Journal of Veterinary Diagnostic Investigation

http://vdi.sagepub.com/ Mycoplasma Infection in a Commercial Goat Dairy Caused by Mycoplasma Agalactiae and Mycoplasma Mycoides Subsp. Mycoides (Caprine Biotype)
Hailu Kinde, Al J. DaMassa, Patricia S. Wakenell and R. Petty J VET Diagn Invest 1994 6: 423 DOI: 10.1177/104063879400600404 The online version of this article can be found at: http://vdi.sagepub.com/content/6/4/423

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J Vet Diagn Invest 6:423-427 (1994)

Mycoplasma infection in a commercial goat dairy caused by Mycoplasma agalactiae and Mycoplasma mycoides subsp. mycoides (caprine biotype)
Hailu Kinde, Al J. DaMassa, Patricia S. Wakenell, R. Petty
Abstract. A commercial dairy goat herd of 600 animals experienced sudden onset of arthritis/polyarthritis, clinical mastitis, and sudden death in does. The offending infectious agents were Mycoplasma agalactiae and M. mycoides subsp. mycoides (caprine biotype). The disease syndrome began approximately 4 weeks following the 1) introduction into the herd of a lactating doe with no apparent clinical signs and 2) a breakdown of proper hygienic conditions in the milking parlor. Over a period of 3 weeks, 90 does (15%) either died or were culled because of arthritis/polyarthritis and mastitis. A management decision resulted in only the does affected with M. mycoides subsp. mycoides being submitted for necropsy; those affected with M. agalactiae, which were in a different string, were not submitted for evaluation. Gross necropsy of the does affected with M. mycoides subsp. mycoides showed purulent discharges from the udders, enlarged supramammary lymph nodes, enlarged and firm spleens, and swollen livers. Microscopic findings were characterized by a loss of vascular integrity and diffuse fluid leakage in multiple organs. Antibiotic therapy with tylosin was attempted but was not successful. The outbreak was terminated following the removal or segregation of affected does and implementation of hygienic conditions in the milking parlor.

Several severe outbreaks of caprine mycoplasmosis have been reported in the United States, primarily from the Central Valley of California.2,6,9,11 The most important offending mycoplasmas have been Mycoplasma capricolum subsp. capricolum, M. mycoides subsp. mycoides, and M. putrefaciens. Of those agents, the most prevalent caprine mycoplasma in the United States (and probably worldwide) is M. m. mycoides. In the United States, disease outbreaks associated with M. m. mycoides have produced 90% morbidity and mortality in kids6 and high mortality and culling rates.11 Most outbreaks are usually initiated by M. m. mycoides, although the single, most important outbreak that required the destruction of an entire herd of 700 goats was caused by M. putrefaciens.9 The disease caused by M. agalactiae is of considerable economic importance in goats and sheep worldwide as a cause of arthritis, mastitis, and conjunctivitis.3,10 Mycoplasma agalactiae, although isolated on 2 separate occasions in the United States,5,4 has not until now been associated with disease in either goats or sheep. Clinical signs of mycoplasma infection in milk goats are usually manifested as arthritis/polyarthritis and
From the California Veterinary Diagnostic Laboratory, San Bernardino, CA 92412 (Kinde), the Department of Population Health and Reproduction, University of California, Davis, CA 95616 (DaMassa, Wakenell), and the Chino Valley Veterinary Clinic, 13180 S. Baker Ave., Ontario, CA 91761 (Petty). Received for publication November 4, 1993.

