- Email: [email protected]
Immunohistochemical and ultrastructural characteristics of Mycoplasma capricolum subsp. capricolum in caprine abortion: A case report
ELSEVIER
IMMUNOHISTOCHEMICAL AND ULTRASTRUCTURAL CHARACTERISTICS OF Mycoplasma capricolum subsp. capricolum IN CAPRINE ABORTION: A CASE REPORT J.L. Rodriguez,‘” D.L. Brooks,’ A.J. DaMassa,’ J. Ores’ and A. Fernandez’ ‘Department of Histology and Pathological Anatomy University of Las Palmas de Gran Canaria, Spain ‘Department of Medicine ‘Department of Population Health and Reproduction School of Veterinary Medicine, University of California, Davis, CA 95616 USA Received
for publication: Accepted:
September
Octubt?r>16,
II, 199s 1995
ABSTRACT Described in this study are the immunohistochemical and ultrastructural findings in a case of caprine abortion due to the experimental infection of the dam with strain GM13 of Mvcoplasma capricolum subsp. capricolum. Mycoplasma antigens were seen mainly in choriallantoic trophoblasts and in the lumen of blood vessels in the allantoic membrane. Examination with an electron microscope showed that the chorioallantoic trophoblasts were filled with typical mycoplasma organisms. No other bacteria were observed in any of the samples. Our results confirm by immunohistochemical and electron microscopic techniques that Mycoplasma capricolum subsp. caoricolum can cause caprine abortion and that the process can occur without premonitory signs. Key w&s:
Mycoplasma capricolum, caprinc, abortion. immunohistochemistry, electron microscopy. INTRODUCTION
In the United States, certain caprine mycoplasma, particularly Mvcoolasma mvcoides subsp. mvcoides (Mmm; large colony or caprine biotypes) and M. caoricolum caoricolum (Mcc) can be highly virulent and pathogenic in goat populations. subsp. The former (Mmm) is widespread in the United States while the latter (Mcc) occurs only sporadically. Both can cause acute to peracute disease in caprine species of all ages, although the disease is most severe in very young animals (9). With both subspecies of Acknowledgments: The authors thank Pedro Castro and Ana Afonso for their excellent technical assistance and Mr. R. W. Nordhausen for electron photographic processing. aPresent address: Jose Luis Rodriguez Navarro. Facultad de Veterinaria. U.L.P.G.C. C/ Francisco Inglott Artiles, 12A. 35016. Canary Islands. Spain.
Theriogenology 46:37%385, 1996 0 1996 by Elsevier Science Inc.
0093-691 X/96/$1 5.00 PII SOO93-691X(96)00160-7
Theriogenology
380
mycoplasmas, the usual clinical manifestations that are observed include agalactia, arthritis or polyarthritis, conjunctivitis, mastitis, and septicemia (2, 9, 16). An outbreak in the Central Valley of California was associated with M. mvcoides subsp. mvcoides and resulted in a 90% mortality rate (about 200 young) in the new kid crop (6). To our knowledge, vertical transmission has not been suggested in M. cauricolum subsp. canricolum infection, although this mycoplasma has been isolated from bull semen (3) and vulvar scabs of ewes (13). However, congenital infection has been suggested with mycoplasmas belonging to the Mvcoplasma mvcoides cluster (2,14,19) and Mvcoolasma mvcoides subsp. mvcoides has been isolated from natural aborted caprine fetuses (15). Although there has been no supporting documentation in the USA, abortions in goat herds naturally infected with Mvcoulasma mvcoides subsp. mvcoides have been observed (A. J. DaMassa and D. L. Brooks, unpublished data). The purpose of this study was to examine the effect of Mvconlasma canricolum subsp. cabricolum infection on abortion in the goat. The results indicated infection of the chorioallatoic trophoblasts in a goat experimentally infected with the GM13 strain of Mvcoolasma caoricolum subsp. capricolum. The dam aborted during the course of study involving the pathogenesis of Mvconlasma canricolum subsp. canricolum. MATERIALS AND METHODS Animals An adult, pregnant Nubian goat (pasture bred and at the mid stage of gestation) from a herd maintained at the University of California at Davis was used in the study. This closed goat herd has been observed for more than 30 yr and has had no history of mycoplasma, chlamidia, Brucella, Q fever, campylobacter, leptospira, listeria, salmonella, or toxoplasma infections, all of which are possible infectious causes of caprine abortion (I 8). The experimental animal had been challenged intratracheally with 1 ml of a 48-h culture of the GM13 strain (7) of M. cauricolum subsp. canricolum at a titer of 1 X 10’ color-changing units/ml (about 1 X 10’ colony-forming units/ml). Microbiological and Pathological Methods Throughout the study, mycoplasma solid and liquid media “B” was used for propagation and/or identification trials (10). Identity of the mycoplasmal isolates was confirmed by preliminary biochemical determinations, followed by growth inhibition and/or immunofluorescence procedures (4,ll).
