sinuses, and lactiferous and interlobular ducts. In addition,. M. agalactiae organisms appeared in the cytoplasm of the epithelium of ducts, and in infiltrating ...
J. Vet. Med. B 49, 226–229 (2002) 2002 Blackwell Verlag, Berlin ISSN 0931–1793
Department of Comparative Pathology, University of Las Palmas de Gran Canaria, Gran Canaria, Spain
Immunohistochemical Detection of Mycoplasma agalactiae in Formalin-Fixed, Paraffin-Embedded Tissues from Naturally and Experimentally Infected Goats F. RODRI´GUEZ1,4, G. A. RAMI´REZ1, A. S. RAMI´REZ2, H. J. BALL3, A. ESPINOSA DE LOS MONTEROS1 and A. FERNA´NDEZ1 Addresses of authors: 1Department of Comparative Pathology; 2Section of Epidemiology and Preventive Medicine, Veterinary Faculty, University of Las Palmas de Gran Canaria, Trasmontan˜a, 35416 Arucas, Gran Canaria, Spain; 3Department of Agriculture for Northern Ireland, Veterinary Sciences Division, Stoney Road, Stormont, BT43SD Belfast, Northern Ireland, UK; 4Corresponding author With 6 figures
Received for publication November 21, 2001
Summary Samples from the mammary tissue of 14 lactating goats (12 naturally infected and two experimentally infected) were examined for the presence of Mycoplasma agalactiae. A monoclonal antibody (5G12) was applied to formalin-fixed, paraffin-wax-embedded sections and labelled by the avidin– biotin peroxidase complex (ABC) method. Histological examination of tissue sections revealed strong immunoreactivity in all animals included in the study. Mycoplasma agalactiae antigen was mainly detected in the cellular debris at the periphery of purulent exudates present within lactiferous sinuses, and lactiferous and interlobular ducts. In addition, M. agalactiae organisms appeared in the cytoplasm of the epithelium of ducts, and in infiltrating macrophages and neutrophils within the ducts, alveoli, interstitial tissue and regional lymph node sinuses. It is concluded that this monoclonal antibody-based immunohistochemical technique is an efficient and specific method for the post-mortem detection of M. agalactiae in cases of clinical mastitis as well as being a useful tool for the study of the route of infection and cellular types involved during mastitis caused by this organism.
Introduction Contagious agalactia has been reported on all five continents, is one of the most serious diseases to affect small ruminants and is regarded by health authorities as being endemic in most Mediterranean countries (Lambert, 1987; DaMassa et al., 1992; Hasso et al., 1993; Real et al., 1994; Bergonier et al., 1997). Contagious agalactia in goats is a syndrome caused by various species of Mycoplasma which share a similar tissue tropism and cultural and antigenic features. These organisms include Mycoplasma agalactiae (considered to be the main causal agent and to be involved in about 90% of the outbreaks of the syndrome), M. mycoides ssp. mycoides, M. mycoides subsp capri, M. capricolum subsp capricolum and M. putrefaciens (Lambert, 1987; Real et al., 1994). Mycoplasma agalactiae primarily affects the mammary gland, joints, the eye and to a lesser extent the respiratory tract (Ojo and Kede, 1976; Bergonier et al., 1997). Most economic losses are the result of infections in the udders of lactating animals resulting in a rapid drop in milk production with high numbers of organisms being shed in the milk (DaMassa et al., 1992; Bergonier et al., 1997). U. S. Copyright Clearance Center Code Statement:
Diagnosis by culture methods is sensitive and specific but time-consuming (DaMassa et al., 1992; Poveda and Nicholas, 2000) while serological tests may produce cross-reactions between M. agalactiae and the other mycoplasma species involved in caprine contagious agalactia (Lambert, 1987; Real et al., 1994; Poveda and Nicholas, 2000). Immunoenzymatic techniques used to detect mycoplasma antigens in fixed tissues have eliminated many problems associated with other identification methods (Adegboye et al., 1995; Rodrı´ guez et al., 1996) but detection of M. agalactiae in caprine mastitis by immunohistochemistry has not been reported in the literature. This study was designed to develop an immunohistochemical procedure using a monoclonal antibody to detect M. agalactiae in formalin-fixed, paraffin-embedded tissues of naturally and experimentally infected caprine udders, and to compare this procedure with mycoplasma isolation in order to evaluate its potential for routine diagnostic use.
