Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations

1993 Respiratory infection of lambs with ovipneumoniae Mumtaz Ahmad Khan Iowa State University

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Respiratory infection of lambs with Mycoplasma ovipneumoniae

Khan, Mumtaz Ahmad, Ph.D.

Iowa State University, 1993

UMI 300 N. ZeebRd. Ann Arbor, MI 48106

Respiratory Infection of lambs with Mycoplasma ovipneumoniae

by

Mumtaz Ahmad Khan

A Dissertation Submitted to the Graduate Faculty in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY

Department : Microbiology, Immunology and Preventive Medicine Major: Veterinary Microbiology

Approved :

Signature was redacted for privacy. In/"Chargearge of Major Work

Signature was redacted for privacy. For the Major

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Iowa State University Ames, Iowa 1993 ii TABLE OF CONTENTS

GENERAL INTRODUCTION 1 LITERATURE REVIEW 4

PAPER I. DETECTION OF MYCOPLASMA OVIPNEUMONIAE IN LAMBS WITH A COUGHING SYNDROME 54 SUMMARY 56 INTRODUCTION 58 MATERIALS AND METHODS 60 RESULTS 69 DISCUSSION 99 REFERENCES 107 ACKNOWLEDGMENTS 113

PAPER II. MITOGENIC ACTIVITY OF MYCOPLASMA OVIPNEUMONIAE FOR OVINE LYMPHOCYTES 114 SUMMARY 116 INTRODUCTION 118 MATERIALS AND METHODS 122 RESULTS 128 DISCUSSION 135 REFERENCES 139

ACKNOWLEDGMENTS 142 ill

PAPER III, HETEROGENEITY OF ISOLATES OF MYCOPLASMA OVIPNEUMONIAE 143 SUMMARY 145 INTRODUCTION 147 MATERIALS AND METHODS 149 RESULTS 154 DISCUSSION 168 REFERENCES 172 ACKNOWLEDGMENTS 176

PAPER IV. EXPERIMENTAL INFECTION OF LAMBS WITH MYCOPLASMA OVIPNEUMONIAE AND A GRAM- NEGATIVE HEMOLYTIC COCCOBACILLUS 177 SUMMARY 179 INTRODUCTION 181 MATERIALS AND METHODS 184 RESULTS 189 DISCUSSION 197 REFERENCES 202 ACKNOWLEDGEMENTS 206 GENERAL SUMMARY 207 LITERATURE CITED 210

ACKNOWLEDGMENTS 226 1

GENERAL INTRODUCTION

Pneumonia is one of the most common diseases of lambs, in all husbandry conditions, throughout the world. Traditionally, it has been classified based on lesion types as either acute exudative or chronic proliferative pneumonia (Stamp and Nisbet, 1963) . Although economic losses from fatal pneumonia are substantial, of even greater importance are those due to unthriftiness, weight loss, reduced feed conversion, delayed marketing, and treatment or prevention costs. These significant adverse effects of respiratory disease on sheep production have also been reported in surveys conducted in North America (Elazhary et al., 1984; Rook, 1988; Hartwig and Eness, 1989) . The etiology of pneumonia in lambs is considered to be extremely complex and relates to synergistic effects of both management practices and infectious agents. A wide variety of microorganisms have been recovered from the respiratory tract of pneumonic sheep (Stevenson, 1969) but the etiological significance of many of them is in doubt. Pasteurella sp. and were the most common organisms isolated from pneumonic as well as normal sheep. Challenge experiments indicated that these organisms alone have limited ability to induce pneumonia and require predisposing factors. A number of viruses, inclement weather, malnutrition, parasitism. 2 fatiguing activities, and other infections such as Chlamydia, were thought to serve this role (Stevenson, 1969; Jones et al., 1982; Davies, 1985). Many species of mycoplasma have been associated with respiratory disease in lambs in reports from Australia, New Zealand, the United , Norway and other countries. Mycoplasma ovipneumoniae is the most commonly isolated mycoplasma from the ovine respiratory tract (Sullivan et al., 1973; Alley et al., 1975; Jones et al., 1979; Cottew and Yeats, 1981; Bakke, 1982; Pfeffer et al., 1983; Goltz et al., 1986) . It is usually isolated from sheep with atypical proliferative type pneumonia but has also been isolated from cases of exudative or chronic pneumonia primarily caused by other organisms, and from normal animals (Alley and Clarke, 1979; Bakke, 1982; Pfeffer et al., 1983; Sullivan et al., 1973). Study of the agent and its association with respiratory disease in the United States is extremely limited. However, there is evidence for its prevalence and its association with outbreaks of a respiratory disease in lambs (Kaeberle and Eness, 1988; Eness et al., 1990). Current knowledge of the nature of this microorganism and its pathogenic potential is quite limited. A marked heterogeneity in isolates of M. ovipneumoniae has been reported and experimental reproduction of disease has been inconsistent. The latter was thought to be due to variation in 3 pathogenicity of different strains, nature of the experimental lambs and procedures utilized for evaluation of pathogenicity (Jones et al., 1976; Gilmour et al., 1982; Mew et al., 1985; Thirkell et al., 1990; lonas et al., 1991). The association of M. ovipneumoniae with respiratory diseases of lambs and lack of information relative to the mechanisms of pathogenesis were the basis for this research. There was a need to isolate and characterize the mycoplasmas present in the sheep population and to evaluate their pathogenic potential.

Explanation of Dissertation Format This dissertation contains a general introduction, review of literature, four papers representing unpublished manuscripts, and a general summary. References cited in the introduction, literature review, and general summary are listed following the general summary. Tables and figures are numbered independently in each paper. References cited for each paper follow the corresponding paper. Investigations presented in the manuscripts were planned and executed, and the manuscripts written, primarily by Mumtaz A. Khan, a Ph.D. candidate, with the advice of the major professor. Dr. Merlin L. Kaeberle, and other co-authors of the manuscripts. 4 LITERATURE REVIEW

Ovine pneumonia is one of the most important diseases affecting the profit and loss ratio of the sheep industry throughout the world. The etiology of pneumonia is considered multifactorial and relates to synergistic effects of microorganisms and predisposing factors. The classification of pneumonias defined in the literature was based on the series of lesion types rather than their etiology. A variety of microorganisms associated with ovine pneumonia have been identified. PaBteurella sp. are the most common organisms isolated from pneumonic sheep, but their presence in normal animals has also been reported (Davies, 1985) . Among a variety of species of ovine mycoplasmas, M. ovipneumoniae is the most commonly isolated mycoplasma from the respiratory tract of pneumonic as well as normal sheep. There is considerable controversy concerning the experimental production of the disease with this mycoplasma, and fulfillment of the Koch's postulates has been difficult. General information about M. ovipneumoniae and its various strains, virulence factors and their effects, pathogenesis and pathology, or immune mechanisms and immunity is limited and confusing. Therefore, this literature review will focus mainly on mycoplasmas associated with the respiratory system and their associated mechanisms of pathogenesis in general, and M. ovipneumoniae 5 associated with sheep, in particular.