381
Theriogenology At necropsy, in paraffin-wax.
tissue samples Sections
immunohistochemical buffered
formaiin
were
technique. for transmission
were fixed in 10% buffered stained
with
hematoxylin-eosin,
Some placentome electron
formalin
samples
and embedded
giemsa
and by an
were also fixed in 10%
microscopy.
After necropsy, the placenta of the dam and fetal tissues were forwarded to the California Veterinary Diagnostic Laboratory System (Davis) for possible exclusion of clamidia, toxoplasma, campylobacter, salmonella, Brucella, Q fever, leptospira and listeria organisms. Polyclonal
Antibodies
Polyclonal antibodies against Mcc strains GM 13 and California Kid were produced at the University of California at Davis. These antibodies were produced in rabbits by a procedure described elsewhere (17). Species specificity was confirmed by a disc growth-inhibition test. Immunohistochemical
Methods
Mycoplasma
antigens
were demonstrated
using an immunohistochemical
method
(Dako LSAB kit, Dako Inc, Burlingame, CA, USA) as previously described (12). Briefly, 3-mm-thick sections were adhered to polylysine-coated glass slides, then they were deparaffinized in xylene and rehydrated. Endogenous peroxide was inhibited by incubation of the sections in a freshly prepared solution of 3% hydrogen peroxide in absolute methanol for 20 min. Sections were washed in phosphate buffered saline (PBS) and placed into a solution
of 0.1% protease (pH 7.2) for 5 min at room temperature.
The
sections were then washed 3 times in PBS and overlaid with normal goat serum (diluted 1:20) for 30 min to prevent background staining. Primary polyclonal antibody was diluted l:l,OOO and applied on sections and incubated 2 h. Incubation steps were performed
at room temperature
in a humidified
chamber.
Sections
were rinsed
in PBS
and incubated with biotinylated goat anti-rabbit IgG serum from a universal kit (Dako LSAB) (1:5 dilution) for 30 min, then rinsed in PBS and incubated for 30 min with streptavidin-peroxidase (A.E.C.
substrate
complex reagent (Dako LSAB, 1:5 dilution).
system, Dako, Santa Barbara,
Aminoethylcarbazole
CA) was used as a chromogen
and was
incubated for 4 to 5 min. The sections were lightly counterstained with Mayer hematoxylin, and coverslips were applied over an aqueous mounting medium. Routine negative controls, either noninfected tissues or nonrelated antisera, were used. Moreover, the anti-Mcc antibodies were either omitted or substituted with normal goat serum for the negative controls.
Theriogenology
382 RESULTS During clinical
the first 22 d following intratracheal
signs of disease.
An examination
infection revealed no clinical abnormalities, goat had aborted. The goat was immediately
challenge,
the study goat did not show
of the goat on the morning
of Day 23 post
but 2 h later, on the next observation, the euthanized with an overdose of intravenous
barbiturate solution. At necropsy, gross changes were confined to the uterus and the The lesions were mainly in one uterine horn and were characterized by placenta. necrosis and congestion of the caruncle surface (Figure 1). The cotyledons were homogeneously congested and were necrotic. Histopathological examination indicated diffuse, suppurative, necrotic placentitis. The most prominent placental lesions consisted
of diffuse necrosis and ulceration of the chorioallantoic membrane but the endometrial epithelium was intact. Giemsa staining showed that the chorionic trophoblasts were filled with intracellular coccoid bodies suggestive of mycoplasma. Immunohistochemical examination showed that the cytoplasm chorioallantoic trophoblasts and the lumen of blood vessels in the membrane were filled with strong punctiform reactions (Figure 2). intensive immunoreaction was observed in the chorionic villi and in the areas of the carucle surface. Immunohistochemical study of the fetus possible because the fetus was dead at abortion and highly autolyzed.