Materials and Methods A commercial dairy goat herd in the island of Gran Canaria, Spain, comprising 220 nanny-goats showed a sudden onset of clinical mastitis. Mammary glands were hot, swollen, tender and painful, milk production dropped by around 75% and the milk contained clots or appeared as a yellowish watery secretion. A total of 30 goats were culled within 3-week period due to the progression of the clinical signs, the lack of improvement after tylosin-treatment and/or mycoplasmapositive milk cultures. Milk samples, mammary tissues and supramammary lymph nodes were collected from 12 (nos 1–12) goats at the abattoir. Mammary tissues from two (nos 13, 14) lactating goats in which M. agalactiae mastitis had been experimentally induced were also used. They were inoculated by intramammary infusion into the right teat with approximately 6.3 · 109 colony forming units (cfu) of a field isolate of M. agalactiae (123/96) grown in 10 ml of mycoplasma broth for 5 days. The left mammary glands of both goats inoculated with sterile mycoplasma broth served as control. Goats were clinically examined each day and humanely killed 14 days after inoculation. Hayflick and SP4 media were used to isolate mycoplasmas (Real et al., 1994; Poveda and Nicholas 2000). Each sample was also cultured on blood agar, McConkey’s agar, and in thioglycolate liquid medium. The biochemical profile of the
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Immunohistochemical Detection of M. agalactiae in Goat Tissue mycoplasma isolate, cloned three times, the growth-inhibition test, the metabolism-inhibition test and immunofluorescence labelling with antisera prepared in rabbits against the reference strains of caprine and ovine mycoplasma were used to identify mycoplasmas (Real et al., 1994; Poveda and Nicholas, 2000). Tissue samples were fixed in 10% neutral-buffered formalin, dehydrated through graded alcohols and embedded in paraffin wax. Sections were cut at 4-lm thickness and stained with haematoxylin and eosin (H & E). For immunohistochemistry, the avidin–biotin peroxidase complex (ABC) method was used. Tissues were dewaxed and rehydrated, and endogenous peroxidase activity was blocked by incubation with 0.3% hydrogen peroxide in methanol for 30 min at room temperature. Tissue sections were then treated with pronase (Sigma Chemical Co, St Louis, MO, USA) diluted 0Æ1% in phosphatebuffered saline (PBS), pH 7.2, for 5 min at room temperature. Sections were then covered with 10% normal horse serum (Vector Laboratories, Burlingame, CA, USA) in PBS for 30 min at room temperature. The primary monoclonal antibody 5G12 (VSD, Stormont, Belfast) was then applied at a dilution of 1 : 2000 in PBS for 18 h at 4C. A biotinylated horse anti-mouse immunoglobulin G (IgG; Vector Laboratories) diluted 1 : 200 in PBS containing 1% normal horse serum, was applied for 30 min at room temperature as secondary reagent. The ABC method (Vector Laboratories) was then applied as the third reagent. To develop the immunoreaction, either one of two chromogens was used: 3-3¢diaminobenzidine tetrahydrochloride (DAB) or 3-amino9-ethylcarbazole (AEC) (Sigma). Sections were lightly counterstained with Harris’s haematoxylin for 1 min, washed in tap water, dehydrated, and mounted. Sections in which the specific primary antibodies were replaced by PBS, normal horse serum, or inappropriate antibodies were included as negative controls and run simultaneously with the test slides. Sections from mammary gland from 32 cases of natural or experimentally induced mastitis in goats by M. bovis, M. mycoides subsp. mycoides, M. mycoides subsp. capri, M. putrefaciens, Escherichia coli, Actinomyces pyogenes, Streptococcus agalactiae, or Staphylococcus aureus (four animals infected with each mycoplasma or bacteria species) were used as negative controls.
Results Milk from nine of 12 lactating goats was culture positive for M. agalactiae and contained from 1 · 105 to 1 · 107 cfu/ml. Mammary gland and supramammary lymph nodes of those animals were also positive for M. agalactiae by isolation. Mycoplasma agalactiae was recovered from mammary secretions, udder tissues and supramammary lymph nodes of the glands on the right of experimentally infected goats, but it was not isolated from similar samples from the left-hand glands, which had been infused with sterile mycoplasma broth. Macroscopically lactiferous sinuses and ducts were dilated and contained yellowish-creamy material. The lesions were mainly bilateral, although of variable intensity, in naturally infected animals. In experimentally infected goats similar changes were found only in the right-hand glands. Supramammary lymph nodes were markedly enlarged and were moderately hyperaemic and oedematous in all goats. Histologically, purulent exudate was observed in the lactiferous sinuses and ducts and in the interlobular ducts (Fig. 1). There was proteinaceous material and aggregates of leucocytes
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within the lumina of many alveoli and associated with necrosis of the epithelium lining of the alveoli (Fig. 2). There was also a marked infiltration of lymphocytes and plasma cells in the intralobular stroma and surrounding the collecting ducts (Fig. 3). In the interstitial tissue, interlobular septa and around the lactiferous ducts, infiltration of numerous macrophages, lymphocytes and plasma cells was observed together with an incipient proliferation of connective tissue. In naturally infected goats some lobules showed an intense fibrous tissue proliferation in combination with an atrophy of many acini. Lymph nodes had non-specific reactive lymphadenopathy with lymphoid follicular hyperplasia and abundant plasma cells and macrophages in the sinuses. The immunoreaction with the anti-mycoplasma antibody 5G12 was observed to be mainly associated with the exudate in the lumina of ducts as a finely punctate reaction (Fig. 4) in all animals. An intense granular, cytoplasmic immunoreaction was observed in neutrophils and macrophages present within ducts (Figs 4, 6) and in the interstitial tissue (Fig. 5). A positive reaction product was also observed in some macrophages in the sinuses of the supramammary lymph nodes in all but two (nos 3, 9) of the naturally infected animals. Mycoplasma agalactiae antigen was also visualized in the cytoplasm of epithelial cells lining the lactiferous and interlobular ducts (Fig. 6). No immunoreaction was detected in tissues infected with other mycoplasma species or with the other bacteria used as control.