General Characteristics of Class Mycoplasmas are included under the class Mollicutes (mollis, soft; cutis, skin) and order Mycoplasmatales. Subdivision into families is based on sterol requirement for growth, genome size, and localization of NADH oxidase. Further division into genera and species is based on the criteria of energy requirements and supplemented by additional biochemical properties and serological relatedness (Table 1). Mycoplasmas are the smallest (0.33-1.0 um in diameter) and simplest self- replicating and free living known; they lack several of the capabilities expressed by other . The lack of a cell wall has been used as the primary basis for inclusion of an organism in this class. This feature is also the underlying reason for typical "fried egg" colonial morphology on solid media. The mycoplasma cell contains only the minimum set of organelles essential for cell growth and replication. A plasma membrane separates the cytoplasm from the external environment. It is composed mainly of proteins, which is two- thirds of the mass, and the remainder is lipid. In addition, mycoplasmas are resistant to penicillin and other antibiotics which interact with cell wall proteins. However, they are susceptible to antibiotics which interfere with protein 6 Table 1. of the Class Mollicutes®

Class: Mollicutes Order I : Mycoplasmatales Family I : . Sterol required for growth . NADH oxidase localized in cytoplasm Genus I : Mycoplaama . Genome size 580-1300 kbp . About 92 species . 23-41 % (G + C) content ratio of DNA . Urease negative Genus II: Ureaplasma . Genome size 730-1160 kbp . About 5 species with serotypes . 27-30 % (G + C) content ratio of DNA . Urease positive Family II: Spiroplasmataceae . Helical during some phase of growth . Sterol required for growth . NADH oxidase localized in cytoplasm Genus I : Spiroplaama . Genome size 1350-1700 kbp . About 11 species . 25-31 % (G + C) content ratio of DNA 7 Table 1 (continued)

Order II: Acholeplasmatales Family I : Acholeplasmataceae . Sterol not required for growth . NADH oxidase localized in membrane Genus I: Acholeplasma . Genome size about 1600 kbp . About 12 species . 27-36 % (G + C) content ratio of DNA Order III: Anaeroplasmatales Family I: Anaeroplasmataceae Genus I : Anaeroplaama . Sterol required for growth . Genome size about 1600 kbp . About 4 species . 29-33 % (G + C) content ratio of DNA . Oxygen sensitive obligate anaerobes Genus II: Aateroleplasma . Sterol not required for growth . Genome size about 1600 kbp . 40 % (G + C) content ratio of DNA Uncultivated, unclassified MLOs: . Genome size 500-1185 kbp . 23-29 % (G + C) content ratio of DNA

° Razin, 1992. 8 synthesis, such as chloramphenicol and tetracyclines (Razin, 1978; Howard and Taylor, 1985). The mycoplasma genome is typically prokaryotic consisting of a circular double stranded DNA molecule, but it differs from the genomes of other prokaryotes in its small size (5 x 10® daltons for Mycoplasmataceae corresponding to 650 genes) that is one-sixth the size of the Escherichia coli genome. The organisms in the other three families were considered to have genomes double this size (Razin et al., 1983a). The percentage of guanine plus cytosine content is relatively very low (23-40 mol %). As a consequence of this limited genetic potential, they usually require intimate association with mammalian cell surfaces and manifest complex nutritional requirements for in vitro growth. The growth media must contain an adequate level of a protein source, supply the necessary osmolarity, glucose, arginine, or urea for energy, and many also require an external source of sterols and fatty acids for membrane synthesis (Razin, 1978; Annon, 1979). Members of family Spiroplasmataceae are plant pathogens and inhabit mainly plants and arthropods. Members of the family Acholeplasmataceae are commensals of animals and so are nonpathogenic. The family Mycoplasmataceae is comprised of the genera Mycoplasma and Ureaplasma and contain species pathogenic for both animals and humans. 9 Mycoplasma Identlflcablon Glucose cataboliam and arginine and urea hydrolysis are obligatory tests in identification of mollicutes. Considering the nutritional tests, determination of a cholesterol requirement is an important test to place a strain into the order Acholeplasmatales or in the anaerobic genus Aateroleplaama (Razin, 1989). Immunofluorescence, growth and metabolism inhibition tests are standard serological techniques used to define separate species of mycoplasmas. Over the years new techniques have evolved and older methods modified and refined by research workers in the field of mycoplasmology. Immunohistochemical (IH) staining is one of those methods which promised the demonstration of mycoplasma (antigens) in tissue sections. This technique required the use of specific antibodies that are labeled, so as to visualize antibody attachment sites microscopically. Immunohistochemical staining has also been used for the diagnosis of diseases associated with autoantibodies, tumors, hormones, immune complex deposition, and for detection of other microorganisms (Colvin et al., 1988; Jannette, 1989; Haines and Chelack, 1991). The first IH staining described was immunofluorescence (IF) (Coons et al., 1941), and the tissue stained with fluorescein-labeled antibodies was viewed under an ultraviolet light microscope. Immunofluorescent stains were used extensively for disease 10 diagnosis, but loss of tissue detail for histological observations and rapid fading of dye were some of their limitations. The alternative method to overcome these limitations was the use of antibodies labeled with enzymes (Nakane and Pierce, 1967). In this method enzyme-labeled antibodies were applied to suspected tissues, followed by an enzyme substrate to stain the tissue at the site of antibody binding. The reaction was visible with an ordinary light- microscope and produced a permanent stain. In addition, treated tissue sections could be further stained for routine histological examination. One of the most commonly used enzymes in IH staining is peroxidase. The initial binding of peroxidase-labeled antibodies to tissue antigens, followed by the peroxidase substrate diaminobenzidine (DAB), resulted in rich chocolate brown deposits in the tissues. One staining method in which the primary antibody (antibody specific for the of interest) is directly labeled with the enzyme or fluorescent dye is called a direct method, whereas in indirect techniques the primary antibody is unlabeled. Here the binding of the primary antibody to the tissues is detected by a second antibody to immunoglobulin labeled with the enzyme or fluorescent dye. The indirect methods are somewhat complex and time consuming but were a means of increasing the sensitivity of the staining method. Since approximately 5-7 second antibodies may bind to each primary antibody, this 11 results in a 5-7 fold enhancement of the visible fluorescence or enzyme signal. Another IH technique that results in even greater amplification of the visible signal than provided by indirect methods is the avidin-biotin complex (ABC) technique. This relies upon the high affinity of the B-vitamin biotin, for the egg white glycoprotein, avidin. In ABC stains, the tissue antigen is bound by an unlabeled primary antibody followed by a second antibody to immunoglobulin labeled with biotin. The biotinylated antibody is then detected by application of preformed complexes of avidin and biotin. Each avidin has binding sites for four biotin molecules, and the complexes are generated with free biotin binding sites which ensure attachment of the complexes to the biotinylated antibody. The biotin molecules are labeled with an enzyme, usually peroxidase. Binding of the large avidin-biotin complexes, containing many peroxidase molecules, results in an amplification which enables detection of much smaller amounts of antigen in tissues. The ABC stain was estimated to be 1000- fold more sensitive than a direct immunoenzyme staining method (Bains and Miller, 1988). All IH stains are dependent upon the ability of the primary antibody to bind to the antigen of interest in the tissue specimen. In the past fresh or frozen tissue specimens have been used for IH staining procedures, but now fixed 12 tissue sections can be utilized. Fixation with agents such as 10% neutral buffered formalin almost invariably results in diminished antigenicity of the tissues. This might be due to reduced permeability and/or cross linking of protein epitopes in fixed tissues, thus a reduction in their recognition by antibodies. The loss of antigenicity varies with the type and duration of fixation and with the nature of the epitopes detected by the antibodies. An optimum temperature is also important. An increase in temperature to more than 60 °C during processing of the tissues, may destroy or damage the antigens, particularly at the edges of the tissue sections. For this purpose, processing of tissues in paraffin blocks helps to store them indefinitely at room temperature without further loss of antigenicity (Hsu et al., 1981; Richert and Maliniak, 1989). The digestion of tissue sections with proteolytic enzymes, such as trypsin, proteinase, pronase, pepsin, or ficin, generally has improved the reactivity of antibodies with antigens in fixed tissues. It might be because of increasing permeability of tissue for antibody binding and/or a breaking down of the formalin-induced cross linkage of tissue proteins (Huang et al., 1976). This technique proved convenient with respect to submission of formalin-fixed specimens for diagnosis of infectious diseases. It also negates the requirements for special transport media or rapid transport of the specimens. 13 This technique enables the pathologist to associate the IH stain, demonstrating the infectious disease agent, with the lesions present in the tissues. This association may be desirable when diagnosing disease due to an organism such as M. ovipneumoniae, which is ubiquitous and might be isolated from an unaffected animal. The detection of the organism in the lesion could help to confirm its involvement in the pathogenesis of the disease and not simply an irrelevant isolate. Immunohistochemical stains might also be an efficient means of detecting organisms which are inherently slow or difficult to diagnose by isolation or growth methods. An additional advantage of using formalin-fixed tissues for diagnosis of infectious diseases is that this minimizes the risks of handling organisms which are potential human pathogens (Haines and Chelae k, 1991).