Figure
1.
Uterus. Necrosis and congestion (arrows) of the caruncle surface mainly in one uterine horn.
of the allantoic A less necrotic was not
383
Theriogenology
Figure 2.
Chorioallantoic membrane. (LSAB method). Mycoplasma antigens were observed mainly in the cytoplasm of remaining trophoblasts (large arrow) and in the lumen of blood vessels (small arrows). (x1000)
Figure 3.
membrane. Transmission electron Choriallantoic photomicrograph of a trophoblast cell containing numerous mycoplasma organisms within the cytoplasm. (Bar= 0.9 micrometers)
384
Theriogenology
Under electron microscopy, the chorioallantoic trophoblasts were filled with typical mycoplasmas spp. (Figure 3). No other bacteria were seen in the samples observed. Mvcoplasma caoricolum subsp. caoricolum was reisolated from the uterus, placenta and from fetal tissues. However, attempts to isolate chlamidia, toxoplasma, campylobacter, salmonella, Brucella, Q fever, leptospira and listeria were negative. DISCUSSION The lack of observed clinical signs in our study animal suggests a disparity in the severity of the GM13 strain compared with that of previous trials in goats (5,6,8). These differences may indicate an age-dependent susceptibility in the goat, the route of administration or the infection dosage (5,6). Based on immunohistochemical results, the erythrophagocytic and chorioallantoic trophoblasts may play a role in the entry and spread of mycoplasma. During bacterimia, the mycoplasmas may be phagocytosed by erythrophagocytic trophoblasts along with the maternal erythrocytes (1). However, neither the endometrial stroma nor the epithelium appeared to be important in placental infection since the lesions were minimal in these areas and were negative following immunohistochemical examination. Hence, we believe that mycoplasma may initially infect placentomal trophoblasts through the vascular system, as occurs in caprine bacterial abortions (1). Further experimental studies are required to confirm this statement. The results of our study appear to confirm, by immunohistochemical and electron microscopy techniques, that M. canricolum subsp. cauricolum can cause caprine abortion and that the process can occur without premonitory signs. REFERENCES 1.
2. 3.
4.
Anderson TD, Meador VP, Cheville NE Pathogenesis of placentitis in the goat inoculated with ~Brucella -. abortus I. Gross and histologic lesions. Vet Path01 1986;23:219-226. Bar-Moshe B, Rapaport E. Observations on MvcoDlasma mvcoides subsp. mvcoides in Saanen goats. Isreli J Med Sci 1981; 17:537-539. Breard A, Poumarat F. Isolement de MvcoDlasma capricolum a partir d ‘un sperme de taureau. (Isolation of Mycoplasma capricolum from bull semen). Rev Elev MM Vet Pays Trop 1988;41: 149-150. Clyde WA. Mycoplasma species identification based upon growth inhibition by specific antisera. J Immunol 1964;92:958-965.
Theriogenology 5. 6.
7.
8. 9. 10. 11. 12.
13. 14.
15.
16. 17. 18.
19.
