trachea of kitten no. 4 but not from the other organ samples of all the other animals. M. gateae was isolated from throat swabs of all the dams before inoculation. In addition, M. arginini was recovered from throat swab of the dam in Experiment I at necropsy.
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Serology - Table IV shows the results of indirect haemagglutination (IHA) and complement fixation (CF) tests performed on kitten sera which were obtained at weekly intervals. No antibodies were detected in all preinoculation kitten sera. The postinoculation sera of all control kittens also had no detectable IHA and CF antibodies against M. arginini. Neither were antibodies against M. arginini detected in all postinoculation sera of kittens nos. 3 and 4 following inoculation into the eyes and nostrils. However, low levels of both IHA and CF antibodies were detected in all postinoculation sera of kittens nos. 7, 8, 10 and 11 following intraperitoneal or intrapulmonary inoculation. Necropsy - All the animals were killed and necropsied at day 21 postinoculation. No pathological abnormalities were found in either the control or infected animals.
DISCUSSION A total of 81 strains of M. arginini was isolated from swabs and tissues cultured from a series of 555 cats (see Table I). The majority of these strains (84%) were recovered from the oropharyngeal regions of cats. This suggests that M. arginini
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has an affinity for the mucous membrane of the oropharyngeal regions. The recovery of M. arginini from the lungs of two necropsied sick cats suggests its possible involvement in cat pneumonia. Its recovery from the brain of a cat with ataxia also suggests that M. arginini could be involved in certain disease syndromes of the central nervous system. Unfortunately, no histopathological studies were available for direct correlation with isolation studies. Similar organisms were recovered from brain tissues of a scrapie infected sheep and mouse (3) and from a lioness (7). However, these authors could not establish an etiological role for M. arginini isolated from brains of animals. The isolation of 'M. felis from cats in New Zealand has been reported and characterized (13, 16). Its pathogenic role in young kittens has been described previously (11). The isolations of A. laidlanvii and feline ureaplasmas have been reported elsewhere (12, 15, 18). Their significance in the cat hosts is the subject of another paper (Unpublished data). Results of experimental infections of M. arginini in kittens suggest that our strain MT 293 is not pathogenic for cats. There was no clinical manifestation in two kittens (nos. 3 and 4) following inoculation into the eyes and nostrils. However, kittens nos. 7, 8, 10 and 11 developed a slight pyrexia and mild anorexia. From the authors' own experience, pyrexia and anorexia are not unexpected in animals given a large dose of mycoplasma antigens. In this case, it could probably be due to the pyrogenic properties of M. arginini. The case with which M. arginini could be recovered from the eyes and throats of kittens following intraocular and intranasal inoculations of the 20X concentrated antigens clearly demonstrates the predilection of the organisms for mucous membranes of the eyes and nasopharynx. Its persistence in these sites suggests rapid colonization without any ill effects on the host. Like A. laidlawii, M. arginini also has a wide distribution in nature (3, 4, 9, 10). It was first reported in sick cats (including Siamese and Burmese pedigrees) by Tan and Miles (17). Since then, it has not been reported by workers in other countries, although Heyward et al (7) isolated a strain from the lung and brain of a lioness which died of encephalitis. That isolate was originally named M. leonis (7).
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Subsequently, it was found to be synonymous with M. arginini by Tully et al (19). The relatively high occurrence of M. arginini in cats in this study suggests that M. arginini might be a common inhabitant in this animal host, although it has only been recorded in New Zealand and also here in Singapore.
ACKNOWLEDGMENTS We are greatly indebted to Drs. J. Tully and W. R. Dowdle of the National Institutes of Health, Bethesda, Maryland, U.S.A., for providing us with the strains M. felis, M. gateae and M. arginini. REFERENCES 1. AL-AUBAIDI, J. M., W. D. TAYLOR, G. R. RIJBASH and A. H. DARDIN. Identification and characterization of M. arginini from Bighorn sheep (Ovis canadensis) and goats. Am. J. vet. Res. 33: 87-90. 1972. 2. BARILE, M. F., R. T. SCHIMKE and D. B. RIGGS. Presence of arginine dihydrolase pathway in mycoplasmas. J. Bact. 91: 189-192. 1966. 3. BARILE, M. F., R. A. DELGUIDICE, T. R. CARSKI, C. J. GIBBS and J. A. MORRIS. Isolation and characterization of Mycoplasma arginini: spec. nov. Proc. Soc. exp. Biol. Med. 129: 489-494. 1968. 4. BARILE, M. F. and J. KERN. Isolation of M. arginini from commercial bovine sera and its implication in contaminated cell cultures. Proc. Soc. exp. Biol. Med. 138: 432-437. 1971. 5. CLYDE, W. A. Jr. Mycoplasma species identification based upon growth inhibition by specific antisera. J. Immun. 92: 958-965. 1964. 6. HERBERT, W. J. Indirect haemagglutination. In Handbook of Experimental Immunology. D. M. Weir, Ed. pp. 720-744. Oxford and Edinburgh: Blackwell Scientific Publications. 1967. 7. HEYWARD, J. T., M. Z. SABRY and W. R. DOWDLE. Characterization of mycoplasma species of feline origin. Am. J. vet. Res. 30: 615-622. 1969. 8. HILL, A. The isolation of Mycoplasma arginini from captive wild cats. Vet. Rec. 91: 224-225. 1972. 9. LEACH, R. H. The occurrence of Mycoplasma arginini in several animal hosts. Vet. Rec. 87: 319-320. 1970. 10. STEWART, S. M. and D. YOUNG. Isolation of Mycoplasma arginini from tissue cultures. Lancet 1: 347. 1971. 11. TAN, R. J. S. Susceptibility of kittens to Mycoplasma felis infection. Jap. J. exp. Med. 44: 235-240. 1974. 12. TAN, R. J. S. and J. G. MARKHAM. Feline Tstrain mycoplasmas. Jap. J. exp. Med. 41: 247-248. 1971. 13. TAN, R. J. S. and J. G. MARKHAM. Isolation of mycoplasma from cats with conjunctivitis. N. Z. vet. J. 19: 28. 1971. 14. TAN, R. J. S. and J. A. R. MILES. Further studies on feline respiratory virus diseases. I. Vaccination experiments. N. Z. vet. J. 19: 15-18. 1971. 15. TAN, R. J. S. and J. A. R. MILES. Mycoplasma isolations from clinically normal cats. Br. vet. J. 128: 87-90. 1972. 16. TAN, R. J. S. and J. A. R. MILES. Characterization of mycoplasmas isolated from cats with conjunctivitis. N.Z. vet. J. 21: 27-32. 1973. 17. TAN, R. J. S. and J. A. R. MILES. Incidence and significance of mycoplasmas in sick cats. Res. vet. sci. 16: 27-34. 1974. 18. TAN, R. J. S. and J. A. R. MILES. Possible role of feline T-strain mycoplasmas in cat abortion. Aust. vet. J. 50: 142-145. 1974. 19. TULLY, J. G., R. A. DELGUIDICE and M. F. BARILE. Synonymy of Mycoplasma arginini and Mycoplasma leonis. Int. J. syst. Bact. 22: 47-49. 1972.
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