Infections Associated with Mycoplasmas Mycoplasmas are host dependent extracellular parasites. Survival outside the host is extremely limited. The organisms are highly susceptible to heat, detergents, and commonly used disinfectants. Mycoplasmas can be highly host specific in terms of disease production. Some mycoplasmas are pathogenic in one host species but colonize other hosts without expressing pathogenicity. Pathogenic mycoplasmas usually have a predilection for the respiratory, ocular, or genital mucosa 14 and generally establish persistent superficial infections. A few species of Mycoplasmas have tropism for one anatomical site, and others may be isolated from a variety of sites. This adaptation may be based on specific colonization factors for which the host provides the receptors, or it may be based on the inability of the natural host to recognize and respond to the parasite (Rosendal, 1986) . Some mycoplasma species have invasive properties, but in others, systemic dissemination and articular involvement may follow mucosal colonization, particularly in hosts with debilitated defenses (Thomas et al. 1987) . The common diseases associated with mycoplasmas are septicemia, serositis, arthritis, and diseases of , genital tract, mammary gland, and conjunctiva (Table 2). Mycoplasmal diseases are multifactorial in nature. Factors such as intercurrent infections, crowding, inclement climatic conditions, age, genetic constitution and stress from transportation, handling, and experimentation influences the final outcome of infection.

Virulence Factors Associated with Mycoplasmas Studies of mycoplasmas have indicated that experimental production of disease, even with the so-called respiratory pathogens, was not consistent, and the disease rarely kills the host. Conversely, hosts seldom eliminate the organisms quickly. The production of disease involves interaction of 15 Table 2. Mycoplasma Associated Diseases'

Agents Host Disease manifestations

M. mycoidea G s'' Septicaemia, polyarthritis, ssp mycoidesCLC) pneumonia, mastitis, conjunctivitis M. capricolum G S Septicemia, arthritis, mastitis M. hyorhinia Swine Polyarthritis, pneumonia, polyserositis M. aynoviae Chickens Tenosynovitis, air sacculitis, arthritis M. bovia Cattle Arthritis, mastitis, pneumonia M. hyoaynoviae Swine Arthritis M. arthritidia Rats Arthritis M. galliaepticum P° Sinusitis, arthritis M. diapar Cattle Pneumonia U. diveraum Cattle Pneumonia, reproductive diseases M. ovipneumoniae S G Pneumonia M. hyopneumoniae Swine Pneumonia M. pulmonia Mice Pneumonia Rats Pneumonia, salpingitis, endometritis M. mycoides Cattle Pleuropneumonia, arthritis ssp mycoidea (SC) M. sp F-38 G P1europneumoni a M. mycoidea G Pleuropneumonia ssp capri M. hovigenitalium Cattle Seminalvesiculitis, mastitis 16 Table 2 (continued)

M. californicum Cattle Mastitis M. canadenae Cattle Mastitis M. alkalescena Cattle Mastitis M. agalactiae S G Mastitis, septicemia, arthritis M. putrefaciena G Mastitis M. bovoculi Cattle Conjunctivitis M. conjunctivae S G Conjunctivitis M. neurolyticum Mice Conjunctivitis, Rolling disease A. laidlawii Variety Urogenital tract, pneumonia of animals A. oculi Variety Conjunctivitis, urogenital of animals tract, pneumonia