mastitis, but other gross and histopathologic changes also may occur. 10 Among adult goats, these mycoplasmas are not highly contagious, but the reverse is true in kids housed in close confinement with M. capricolum subsp. capricolum and with M. m. mycoides-affected penmates, i.e., normal kids housed in close contact with infected kids readily acquire the infection.7,8 Often, lactating does become infected in the milking parlor because of unsanitary conditions, which result in the introduction of the mycoplasma into the teat canal by the milking machine. Hand milking also can result in the introduction of the mycoplasma into the teat canal if the hands and udder are not properly disinfected before milking. With highly infectious mycoplasma such as M. c. capricolum and M. m. mycoides (and probably M. agalactiae also), kids can readily acquire the infection by the ingestion of the agent through colostrum or milk containing the mycoplasma. 10 Infection of kids via the oral route can also occur with M. putrefaciens.9 The purpose of this report is to describe the clinical, microbiological, pathological, and epidemiologic findings of an unusual outbreak of caprine mycoplasmosis in which Mycoplasma agalactiae and M. m. mycoides were involved. Case history A commercial dairy goat herd of 600 lactating does experienced a sudden onset of arthritis/polyarthritis, clinical mastitis, and death. The herd had been assem-

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bled about 1 year earlier through the purchase of does from several sources throughout California. According to the owner, the disease onset coincided with the introduction into the herd of a clinically normal lactating doe and also with the breakdown of normal hygienic conditions in the milking parlor by newly acquired personnel. A few adult does possessed chronic joint problems (big knees) that were suspected of being caused by the caprine arthritis-encephalitis (CAE) virus, but this suspicion was not confirmed. About 10 days before the beginning of the acute deaths in the does, a single sample of joint fluid from an arthritic doe was submitted to the University of CaliforniaDavis for identification. A glucose- and arginine-negative mycoplasma was recovered from the joint fluid and was identified in this laboratory as Mycoplasma agalactiae, a notable caprine and ovine pathogen responsible for arthritis, mastitis, and other disease syn10 dromes in these hosts. The owner kept about 100 dairy cows on the premises, although he stated that neither cow milk nor colostrum were fed to goats. A total of 90 goats (15%) died or were culled within a period of 3 weeks due to arthritis or polyarthritis and severe mastitis. Forty-six goats died acutely with clinical signs of arthritis or mastitis during a 1-week period. Seven were culled from the milk string because of mycoplasma-positive milk cultures, and 37 were culled because of clinical mastitis and/or arthritis. According to the owner, tylosin at a dose of 10 mg/kg body weight was used to treat goats with clinical mastitis, but the treatment was ineffective. Materials and methods
Specimens submitted. Seven dead lactating goats and 1 dead 2-wk-old kid were submitted to the California Veterinary Diagnostic Laboratory System, San Bernardino Branch, for necropsy examination and diagnosis. In addition, udder secretions and joint fluids from other goats in the hospital pen and 322 composite milk samples from lactating goats were also examined. Necropsy and tissue preparation. Necropsy was performed on all the submitted animals. Sections of lung, spleen, liver, heart, brain, lymph nodes, and supramammary glands were fixed in 10% buffered formalin, sectioned, and stained with hematoxylin and eosin. Microbiology. Specimens of milk, lungs, external ear canals, joint fluids, livers, spleens, brain, and udder secretions were cultured in modified medium B12 and on trypticase soy agar supplemented with 5% defibrinated ovine blood. Ability of the mycoplasma to ferment glucose, hydrolyze arginine, form film and spots, produce phosphatase, and reduce 2,3,5,triphenyltetrazolium chloride was determined by methods described previously.4 Viable mycoplasma counts were determined as described elsewhere. 16 Mycoplasma identification. Mycoplasmas were identified at the University of California-Davis by a growth-inhibition procedure,1 modified by using agar wells instead of antise-

rum-impregnated discs. All isolates were examined further by either direct or indirect immunofluorescence. 13 Antiserum. Antiserum used to identify Mycoplasma mycoides subsp. mycoides was prepared in rabbits using the GM126 isolate of the mycoplasma. Antiserum used to identify M. agalactiae originated from 2 sources and was of 2 different animal origins: 1) rabbit antiserum prepared against the GM 1395 isolate of the mycoplasma and 2) a direct fluorescein-isothiocyanate reference antiserum conjugate prepared in horses against the PG2 strain of M. agalactiae (National Institutes of Health reference reagent).a