385
Cordy DR. Septicaemia and pneumonia in Mvcoplasma capricolum infections in young goats. Aust Vet J 1984;61:202. DaMassa AJ, Brooks DL, Adler HE. Caprine mycoplasmosis: widespred infection in goats with Mvcoulasma mvcoides subsp. mvcoides (large colony type). Am J Vet Res 1983a;44:322-325. DaMassa AJ, Brooks DL, Adler HE, Watt DE. Caprine mycoplasmosis: acute pulmonary disease in newborn kids given Mvcoplasma capricolum orally. Aust Vet J 1983b;60: 125-126. DaMassa AJ, Porter TL. Acute disease in lambs caused by a Mvcoolasma species. Vet Ret 1987;121:166-167. DaMassa AJ, Wakenell PS, Brooks DL. Mycoplasmas of goats and sheep. J Vet Diagn Invest 1992;4:101-113. Freundt E A. Culture media for classic mycoplasmas. Methods Mycoplasmol 1983;1:127-135. Gardella RS, DelGiudice RA, Tully JG. Immunofluorescence. Methods Mycoplasmol 1983;1:431-439. Hsu SM, Raine L, Fanger H. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody PAP) procedure. J Histochem 1981;29:577-588. Jones GE, Rae AG, Holmes RG. Isolation of exotic mycoplasmas from sheep in England. Vet Ret 1983;113:540. Kapoor PK, Garg DN, Mahajan SK. Isolation of Mvcoplasma mycoides subsp. mvcoides (LC variant, Y-goat) from naturally aborted bovine fetuses. Theriogenology 1989;32: 683-69 I. Rodriguez JL, Espinosa de 10s Monteros A, Herraez P, Poveda JB, Fernandez A. Isolation of Mvcoplasma mvcoides subsp. mvcoides (LC Variant) from two naturally aborted caprine fetuses. Theriogenology 1995;44: 1003- 1009. Rosendal S. Experimental infection of goats, sheep, and calves with the large colony type of Mycoplasma mycoides subsp. mvcoides. Vet Path01 1981;18:71-81. Senterfit, LB. Preparation of antigens and antisera. Methods Mycoplasmol 1983;1:401-404. Smith MC, Sherman DM. Reproductive system. In: C.C. Carnn, S. Hunsberger, R. Lukens and M. Denardo (Eds). Goat Medicine. Lea and Febiger, N.Y. 1994;411-463. Villalba EJ, Poveda JB, Ferntindez A, Rodriguez JL, GutiCrrez C, GhmezVillamandos JC. An outbreak caused by Mvcoolasma mvcoides species in goats in the Canary Islands. Vet Ret 1992; 130:330-331.
IMMUNOHISTOCHEMICAL AND ULTRASTRUCTURAL CHARACTERISTICS OF Mycoplasma capricolum subsp. capricolum IN CAPRINE ABORTION: A CASE REPORT J.L. Rodriguez,‘” D.L. Brooks,’ A.J. DaMassa,’ J. Ores’ and A. Fernandez’ ‘Department of Histology and Pathological Anatomy University of Las Palmas de Gran Canaria, Spain ‘Department of Medicine ‘Department of Population Health and Reproduction School of Veterinary Medicine, University of California, Davis, CA 95616 USA Received
for publication: Accepted:
September
Octubt?r>16,
II, 199s 1995
ABSTRACT Described in this study are the immunohistochemical and ultrastructural findings in a case of caprine abortion due to the experimental infection of the dam with strain GM13 of Mvcoplasma capricolum subsp. capricolum. Mycoplasma antigens were seen mainly in choriallantoic trophoblasts and in the lumen of blood vessels in the allantoic membrane. Examination with an electron microscope showed that the chorioallantoic trophoblasts were filled with typical mycoplasma organisms. No other bacteria were observed in any of the samples. Our results confirm by immunohistochemical and electron microscopic techniques that Mycoplasma capricolum subsp. caoricolum can cause caprine abortion and that the process can occur without premonitory signs. Key w&s:
Mycoplasma capricolum, caprinc, abortion. immunohistochemistry, electron microscopy. INTRODUCTION
In the United States, certain caprine mycoplasma, particularly Mvcoolasma mvcoides subsp. mvcoides (Mmm; large colony or caprine biotypes) and M. caoricolum caoricolum (Mcc) can be highly virulent and pathogenic in goat populations. subsp. The former (Mmm) is widespread in the United States while the latter (Mcc) occurs only sporadically. Both can cause acute to peracute disease in caprine species of all ages, although the disease is most severe in very young animals (9). With both subspecies of Acknowledgments: The authors thank Pedro Castro and Ana Afonso for their excellent technical assistance and Mr. R. W. Nordhausen for electron photographic processing. aPresent address: Jose Luis Rodriguez Navarro. Facultad de Veterinaria. U.L.P.G.C. C/ Francisco Inglott Artiles, 12A. 35016. Canary Islands. Spain.