® From Rosendal (1986), Sheep & Goats, ° Poultry multiple factors in association with infection with pathogenic mycoplasmas. In most of the mycoplasmal diseases, the pathogenic effect involves the adherence and colonization of mycoplasma on the epithelial surfaces, although some species are invasive. The pathogenic mycoplasmas adhere to or colonize the epithelial lining, through binding sites on the mycoplasmal cell membrane and receptors on the host cell membrane. The establishment of a close association with eukaryotic cells is also reflected in an often strong hemagglutinating and hemadsorptive capacity of certain 17 mycoplasmas. The ability to attach to host epithelium is best studied with the respiratory pathogens (Razin, 1978) . The attachment processes of M. pneumoniae and M. gallisepticum are involved in the initial and critical event in host-parasite interaction with a receptor site on the external membrane surface of the host cell. The specialized attachment organelles of these mycoplasmas, called Tip or Bleb respectively, are concentrated at one site of these mycoplasma cells (Collier, 1979; Tajima et al., 1979). These attachment organelles adhere with the aid of sialic acid residues on the host cells, but there is evidence that other components of cell membrane may have a role in this process. In vitro studies indicated that M. pneumoniae organisms located on non- ciliated cells lie parallel to the plasma membrane of those cells, while those on ciliated cells are oriented vertically (Gabridge, 1982). This suggested that the specialized tip structure of this organism may not be mandatory for attachment. Other studies indicated that only metabolically active mycoplasma cells were able to adhere to respiratory ciliated epithelium, and this adhesion capacity could be decreased or even nullified by energy metabolism inhibitors, ionophores, low temperature, and aging of the culture (Banai et al., 1980; Amar et al., 1978). The site to which these mycoplasmas attach, a sialoglycoprotein, could readily be inactivated by neuraminidase, is partially sensitive to 18 pronase, and resistant to trypsin, indicating its protein nature (Gabridge, 1982; Kahane et al., 1982). This binding protein (PI) of M. pneumoniae, is a 165 Kd protein, is immunogenic, and antibodies to this protein prevent attachment and pathogenicity of M. pneumoniae in laboratory animals (Brunner et al., 1985). Similarly, pre­ incubation of M. gallisepticum with glycophorin, a major sialoglycoprotein on human RBCs, could completely abolish attachment of this mycoplasma to red blood cells (Banai et al., 1978). Mycoplasma pneumoniae treated with trypsin subsequently regenerated the binding protein and the adherence ability on reculture in fresh medium, but erythromycin-treated mycoplasma could not regenerate this ability (Hu et al., 1977). However, the significance of the PI organelle in vivo is not clear since avirulent strains of M. pneumoniae do have the PI structure but were unable to attach to host cells. In chickens the attachment of M. gallisepticum to tracheal epithelium via the bleb was similar to M. pneumoniae. A glycoprotein was reported to be its specific receptor but the binding protein had not been analyzed (Uppal and Chu, 1977; Banai et al., 1978). Capsule producing mycoplasmas, e.g. M. hyopneumoniae and M. dispar, adhere to respiratory epithelium of pigs and calves respectively. These mycoplasmas do not have any specialized attachment organelles but a capsule-like material was 19 prominent in virulent strains of these species and is speculated to play a role in colonization and disease processes (Taylor-Robinson et al., 1981). Species of mycoplasma including M. bovoculi, M. bovirhinia, M. arginini, M. dispar, and M. hyopneumoniae colonize mucosal surfaces and were found to produce superoxide dismutases (SOD). Mycoplasmas that are associated with systemic infection do not produce these enzymes. These observations led to speculation that SOD has some role in colonization by some mycoplasmas (Rosenbusch, 1992). The intimate association between the mycoplasmas and the ciliated epithelial cells of the host enables mycoplasmas to avoid clearance by the mucociliary escalator. Also this allows biochemical changes and cell damage by the release of toxic products in close proximity to the host cells (Hu et al., 1975) . The intimate association of the mycoplasmas with epithelial cells could also contribute their persistence in the respiratory tract and in this position phagocytic clearance might be ineffective. However, the reduction in severity of pneumonia in immunosuppressed animals indicated that the host's immune response was also important in the development of respiratory disease (Denny et al., 1972; Taylor et al., 1974) . 20 Pathogenic Factors of Mycoplasmas In order to establish a respiratory infection, disease producing microorganisms must overcome the host defenses. These include a mucous blanket, mucociliary transport mechanism, phagocytic cells, local or systemic antibodies, and other secretions (Liggit, 1985). Mycoplasmas associated with pneumonia have been known to adhere to ciliated epithelium and to induce ciliostasis, followed by loss of cilia and other cytopathic changes in epithelial cells. These cytopathic changes were associated with various pathogenic factors associated with mycoplasmas. In addition, a number of biochemical changes (decrease in ATP, cAMP, carbohydrate utilization, amino acid transport, and molecular synthesis), as well as oxidative metabolism, could contribute to cell damage with resultant morphological changes. The host immune response may also play a major role in tissue injuries (Gabridge, 1975; Hu et al., 1975). Attachment of mycoplasmas to host cells is crucial for their survival within a host (Razin, 1978) and also a critical step in tissue colonization and subsequent disease pathogenesis by virulent microorganisms (Baseman et al., 1982). Adherence of mycoplasmas occurs through the interaction of adhesins, surface protein(s), on the organisms to receptor sites on the surface of a host cells. The consequences of attachment of virulent mycoplasmas are often injury to the 21 host cells. Ciliostasis was a common result of infection of tracheal organ cultures with respiratory pathogens such as AT. pneumoniae, M. pulmonis, M. ovipneumoniae, and M. gallisepticum and has been a widely used experimental procedure (Collier, 1979; Cassell et al., 1985; Jones et al., 1985) . Loss of ciliary motion due to mycoplasmal activity seemed to relate to pathogenicity and there was appreciable host specificity (Araake, 1982). Hydrogen peroxide and superoxide anion produced by certain mycoplasmas have been incriminated as pathogenic factors. These have been demonstrated to be responsible for lysis of erythrocytes in vitro and also could potentially damage tissue of the respiratory tract (Clyde, 1963) . However, the production of hydrogen peroxide did not itself determine pathogenicity, since nonpathogenic A. laidlawii did produce it and loss of virulence in M. pneumoniae was not accompanied by a decrease in hydrogen peroxide production (Razin, 1969; Kahane, 1984). The superoxide anions and hydrogen peroxide produced by M. pneumoniae and other mycoplasma species are quickly destroyed by the host catalase and peroxidase activity. For the hydrogen peroxide to exert its toxic effect, either host cell catalase is depleted or mycoplasmas must adhere close enough to the host cell surface to maintain a toxic, steady state concentration of hydrogen peroxide sufficient to cause direct damage to the cell membrane (Cohen 22 and Somerson, 1969). Almagor et al. (1984) reported the ability of superoxide radical excreted by M. pneumoniae to inhibit host cell catalase activities. The cytotoxic changes observed in tracheal epithelium of hamsters infected with M. pneumoniae was due to membrane peroxidation (Kahane, 1984) . The mechanisms of cell injury for hydrogen peroxide-producing M. gallisepticum and M. pulmonis were likely similar, but not proven. Ammonia is an end product of ureaplasma metabolism, so is also produced by mycoplasma possessing the arginine dihydrolase pathway. Urea produced in large quantity, may have a role in pathogenicity. However, it is the depletion of essential amino acids in infected host cells which is considered the main cause of cell damage by those mycoplasmas (Schneider and Stanbridge, 1975). Proteases of certain mycoplasmas have the ability to liquefy gelatin. Increased collagenase activity in certain isolates of M. arthritidia was observed to have some role in the development of arthritis in mice (Kluve et al., 1981). Numerous proteins and other enzymatic activities associated with the mycoplasma membrane were reported to have nonsignificant effects on pathogenicity (Razin and Rottem, 1976). However, there are some new openings to explore for future research workers in this regards. A phenol water extract of M. mycoidea ssp mycoidea small colony (SC) type, capsular galactan, has endotoxic activities. 23 i.e., it is pyrogenic in rabbits, lethal to chick embryos, and able to produce leukopenia and promote lesions in cattle (Villemot et al., 1962). Injection into calves, sheep, and goats produced endotoxic effects as indicated by an increased pulmonary resistance, pulmonary edema, hemorrhage, and in some instances, pulmonary capillary thrombosis. These effects might be due to binding of galactan to receptor sites on either blood elements or blood vessels and the secondary effect of released biogenic amines in the (Buttery et al., 1976; Lloyd et al., 1971). The phenol-water extract of large colony (LC) type of Mycoplasma mycoidea ssp mycoidea induced an inflammatory response, and whole cells of this mycoplasma had a cytopathic effect on the vascular endothelium of goats. This pathogenic role of capsular galactan is similar to endotoxin of most Gram-negative bacteria. However, it is different from classical lipopolysaccharides. Similarly, endotoxic effects of lipoglycans, extracted from many Acholeplaama species, match E. coli endotoxin in potency, but it cannot be regarded as a toxic factor because Acholeplaama are considered nonpathogenic (Villemot et al., 1962; Lloyd et al., 1971; Seid et al., 1980). A true exotoxin (200 Kd protein) with neurotoxic properties was isolated from the supernatant of M. neurolyticum (strain A) cultures (the agent of rolling disease). Intravenous inoculation of the material into mice 24 and rats had a lethal effect in mice (not in rats) along with vascular damage in the brain and perivascular astrocytic hypertrophy. The toxin bound to receptors in the brain (gangliosides), causing swelling of the capillary endothelium and partial to total occlusion of capillary lumen (Tully, 1964). A similar neurotoxin, for turkey poults, was isolated from M. galliaepticum (Thomas et al., 1966). The neurotoxicity was associated only with intact mycoplasma cell, as heating at 50 °C for 1 hour or repeated freezing and thawing destroyed the toxic activities. The toxic components of these mycoplasmas causing neurologic symptoms and death might be different from toxins involved in arthritic lesions. In this regard, the challenge experiments with either of the mycoplasmas showed an alteration in permeability, deposition of fibrinoid material, and swelling of capillary endothelium. This resulted in occlusion of capillaries and subsequent polyarthritic lesions (Clyde and Thomas, 1973) . An inflammatory toxin, a 73 Kd polysaccharide, composed of glucose, fucosamine, galactosamine, and a heptose, extracted from membrane of M. hovis elicited an inflammatory response when injected into the skin of guinea pigs and the mammary gland of cows. This inflammatory toxin is linked to proteins in the membrane of M. bovia (Geary et al., 1981). Two membrane components of M. hyopneumoniae were cytotoxic for porcine pulmonary fibroblasts and could be neutralized by specific 25 antibodies (Rosendal, 1986). Colonization of mycoplasmas on the epithelial lining of the infected animals did provide a nutritional advantage to the parasites. Mycoplasmas obtain their nucleic acid precursor requirements from the host cell and deprive them for their own utilization (Stanbridge, 1971). The attached mycoplasmas not only enjoyed the higher concentration of nutrients adsorbed onto their host cell membrane, but also utilized the fatty acids and cholesterol of the host membrane. Arginine utilizing mycoplasmas are known to possess the arginine dihydrolase pathway for generation of ATP from their environment. Studies with lymphocyte cultures have indicated that these mycoplasmas suppressed lymphocyte function by depletion of arginine from the media. This supported the notion that these mycoplasmas could induce cytonecrosis in infected cells through depletion of essential nutrients in tissues as well as build up a local concentration of toxic metabolites; both could seriously jeopardize tissue survival. Although at present a precise biochemical pathway of the effect of pathogenic mycoplasmas is not clear, further studies concerned with mechanisms of pathogenesis, pathology, immunology, and characterization of etiologic agents are required to establish correlation on a cause-effect basis. Mycoplasmas are known to activate complement via both the classical pathway (M. mycoides LC type and glycoprotein of M. 26 bovis) and alternative pathways (W. hovia) . This results in the release of C3a and C5a (anaphylatoxins) components of complement, which are known to initiate inflammation and cell damage (Bredt et al., 1977; Rosendal, 1984). Activation of complement could also lead to opsonization and killing of mycoplasma and thus is beneficial to hosts. Mycoplasma mycoidea ssp mycoides LC type also activates the coagulation cascade as reported in septicemic goats and this causes aggregation of platelets on exposed subendothelial collagen. Subsequent release of coagulation factors promotes coagulation and thrombus formation (Rosendal, 1986).