Results Microbiological findings. The milk of all does submitted for necropsy were mycoplasma positive on B mycoplasma agar. The following sites were also mycoplasma positive in all necropsied goats: livers, lungs, spleens, joint fluids (atlanto-occipital, coxofemoral), and external ear canals. Only the spleen and external ear canal were mycoplasma positive in the kid. Two distinct mycoplasma colony types were recovered, designated here as types A and B. Type A colonies were recovered only from the goats submitted for necropsy, i.e., from ear canals, livers, lungs, spleens, joint fluids, and milk. They grew rapidly on mycoplasma agar and attained a colony diameter of about 1.5-2.0 mm after 48-72 hours of incubation at 37 C. They were glucose positive, arginine negative, and phosphatase positive, did not form film and spots, and did not reduce 2,3,5,triphenyltetrazolium chloride. Type A colonies grew well on ovine blood agar with the production of a viridans type (=alpha) hemolysis that later changed to a beta type after an additional 2-4 days of incubation. Type B colonies were recovered only from nonnecropsied goats: 1) 1 sample of joint fluid from an individual goat and 2) 7 out of 322 milk samples. These colonies grew more slowly (in comparison to type A colonies) and attained a diameter on mycoplasma agar of about 1.0 mm after 4-5 days of incubation at 37 C. Glucose was not fermented, and arginine was not hydrolyzed. Film and spots were formed on B agar. Phosphatase was produced and 2,3,5,triphenyltetrazolium chloride was reduced. Type B colonies grew poorly on ovine blood agar, requiring several days (5-7) for growth to be discernible. Mycoplasma identification. Type A colonies from the milk of goats submitted for necropsy were inhibited markedly in the growth-inhibition test (6-8 mm) by rabbit antiserum to M. mycoides subsp. mycoides and were further confirmed to be that species by indirect immunofluorescence. Type B colonies (from nonnecropsy specimens, samples B) reacted in the growthinhibition test (4-6 mm inhibition) using antiserum to M. agalactiae strain GM139 and further reacted strongly in the direct immunofluorescence test using fluorescein-labeled antibody to M. agalactiae strain

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Figure 1.

Section of liver of a goat with mycoplasmosis showing focal necrosis and inflammatory cell infiltration.

PG2. For purposes of reference, a colony identified as M. m. mycoides was chosen as the representative type and was designated GM 1085B; for M. agalactiae, the representative type (from samples B) was designated GM1085G. Mycoplasma enumeration in milk samples. Milk samples from goats affected with M. m. mycoides contained 1 106-1 109 colony-forming-units (CFU)/ ml of the mycoplasma. The corresponding values in the milk of goats affected with M. agalactiae (milk samples B) were 1 x 106-1 x 108 CFU/ml. Gross necropsy. The lungs of the adult goats had gray, patchy, emphysematous areas in some animals but were plum colored and atelectic in others. Spleens were swollen and firm. Udders were firm and swollen and contained thick yellowish secretions. The lesions in the kid were fibrinous pleuritis and peritonitis. A soft nodule (0.5 cm diameter) was present on the apical lobe of the left lung. Microscopic findings. The primary changes in the tissues of the adult goats were loss of vascular integrity and diffuse fluid leakage in multiple organs. Within the livers there were microthrombi, erythrophagocytosis, fatty vacuolation of the hepatocytes, Kupffer cell hy-

perplasia, and occasional foci of necrosis (Fig. 1). The spleen showed mild lymphoid depletion and erythrophagocytosis. The alveoli of the lungs were filled with proteinaceous fluid. Within the mammary glands there was fibrinocaseous exudation in the ductal lumen, interstitial fibrosis, ulceration of the ductal lumen (Fig. 2), and loss of architecture due to large granuloma formation. Lesions in the lungs of the kid were more severe than in the lungs of the adults and were characterized by pulmonary edema, multifocal caseating granulomas, and chronic peribronchial inflammation. Fatty vacuolations of the hepatocytes and focal accumulations of mononuclear cells in the meninges (meningitis) were also noted. Other tissues were unremarkable. Discussion The results of this investigation clearly show that M. agalactiae and M. mycoides subsp. mycoides were involved in this mycoplasmosis outbreak, although affected animals harbored only 1 mycoplasma species, i.e., no animals examined were infected with both mycoplasma species. The outbreak is unusual from the point of view that mycoplasmosis outbreaks in goats

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Figure 2. fibrosis.