Theriogenology 46:37%385, 1996 0 1996 by Elsevier Science Inc.
0093-691 X/96/$1 5.00 PII SOO93-691X(96)00160-7
Theriogenology
380
mycoplasmas, the usual clinical manifestations that are observed include agalactia, arthritis or polyarthritis, conjunctivitis, mastitis, and septicemia (2, 9, 16). An outbreak in the Central Valley of California was associated with M. mvcoides subsp. mvcoides and resulted in a 90% mortality rate (about 200 young) in the new kid crop (6). To our knowledge, vertical transmission has not been suggested in M. cauricolum subsp. canricolum infection, although this mycoplasma has been isolated from bull semen (3) and vulvar scabs of ewes (13). However, congenital infection has been suggested with mycoplasmas belonging to the Mvcoplasma mvcoides cluster (2,14,19) and Mvcoolasma mvcoides subsp. mvcoides has been isolated from natural aborted caprine fetuses (15). Although there has been no supporting documentation in the USA, abortions in goat herds naturally infected with Mvcoulasma mvcoides subsp. mvcoides have been observed (A. J. DaMassa and D. L. Brooks, unpublished data). The purpose of this study was to examine the effect of Mvconlasma canricolum subsp. cabricolum infection on abortion in the goat. The results indicated infection of the chorioallatoic trophoblasts in a goat experimentally infected with the GM13 strain of Mvcoolasma caoricolum subsp. capricolum. The dam aborted during the course of study involving the pathogenesis of Mvconlasma canricolum subsp. canricolum. MATERIALS AND METHODS Animals An adult, pregnant Nubian goat (pasture bred and at the mid stage of gestation) from a herd maintained at the University of California at Davis was used in the study. This closed goat herd has been observed for more than 30 yr and has had no history of mycoplasma, chlamidia, Brucella, Q fever, campylobacter, leptospira, listeria, salmonella, or toxoplasma infections, all of which are possible infectious causes of caprine abortion (I 8). The experimental animal had been challenged intratracheally with 1 ml of a 48-h culture of the GM13 strain (7) of M. cauricolum subsp. canricolum at a titer of 1 X 10’ color-changing units/ml (about 1 X 10’ colony-forming units/ml). Microbiological and Pathological Methods Throughout the study, mycoplasma solid and liquid media “B” was used for propagation and/or identification trials (10). Identity of the mycoplasmal isolates was confirmed by preliminary biochemical determinations, followed by growth inhibition and/or immunofluorescence procedures (4,ll).
381
Theriogenology At necropsy, in paraffin-wax.
tissue samples Sections
immunohistochemical buffered
formaiin
were
technique. for transmission
were fixed in 10% buffered stained
with
hematoxylin-eosin,
Some placentome electron
formalin
samples
and embedded
giemsa
and by an
were also fixed in 10%
microscopy.
After necropsy, the placenta of the dam and fetal tissues were forwarded to the California Veterinary Diagnostic Laboratory System (Davis) for possible exclusion of clamidia, toxoplasma, campylobacter, salmonella, Brucella, Q fever, leptospira and listeria organisms. Polyclonal
Antibodies
Polyclonal antibodies against Mcc strains GM 13 and California Kid were produced at the University of California at Davis. These antibodies were produced in rabbits by a procedure described elsewhere (17). Species specificity was confirmed by a disc growth-inhibition test. Immunohistochemical
Methods
Mycoplasma
antigens
were demonstrated
using an immunohistochemical
method
(Dako LSAB kit, Dako Inc, Burlingame, CA, USA) as previously described (12). Briefly, 3-mm-thick sections were adhered to polylysine-coated glass slides, then they were deparaffinized in xylene and rehydrated. Endogenous peroxide was inhibited by incubation of the sections in a freshly prepared solution of 3% hydrogen peroxide in absolute methanol for 20 min. Sections were washed in phosphate buffered saline (PBS) and placed into a solution
of 0.1% protease (pH 7.2) for 5 min at room temperature.