Pathologic Changes The respiratory mycoplasmas, e.g., M. pneumoniae, mainly colonize the ciliated epithelial cells lining the respiratory tract. The organisms are usually observed nestling between the cilia where they persist in the face of the host defenses. Generation of mycoplasmal products, immune mediated responses, and tissue damage resulted in the characteristic lesions, including a lymphocyte infiltration to form peribronchial and perivascular accumulations. An endobronchial exudate comprised mainly of polymorphonuclear leukocytes was also evident (Fernald, 1979). The lesions associated with M. pulmonis ranged from mild to focal, to severe or diffuse peribronchial lymphoid cuffing and are related to the challenge dose, 27 virulence of the organisms, and enhancing factors involved in the disease process (Cassell et al., 1973; Cassell et al., 1985). Lymphoid accumulations and colonization of bronchial epithelium are also the features of M. hyopneumoniae infection in pigs (Whittlestone, 1976). Mycoplasma diapar has been isolated from cuffing pneumonia of calves and this mycoplasma attaches to and is toxic for ciliated epithelium of the respiratory tract (Gourlay et al., 1970). The site and type of lesions induced by M. bovis differ from those noted above as it grows in the sub-epithelial tissues of respiratory parenchyma and cause necrotic nodules surrounded by accumulation of mononuclear cells (Gourlay and Howard, 1978) . The respiratory lesions of M. mycoides ssp. mycoides (SC) in cattle are unlike those of most respiratory mycoplasmas. Hudson (1971) reported the production of exudation in air ways, necrosis and edema in regional lymph nodes, edema of interlobular septa, and serous exudation in the pleural cavity. These changes intensify and develop into copious serofibrinous pleuritis, consolidation of lungs, and necrosis of regional lymph nodes. The respiratory diseases caused by with the LC type of M. mycoides ssp mycoides, M. mycoides ssp capri and strain F38 in goats resemble those of cattle caused by the SC type of Mycoplasma mycoides ssp mycoides except that polyarthritis is common in goats (Cottew, 1984; Okoh and Ocholi, 1986). 28