Section of a mammary gland of a goat with mycoplasmosis showing fibrinocaseous exudate in ductal lumen and interstitial

attributed to more than 1 mycoplasma species at 1 time is highly unusual. The current outbreak is also important epidemiologically because it incriminates M. agalactiae in a disease process for the first time in the United States; it was recovered in pure culture from mastitic milk with viable counts ranging from 1 x 106 to 1 x 108 CFU/ml. Although M. agalactiae was reported previously on 2 occasions in the United States,5,14 neither report incriminated the mycoplasma in a disease process. In this country, M. agalactiae was first isolated from the joint fluid of an emaciated goat doe necropsied because of Johnes disease in 1969,14 and in 1983 the mycoplasma was recovered for the second time from the milk of a goat with mastitis,5 although it could not be ascertained at that time if the agent was the specific cause of the clinical disease. The isolation of M. agalactiae in this report and its implication as a cause of overt disease (mastitis) and the isolation of the agent from the joint fluid of a goat in this study implies that this agent is pathogenic and is firmly entrenched in the goat population of this country. From an epidemiological viewpoint, it is unfortu-

nate that no goats from which M. agalactiae was isolated were submitted for necropsy. The reasons for this are obscure, but the most likely explanation may be that after submission of 8 goats for necropsy, the dairy management considered that no further submissions were necessary. All the goats on the premises shared a common milking parlor, although they were in 2 different strings and were managed by the same personnel. The reasons for the absence of concurrent infection of individual animals by both species of mycoplasma is unknown. The gross and histopathologic changes in the goats affected with M. m. mycoides were consistent with previously reported results6,8,10,17,18 and included purulent discharges from the udders, excess joint fluid in some animals, enlarged supramammary lymph nodes, and evidence of septicemia with enlarged, firm spleens and swollen livers. Histopathologic changes were characterized by an interstitial pneumonia, loss of vascular integrity and diffuse fluid leakage in multiple organs, and occasional granuloma formation. Lesions in the kid were similar to those in previous studies and lend supporting, additional evidence that in young animals

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infected with this agent a meningitis is usually present. Inflammation of the meninges is not normally present in adults infected with M. m. mycoides. The source of the infection in this outbreak is unclear. There is no doubt that mycoplasma can be spread in the milking parlor, both in bovine and caprine facilities. However, it is not known whether the introduction of a new lactating doe into this herd was the source of the infection. In this case, the outbreak began about 4 weeks after a new doe was introduced, but prior studies have shown that mastitis begins within 1-3 days following the introduction of caprine mycoplasma into the teat canal. A more plausible reason for the outbreak is a breakdown in the sanitary conditions, which has been the reason for several past mycoplasmosis outbreaks in goat herds. The presence of about 100 dairy cows on the premises is probably of no consequence in this outbreak because M. agalactiae is not known to be harbored by cows. The possible presence of the caprine CAE virus in this herd also appears to be unimportant in this outbreak. Several features distinguish this mycoplasmosis outbreak from CAE, including 1) recovery of specific mycoplasma in high concentration from the milk of affected goats, 2) sudden acute deaths, and 3) recovery of the mycoplasma from multiple organs of affected goats (septicemia). Further, the gross and histopathologic changes were consistent with previously reported data from mycoplasmosis outbreaks. Occasionally, chlamidia are reported as a cause of polyarthritis in goats. 15 However, these reports have not been substantiated; there is no documented evidence of experimental reproduction of polyarthritis by chlamidial agents. In this study, chlamidia were not considered because of the presence of other agents, i.e., mycoplasmas that are known to be a cause of disease. In the United States, M. agalactiae is classified by the US Department of Health and Human Services and the Centers for Disease Control19 as a nonindigenous pathogen of domestic livestock and that the importation, possession, or use of [M. agalactiae] is prohibited or restricted by law or by U. S. Dept. of Agriculture Regulations or administrative policies. In view of the occurrence of M. agalactiae in an active disease process in this study and the recorded occurrence of this agent in the goat poputation of the United 5,14 States since 1969, the pathogenicity of M. agalactiae isolates in the United States needs to be further defined, because this species continues to create confusion for regulatory agencies. Sources and manufacturers
a. Dr. J. G. Tully, National Institute of Allergy and Infectious Diseases, Frederick, MD.