The
sections were then washed 3 times in PBS and overlaid with normal goat serum (diluted 1:20) for 30 min to prevent background staining. Primary polyclonal antibody was diluted l:l,OOO and applied on sections and incubated 2 h. Incubation steps were performed
at room temperature
in a humidified
chamber.
Sections
were rinsed
in PBS
and incubated with biotinylated goat anti-rabbit IgG serum from a universal kit (Dako LSAB) (1:5 dilution) for 30 min, then rinsed in PBS and incubated for 30 min with streptavidin-peroxidase (A.E.C.
substrate
complex reagent (Dako LSAB, 1:5 dilution).
system, Dako, Santa Barbara,
Aminoethylcarbazole
CA) was used as a chromogen
and was
incubated for 4 to 5 min. The sections were lightly counterstained with Mayer hematoxylin, and coverslips were applied over an aqueous mounting medium. Routine negative controls, either noninfected tissues or nonrelated antisera, were used. Moreover, the anti-Mcc antibodies were either omitted or substituted with normal goat serum for the negative controls.
Theriogenology
382 RESULTS During clinical
the first 22 d following intratracheal
signs of disease.
An examination
infection revealed no clinical abnormalities, goat had aborted. The goat was immediately
challenge,
the study goat did not show
of the goat on the morning
of Day 23 post
but 2 h later, on the next observation, the euthanized with an overdose of intravenous
barbiturate solution. At necropsy, gross changes were confined to the uterus and the The lesions were mainly in one uterine horn and were characterized by placenta. necrosis and congestion of the caruncle surface (Figure 1). The cotyledons were homogeneously congested and were necrotic. Histopathological examination indicated diffuse, suppurative, necrotic placentitis. The most prominent placental lesions consisted
of diffuse necrosis and ulceration of the chorioallantoic membrane but the endometrial epithelium was intact. Giemsa staining showed that the chorionic trophoblasts were filled with intracellular coccoid bodies suggestive of mycoplasma. Immunohistochemical examination showed that the cytoplasm chorioallantoic trophoblasts and the lumen of blood vessels in the membrane were filled with strong punctiform reactions (Figure 2). intensive immunoreaction was observed in the chorionic villi and in the areas of the carucle surface. Immunohistochemical study of the fetus possible because the fetus was dead at abortion and highly autolyzed.
Figure
1.
Uterus. Necrosis and congestion (arrows) of the caruncle surface mainly in one uterine horn.
of the allantoic A less necrotic was not
383
Theriogenology
Figure 2.
Chorioallantoic membrane. (LSAB method). Mycoplasma antigens were observed mainly in the cytoplasm of remaining trophoblasts (large arrow) and in the lumen of blood vessels (small arrows). (x1000)
Figure 3.
membrane. Transmission electron Choriallantoic photomicrograph of a trophoblast cell containing numerous mycoplasma organisms within the cytoplasm. (Bar= 0.9 micrometers)
384
Theriogenology
Under electron microscopy, the chorioallantoic trophoblasts were filled with typical mycoplasmas spp. (Figure 3). No other bacteria were seen in the samples observed. Mvcoplasma caoricolum subsp. caoricolum was reisolated from the uterus, placenta and from fetal tissues. However, attempts to isolate chlamidia, toxoplasma, campylobacter, salmonella, Brucella, Q fever, leptospira and listeria were negative. DISCUSSION The lack of observed clinical signs in our study animal suggests a disparity in the severity of the GM13 strain compared with that of previous trials in goats (5,6,8). These differences may indicate an age-dependent susceptibility in the goat, the route of administration or the infection dosage (5,6). Based on immunohistochemical results, the erythrophagocytic and chorioallantoic trophoblasts may play a role in the entry and spread of mycoplasma. During bacterimia, the mycoplasmas may be phagocytosed by erythrophagocytic trophoblasts along with the maternal erythrocytes (1). However, neither the endometrial stroma nor the epithelium appeared to be important in placental infection since the lesions were minimal in these areas and were negative following immunohistochemical examination. Hence, we believe that mycoplasma may initially infect placentomal trophoblasts through the vascular system, as occurs in caprine bacterial abortions (1). Further experimental studies are required to confirm this statement. The results of our study appear to confirm, by immunohistochemical and electron microscopy techniques, that M. canricolum subsp. cauricolum can cause caprine abortion and that the process can occur without premonitory signs. REFERENCES 1.