Lymphoblastogenesis In vitro cell mediated immune responses to infectious organisms has been studied by examining the development of lymphocyte transformation, delayed type hypersensitivity skin reaction, lymphokine production, and macrophage migration inhibition to their antigens. Mitogens have been reported to stimulate both sensitized and unsensitized lymphocytes causing them to proliferate (Ginsburg and Nicolet, 1973). Mitogen driven lymphoblastogenesis is a common method of evaluating the nonspecific immunological potency of lymphocytes. Phytohaemagglutinin (PHA), concanavalin A (Con-A), and pokeweed mitogen (PWM) were some common mitogens used. Streptolysin S, streptococcal filtrate and lipopolysaccharides of Gram-negative bacteria were among the few mitogens that have been isolated from microorganisms. Many mycoplasma have been reported to behave as mitogens and stimulate a variety of lymphocytes to undergo blast transformation. This mitogenicity of mycoplasmas was not confined to lymphocytes of the original host from which those organisms were isolated and various strains of the same mycoplasma have different mitogenic capabilities (Naot and Ginsburg, 1977). The biochemical nature of mitogenic factors of mycoplasmas was reported to be mainly proteins and to some extent carbohydrates, but lipids have no role. The purified membrane proteins of M. pulmonis have greater mitogenic effect 29 than the same dose of whole mycoplasma proteins (Naot, 1982). When the membrane preparation of M. pulmonis was subjected to freezing and thawing and heat treatment, it was noted that the latter had decreased their mitogenicity, whereas no such effect was observed with other mycoplasmas (Cole et al., 1977; Messier et al., 1990). Since specifically sensitized lymphocyte transformation appeared to develop in a number of different animal species following mycoplasma infection, the mitogenic property of some mycoplasmas has led to difficulties in the interpretation of the results. The significance of response was unclear, because it did not correlate with resistance (Naot, et al., 1978). However, this mycoplasma- lymphocyte interaction provided an important model for understanding the process of disease development.

Suppression Effect. Earlier studies reported in the literature revealed that certain mycoplasmas inhibited the response of lymphocytes to mitogens such as PHA (Roberts et al., 1973). Suppression of the immune response has also been noticed in cattle in the terminal stages of contagious bovine pleuropneumonia, in chickens inoculated with inactivated M. galliBepticum, in calves infected with M. bovis, in humans infected with M. pneumoniae and in rats inoculated with M. arthritidia (Bennet and Jaspar, 1977; Sabato et al., 1981). In vitro suppression of human lymphocyte blast transformation was 30 observed when M. pneumoniae were co-cultured with PHA. The removal of the organisms and supplementation with fresh medium containing PHA reversed the inhibitory effect (Copperman and Morton, 1966). The inhibitory effect on lymphocyte transformation of both viable or killed nonfermenting mycoplasmas was due to rapid depletion of the essential amino acid, arginine, from the medium, as a consequence of the mycoplasmal arginine dihydrolase pathway. Fermentative mycoplasma species which lack this pathway had no inhibitory effect (Barile and Leventhal, 1968). Later studies indicated that arginine utilizing mycoplasmas could retain their mitogenic potential by arginine supplementation of the culture media, or by limiting mycoplasmal growth by antibiotics (Cole, et al., 1977). Other experiments with both arginine and glucose metabolizing strains of certain mycoplasmas demonstrated immunosuppression with a depressed response to PHA. The mechanism was unclear since it was not related to metabolism of arginine (Barile and Leventhal, 1968). This immunosuppressive ability was not an absolute one, as a measurable response was mounted toward all pathogenic mycoplasmas, although, in some instances, it might be slow or weak. Studies with M. arthritidis indicated that infected rats did not produce growth or metabolic inhibiting antibodies, whereas, antibodies could be detected by other tests. Antigens 31 shared between the mycoplasmas and their natural hosts, or mammalian cell lines (biologic mimicry), were believed to be the cause for inability to produce inhibiting antibodies. This biologic mimicry enabled the mycoplasmas to escape from immune recognition. Conversely, it may trigger autoimmune reactions (Biberfeld et al., 1983). The suppressive activity on lymphocytes of both fermentative and nonfermentative mycoplasmas was dependent upon the dose and membrane protein used. This effect was varied and reversible however, depending upon species of mycoplasma and type of lymphocyte used (Cole et al., 1977; Gabridge and Schneider, 1975).

Stimulation of Lymphocytes. The mycoplasmal species stimulated mitosis in different lymphocyte populations of different species of animals. Acholeplasma laidlawii and M. pulmonis stimulated both T and B lymphocytes of humans and rats respectively (Naot et al., 1979 a,b,c). There is a good possibility that mycoplasmas that stimulated B cells might also stimulate T cells (Stanbridge, 1982). It was earlier reported that M. pneumoniae, M. fermentans, M. arthritidis, M. arginini, and M. hominis have mitogenicity for T cells and their activity could be abolished by anti Thy-1 antibody and complement treatment. The T-cell mitogens were reported to be present in a cell-free culture supernatant of M. arthritidis and not significantly associated with the intact cell (Cole et 32 al., 1982). However, studies with M. pneumoniae, M. fermentans, M. arginini, and M. hominia failed to detect any active component in culture supernatants of these mycoplasmas. The mitogenic potential of these mycoplasmas was not restricted to the original hosts from which they were isolated. Studies with M. pneumoniae have indicated that the agent could stimulate lymphocytes from mice and guinea pigs as well as from people (Cole et al., 1977). A similar effect was noted with mycoplasmas from other hosts, indicating that many mycoplasmas shared some common mitogenic potential. Mitogenic potential of some mycoplasmas has also been demonstrated to have a high degree of specificity. Mycoplasmas adhered intimately to lymphocytes and eventually formed a cap, similar to lectin-induced capping process, which could have intense effect on lymphocytic functions. This property of mycoplasmas was related to the presence of multivalent ligands and to capping on the surface of lymphocyte, thereby causing polar redistribution of both mycoplasmas and their receptors. This redistribution of bound receptors pull the mycoplasmas to one pole of the cells with resultant high percentage of transformation into blast cells (Stanbridge and Weiss, 1978). Studies with M. pulmonis indicated that nonviable organisms, their membrane fragments, or culture filtrate retained the capability of crosslinking receptor molecules on lymphocyte membranes and to induce a capping process. The mycoplasmal 33 capping might contribute to histological lesions through the removal of one or more host membrane fragments via the co- capping and shedding process. These membrane fragments might be identified as foreign and induce T or B-cell responses (Stanbridge, 1982). It was evident that the lesions in some mycoplasmal infected hosts were immune injuries and in vitro mitogenicity was shared by a variety of pathogenic mycoplasmas. Mycoplasma pneumoniae induced nonspecific IgM antibody in man by nonspecific B-cell activation (Biberfeld and Nilson, 1978; Biberfeld and Gronowicz, 1978). The release of soluble lymphokines or monokines by mitogen-activated lymphocytes or macrophages, could induce cytotoxicity to host cells. These mediators could also recruit other cells resulting in further differentiation of activated lymphocyte sub-populations (Cole et al., 1985). The experimental production of interstitial pneumonia and tracheitis in rats by nonviable M. pulmonis cells or their purified membranes was indistinguishable from that produced by viable mycoplasma. This indicated that the nonspecific activation of T-lymphocytes was a major cause of pneumonia in rats infected with M. pulmonis (Naot, 1982) .