Discussion Mastitis is an important disease of goats and is a significant constraint on milk production (DaMassa et al., 1992; Bergonier et al., 1997). Numerous outbreaks of contagious agalactia induced by M. agalactiae have been reported in goats in the Canary Islands. M. mycoides subsp. mycoides and M. mycoides subsp. capri are the two other most frequent mycoplasma species involved in cases of mastitis in the herds (Real et al., 1994). Pathological changes ranged from areas of acute galactophoritis and mastitis to chronic fibrosing mastitis with infiltration of lymphocytes and plasma cells and marked atrophy of the glandular parenchyma. These changes closely resemble natural and experimental infections described by others (Ojo and Kede, 1976; Lambert, 1987; DaMassa et al., 1992), and the clinical and pathological findings are similar to those described in mastitis produced in goats by M. mycoides subsp. mycoides, M. mycoides subsp. capri, M. capricolum subsp. capricolum and M. putrefaciens (Ojo and Kede, 1976; Lambert, 1987; DaMassa et al., 1992; Hasso et al., 1993; Bergonier et al., 1997). The observations reported here indicate that M. agalactiae may cause mastitis that leads to agalactia. Pathological changes were characterized by a rapid-onset purulent mastitis with purulent exudate, necrosis of epithelium, fibrosis and mononuclear infiltration in the interstitial tissue and compression and atrophy of the acini. The necrosis and exfoliation of epithelial cells of the acini and the compression of acini due to the proliferation of the connective tissue contributed to a decrease in the mass of the secretory tissue and to agalactia. The results of the present study indicate that immunohistochemical methods using monoclonal antibodies are useful tools for the diagnosis and study of the pathogenesis of
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Fig. 1. Goat no. 4: purulent exudate within mammary ducts; H & E, · 19.4.
Fig. 2. Goat no. 7: numerous neutrophils and fewer macrophages within alveoli, vacuolization of alveolar epithelial cells and infiltration of mononuclear cells in the interstitium; H & E, · 194.
Fig. 3. Goat no. 5: intense infiltration of mononuclear cells in an mammary lobule. Note the incipient fibrosis and atrophy of some alveoli; H & E, · 97.
F. RODRI´GUEZ et al.
Fig. 4. Goat no. 7: intense granular immunoreaction to M. agalactiae antigen associated with the necrotic exudate present in a duct; DAB chromogen with haematoxylin counterstain, · 97.
Fig. 5. Goat no. 8: M. agalactiae antigen in the cytoplasm of macrophages infiltrated in the interstitium of a mammary lobule; AEC chromogen with haematoxylin counterstain, · 194.
Fig. 6. Goat no. 14: immunoreaction to M. agalactiae antigen in the cytoplasm of epithelial cells lining a duct and in intraductal macrophages; AEC chromogen with haematoxylin counterstain, · 242.5.
Immunohistochemical Detection of M. agalactiae in Goat Tissue mastitis caused by M. agalactiae. Immunoperoxidase procedures carried out using polyclonal antisera raised against mycoplasma antigens are difficult to interpret because of non-specific background labelling (Adegboye et al., 1995; Rodrı´ guez et al., 1996). In addition, numerous antigens are shared among mycoplasma involved in contagious agalactia, making it difficult to distinguish between mastitis caused by different mycoplasma species (DaMassa et al., 1992; Poveda and Nicholas 2000). Mycoplasma agalactiae organisms can remain dormant for months in female goats until conditions favouring their development, such as parturition or lactation, induce disease expression (Lambert, 1987). Some macrophages and neutrophils in the mammary interstitium and lymph node sinuses that contained mycoplasma antigen, observed in this study, could act as reservoirs of infection. In addition, this finding suggests that M. agalactiae can invade the mammary parenchyma from where it might to be able to reach other tissues as described by others (DaMassa et al., 1992; Hasso et al., 1993). Although mycoplasmas have been considered as organisms infecting the surface of external mucosae, the presence of M. bovis antigen in the cytoplasm of bronchiolar epithelial cells (Adegboye et al., 1995) and of M. agalactiae in the epithelium of mammary ducts reported in this study, indicate the ability of these closely related mycoplasmas to invade and to damage epithelial cells, inducing the typical bronchiolitis and galactophoritis described in these infections. Immunohistochemical detection of M. agalactiae was more sensitive in our study than microbiological culture, although antibiotic treatment might have been responsible for culture inhibition or the lack of mycoplasma in the milk (Lambert, 1987; Bergonier et al., 1997). In conclusion, the presence of M. agalactiae was consistently demonstrated using a simple, informative, and reproducible immunohistochemical technique on formalin-fixed, paraffin-embedded tissue.
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Acknowledgements This work was supported by grant PI2001–127 from the ‘Consejerı´ a de Educacio´n, Cultura y Deportes del Gobierno de Canarias’.
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