2. 3.
4.
Anderson TD, Meador VP, Cheville NE Pathogenesis of placentitis in the goat inoculated with ~Brucella -. abortus I. Gross and histologic lesions. Vet Path01 1986;23:219-226. Bar-Moshe B, Rapaport E. Observations on MvcoDlasma mvcoides subsp. mvcoides in Saanen goats. Isreli J Med Sci 1981; 17:537-539. Breard A, Poumarat F. Isolement de MvcoDlasma capricolum a partir d ‘un sperme de taureau. (Isolation of Mycoplasma capricolum from bull semen). Rev Elev MM Vet Pays Trop 1988;41: 149-150. Clyde WA. Mycoplasma species identification based upon growth inhibition by specific antisera. J Immunol 1964;92:958-965.
Theriogenology 5. 6.
7.
8. 9. 10. 11. 12.
13. 14.
15.
16. 17. 18.
19.
385
Cordy DR. Septicaemia and pneumonia in Mvcoplasma capricolum infections in young goats. Aust Vet J 1984;61:202. DaMassa AJ, Brooks DL, Adler HE. Caprine mycoplasmosis: widespred infection in goats with Mvcoulasma mvcoides subsp. mvcoides (large colony type). Am J Vet Res 1983a;44:322-325. DaMassa AJ, Brooks DL, Adler HE, Watt DE. Caprine mycoplasmosis: acute pulmonary disease in newborn kids given Mvcoplasma capricolum orally. Aust Vet J 1983b;60: 125-126. DaMassa AJ, Porter TL. Acute disease in lambs caused by a Mvcoolasma species. Vet Ret 1987;121:166-167. DaMassa AJ, Wakenell PS, Brooks DL. Mycoplasmas of goats and sheep. J Vet Diagn Invest 1992;4:101-113. Freundt E A. Culture media for classic mycoplasmas. Methods Mycoplasmol 1983;1:127-135. Gardella RS, DelGiudice RA, Tully JG. Immunofluorescence. Methods Mycoplasmol 1983;1:431-439. Hsu SM, Raine L, Fanger H. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody PAP) procedure. J Histochem 1981;29:577-588. Jones GE, Rae AG, Holmes RG. Isolation of exotic mycoplasmas from sheep in England. Vet Ret 1983;113:540. Kapoor PK, Garg DN, Mahajan SK. Isolation of Mvcoplasma mycoides subsp. mvcoides (LC variant, Y-goat) from naturally aborted bovine fetuses. Theriogenology 1989;32: 683-69 I. Rodriguez JL, Espinosa de 10s Monteros A, Herraez P, Poveda JB, Fernandez A. Isolation of Mvcoplasma mvcoides subsp. mvcoides (LC Variant) from two naturally aborted caprine fetuses. Theriogenology 1995;44: 1003- 1009. Rosendal S. Experimental infection of goats, sheep, and calves with the large colony type of Mycoplasma mycoides subsp. mvcoides. Vet Path01 1981;18:71-81. Senterfit, LB. Preparation of antigens and antisera. Methods Mycoplasmol 1983;1:401-404. Smith MC, Sherman DM. Reproductive system. In: C.C. Carnn, S. Hunsberger, R. Lukens and M. Denardo (Eds). Goat Medicine. Lea and Febiger, N.Y. 1994;411-463. Villalba EJ, Poveda JB, Ferntindez A, Rodriguez JL, GutiCrrez C, GhmezVillamandos JC. An outbreak caused by Mvcoolasma mvcoides species in goats in the Canary Islands. Vet Ret 1992; 130:330-331.