Mycoplasma of Sheep and Goats Several members of the genera Mycoplasma and Ureaplasma have been proven to be pathogens of the respiratory tract of 34 sheep and goats, but fulfillment of Koch's postulates has been difficult (Livingston et al., 1979; Cassell, 1982; Gourlay and Howard, 1982; Mohan et al., 1992). Many mycoplasmas have been isolated from the upper respiratory tract of healthy or pneumonic sheep and goats or from their lungs with or without pneumonia (Jones, 1983; Jones, et al., 1976). Mycoplasma ovipneumoniae has also been recovered from lymph nodes and thymus of experimentally infected kids and lambs (Mohan et al., 1992). A list of mycoplasmas isolated from sheep and goats and their biochemical characteristics is provided in Table 3. However, this list is not complete as some publications have listed some isolates as unclassified or unspeciated mycoplasmas. Moreover, some strains have been isolated only once or a few times, and their role in disease, if any, is unknown. Clinical mycoplasmosis often lacks pathognomonic characteristics, and symptoms could be shared by or can mimic other significant infections. Mycoplasma capricolum and M. mycoides ssp mycoldes (LC) are the most deadly mycoplasmal diseases. The animal can die with these diseases without premonitory signs. In such cases, the histological changes in lung tissues are slight and could easily be misinterpreted (DaMassa et al., 1992). In addition, the designation of diseases is sometimes misleading. For example, contagious agalactia of sheep and goats, caused by M. agalactiae, does 35 Table 3. Biochemical Characteristics of Mycoplasmas of Sheep and Goats*

Mycoplasma Strain G A T P I F D

M. agalactiae PG2 - - + + - + +

M. arginini G230 - + V - - - +

M. bovirhinis PG43 + - + - - - +

M. bovÎB Donetta - - + + - V +

M. capricolum Cal.Kid + + (s) + - (w) + - +

M. conjunctivae HRG581 + - + - - - +

M. gall inarum PG16 - + + - - + +

M. mycoidea ssp capri PG3 + - + - (w) + - + M. m. ssp mycoidea

(se type) PGl + - - - (w) - (w) - +

(LC type) Y(GM12) + - - + + - +

M. ovipneumoniae Y98 + - + - - - +

M. putrefaciens KSI + V s + - - +

M. sp. HRCG145 G145 - + + - - - +

M. sp. A1343 A1343 - - + + - - +

M. sp. 2D 2D - - + + - + +

M. sp. F38 F38 + - + - w - +

M. sp. G G(GM274B) s + + - - V +

M. sp. U U(GM623) - + V + - - +

M. sp. V V(GM257A) + - + - - - +

A. axanthum S743 + - + - - - -

A. granularum BTS39 + - + - - - -

A. laidlawii PG8 + - + - - - -

A. oculi 19L + - + - - - - Ureaplaama sp. NT NT NT NT NT NT NT G = glucose, A = arginine, T = tetrazolium, P = phosphatase, I = digestion of inspissated serum, F = film and spot formation, D = digitonin. Reactions are scored as v = slow, w = weak, NT = not tested. ® DaMassa et al., 1992. 36 not only affect females but also males, and many other mycoplasmas can produce mastitis leading to agalactia. In the United States, reports in the literature have pointed out the prevalence of M. ovipneumoniae and M. arginini in lambs with chronic proliferative interstitial pneumonia (St. George and Carmichael, 1975). However, these mycoplasmas have also been isolated from the respiratory tract of clinically normal sheep (Brogden et al., 1988).

Pneumonia in Sheep Infectious pneumonia is a common disease especially of young lambs in all sheep producing countries of the world. The specific etiology and associated pathological lesions have not been clearly defined in the literature. The etiology of pneumonia is extremely complex and relates to synergistic effects of both management factors and microorganisms. A variety of microorganisms have been isolated from pneumonic ovine lungs including Pasteurella haemolytica (Gilmour, 1978; Gilmour, 1980; Stamp and Nisbet, 1963), Pasteurella multocida (Hamdy et al., 1959; Kaeberle, unpublished data), occasionally other bacteria (Alley et al., 1975; Bakke and Nostvold, 1982; Stevenson, 1969), various mycoplasmas (Alley et al., 1975, Bakke, 1982; Bakke and Nostvold, 1982; Clarke et al,. 1974; Cottew, 1971; Jones et al., 1979; Pfeffer, 1981), chlamydia (Dhingra et al., 1980; Foggie, 1977; Hamdy et al., 1959), 37 adenoviruses (Adair et al., 1982; Belak and Palfi, 1974; Davies, 1985), parainfluenza virus type 3 (Davies, 1980; Hore et al., 1968; Lehmkuhl and Cutlip, 1982), reoviruses (Davies, 1985) and some nonclassified Gram-negative bacteria (Bosworth and Lovell, 1944) . Paateurella sp. and M. ovipneumoniae were the most common organisms isolated from both healthy and pneumonic lambs (Gilmour, 1980; Alley et al., 1975; Bakke, 1982; Davies, 1985; Lukacs et al., 1985; Malone et al., 1988). Paateurella sp. are the most commonly isolated bacteria not only from acute exudative and chronic proliferative pneumonia but also from apparently healthy sheep. Some viruses were reported to cause clinical disease which was mostly self limiting. Chlamydia psittaci caused disease similar to atypical pneumonia, but the organisms did not persist long in the respiratory tract (Davies, 1985). Results of experimental production of pneumonia with pure cultures of any of the above species of organisms were inconsistent, as the organisms either were rapidly cleared from the body or produced only mild clinical disease (Foggie, 1977; Jones et al., 1982). A potential role for mycoplasma in pneumonia of sheep had initially been suggested by experiments conducted by Hamdy et al. (1959), who consistently reproduced pneumonia by stressing lambs by intratracheal inoculation of a Mycoplasma sp. and P. multocida. A number of species of mycoplasma have been isolated from the respiratory tract of sheep from various 38 corners of the world (Alley et al., 1975; Bakke and Nostvold, 1982; Banerjee et al., 1979; Cottew, 1971; Hamdy et al., 1959; Pfeffer et al., 1983). Early investigations in Australia associated M. ovipneumoniae with a severe proliferative pneumonia appearing in lambs between the age of 5-10 weeks. Clinical signs included repeated moist coughing, sneezing, and a copious mucoid nasal discharge. Morbidity was high (90%) and clinical disease persisted over several weeks resulting in poor weight gain. While mortality was low, lungs from animals that died or were slaughtered after 16 weeks of age, showed that nearly all had lesions of proliferative pneumonia. The cytopathogenic agent was cultivated in bovine testicular cell culture and subsequently grown in artificial medium. Later studies confirmed the association of M. ovipneumoniae with this ovine pneumonia (Carmichael et al., 1972; Sullivan et al., 1973). Other mycoplasmas including M. arginini, M. conjunctivae, A. laidlawii and Ureaplaamaa have also been isolated from pneumonic and healthy lungs but are not considered to play a major role in development of disease (Foggie and Angus, 1972; Davies, 1985). Mycoplasma ovipneumoniae and M. arginini have also been isolated from goats with pneumonia and pleuritis, but the latter mycoplasma is not considered a significant pathogen of the respiratory tract of this species (Goltz et al., 1986). 39