References
1. Clyde WA Jr: 1964, Mycoplasma species identification based upon growth inhibition by specific antisera. J Immunol 92:958965. 2. Cordy DR, Adler HE, Yamamoto R: 1955, A pathogenic pleuropneumonialike organism from goats. Cornell Vet 45:25-30. 3. Cottew GS: 1979, Caprine-ovine mycoplasmas. In: The mycoplasmas, vol. II. Human and animal mycoplasmas, ed. Tully JG, Whitcomb RF, pp. 103-132. Academic Press, San Francisco, CA. 4. Cottew GS: 1983, Recovery and identification of caprine and ovine mycoplasmas. Methods Mycoplasmal 1:91-104. 5. DaMassa AJ: 1983, Recovery of Mycoplasma agalactiae from mastitic goat milk. J Am Vet Med Assoc 183:548-549. 6. DaMassa AJ, Brooks DL, Adler HE: 1983, Caprine mycoplasmosis: widespread infection in goats with Mycoplasma mycoides subsp. mycoides (large colony type). Am J Vet Res 44: 322-325. 7. DaMassa AJ, Brooks DL, Adler HE, Watt DE: 1983, Caprine mycoplasmosis: acute pulmonary disease in newborn kids given Mycoplasma capricolum orally. Aust Vet J 60:125-126. 8. DaMassa AJ, Brooks DL, Holmberg CA: 1986, Induction of mycoplasmosis in goat kids by oral inoculation with Mycoplasma mycoides subsp. mycoides. Am J Vet Res 47:2084-2089. 9. DaMassa AJ, Brooks DL, Holmberg CA, Moe AI: 1987, Caprine mycoplasmosis: an outbreak of mastitis and arthritis requiring the destruction of 700 goats. Vet Rec 120:409-413. 10. DaMassa AJ, Wakenell PS, Brooks DL: 1992, Mycoplasmas of goats and sheep. J Vet Diagn Invest 4:101-113. 11. East NE, DaMassa AJ, Logan LL, et al.: 1983, Milkborne outbreak of Mycoplasma mycoides subspecies mycoides infection in a commercial goat dairy. J Am Vet Med Assoc 182:13381341. 12. Freundt EA: 1983, Culture media for classic mycoplasmas. In: Methods in mycoplasmology, ed. Razin S, Tully JG, vol. 1, pp. 127-135. Academic Press, San Francisco, CA. 13. Gardella RS, DelGuidice RA: 1983, Immunofluorescence. Methods Mycoplasmal 1:431-439. 14. Jasper DE, Dellinger JD: 1979, Isolation of exotic mycoplasma from goats. Proc Annu Meet Am Assoc Vet Lab Diagn 22:119124. 15. Matthews JM: 1991, Outline of clinical diagnosis in the goat, p. 87. Wright, London, UK. 16. Rodwell AW, Whitcomb RF: 1983, Methods for direct measurement of mycoplasma growth. Methods Mycoplasmal 1:185196. 17. Rosendal S: 1981, Experimental infection of goats, sheep, and calves with the large colony type of Mycoplasma mycoides subsp. mycoides. Vet Pathol 18:71-81. 18. Rosendal S: 1983, Susceptibility of goats and calves after experimental inoculation or contact exposure to a Canadian strain of Mycoplasma mycoides subsp. mycoides isolated from a goat. Can J Comp Med 47:484-490. 19. US Department of Health and Biomedical Laboratories: 1984, Biosafety in microbiological and biomedical laboratories. Public Health Service, Centers for Disease Control, and National Institutes of Health. US Government Printing Office, Washington, DC. p. 89.

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