Mycoplaama ovipnexunoniae and Pneumonia Mycoplasma ovipnexmoniae associated with naturally- occurring disease characterized by proliferative interstitial pneumonia was first described in the early 1970s by Carmichael et al. (1972). Studies have indicated that M. ovipnexmoniae was the most common microorganism associated with pneumonia of lambs (Alley and Clarke, 1979; Bakke and Nostvold, 1982; Pfeffer et al., 1983). This mycoplasma can be isolated frequently from the lung, trachea, and nose and occasionally from eyes of sheep with pneumonia, but can also be found in the respiratory tract of healthy sheep (Jonas et al., 1985; Brogden et al., 1988). Upper respiratory tract colonization with this mycoplasma can occur as early as 1-2 days after birth but the disease is most commonly seen in lambs of 5-10 weeks of age. The pneumonia is apparently related to the decline of colostral antibody. Factors such as stocking density and management practices influence the incidence within a flock. Clinically the disease is characterized by a moist cough, nasal discharge, exercise intolerance and poor weight gain. These signs decrease with age, but pneumonic lesions of a proliferative type of pneumonia are present. The characteristic lesions of this disease are proliferation of epithelial cells, peribronchiolar and perivascular lymphoid cell cuffing, and nodular lymphoid hyperplasia (Peggie et al., 1976; Alley and Clarke, 1977; 40 Bakke, 1982; Sullivan et al., 1973). However, these lesions are inconsistent, which may be due to the existence of different strains of M. ovipneumoniae (Thirkell et al., 1990) or to the presence of other bacteria (most often P. haemolytica biotype A) which can exacerbate the disease (lonas et al., 1985). Mycoplasma ovipneumoniae has also been isolated from lungs with typical exudative pneumonia, or chronic pneumonias that followed a bacterial or viral infection, and from normal lungs (Stipkovits et al., 1975; Davies, 1985). The presence of M. ovipneumoniae in high titer in pneumonic sheep and absence or presence in low titer in lungs of unaffected controls, suggest its direct or indirect role in ovine pneumonia (Alley et al., 1975). Attempts by various researchers to produce pneumonia in lambs with pure cultures of M. ovipneumoniae have yielded inconsistent results (Davies, 1985) . Nearly all animals developed severe pneumonia in the early Australian studies (Sullivan et al., 1973) but only 25- 50% of the lambs developed mild pneumonia in studies in Scotland (Foggie et al., 1976) and less than 20% developed lesions in experiments conducted in New Zealand (Davies et al., 1981; Alley and Clarke, 1977). However, the lesions that developed in experimental animals were almost identical to those reported for the naturally occurring disease. Later studies demonstrated that M. ovipneumoniae and P. haemolytica serotype A act synergistically to produce chronic atypical 41 pneumonia, principally affecting lambs under 12 months of age (Jones and Gilmour, 1983a). Since an association of M. ovipneumoniae with lamb pneumonia had been observed, failure to reproduce the disease in a high percentage of animals was puzzling. It was suggested that the challenge organisms might have been attenuated during isolation and cloning on artificial medium or that strains varied in virulence. Jones et al. (1982) provided evidence of attenuation of mycoplasma during in vitro cultivation. In this regard it is interesting to note that the experimental production of disease in early Australian studies was accomplished by utilizing tissue culture grown mycoplasma (Sullivan et al., 1973) while other researchers utilized artificial mycoplasma media. Moreover, attempts to produce pneumonia in lambs with a homogenate of pneumonic lungs containing M. ovipneumoniae alone or in combination with P. haemolytica, yielded proliferative pneumonia in most of experimental animals (Jones et al., 1982); this signifies the importance of this hypothesis. One could also expect the prevalence of multiple strains with variations in their virulence of this mycoplasma species in the sheep population. Earlier, metabolic inhibition and growth inhibition tests showed intra-species differences among strains (Jones et al., 1976). Further evidences for strain variation among various isolates of M. ovipneumoniae by using bacterial restriction 42 endonuclease DNA analysis and SDS-PAGE was reported (Mew et al., 1985; lonas et al., 1991b). Another variability in the challenge studies was the nature of the experimental lambs. Naturally reared lambs were more likely to develop pneumonia with M. ovipneumoniae than were specific pathogen-free animals (Foggie et al., 1976). This indicated a variability in natural susceptibility of the hosts. Experimental studies utilizing mixed clones of M. ovipneumoniae indicated the production of more severe disease than with a single clone and did signify the prevalence of multiple strains of this mycoplasma in the sheep population. Reported experimentation suggests that M. ovipneumoniae acts in concert with other agents in the induction of respiratory disease. Intratracheal inoculation of ovine pneumonic lung homogenate containing M. ovipneumoniae and P. haemolytica produced lesions identical to the naturally occurring disease (Alley and Clarke, 1979; Gilmour et al., 1982). However, the inoculation of a broth culture of M. ovipneumoniae alone or in combination with P. haemolytica resulted in a variety of pneumonic lesions in lambs (Foggie et al., 1976; Davies et al., 1981). Other studies indicated that M. ovipneumoniae, isolated from nasal swabs of infected lambs was unable to produce typical clinical disease in experimental lambs, but when injected into the mammary gland of ewes produced clinical mastitis. This suggested that the isolates 43 were capable of inducing an inflammatory response (Buddie et al., 1984) . It was suggested that M. ovipneumoniae should not be regarded as part of the normal flora of the ovine nasal cavity because many disease positive lambs were found negative when routinely tested and only 5% of the ewes examined carried the organisms in their nasal cavity. This nasal presence of M. ovipneumoniae in ewes was probably important and would provide an obvious source from which young lambs could be infected (lonas et al., 1985).

Strain Variability of Mycoplaama ovipneumoniae The members of class Mollicutes comprise a heterogeneous group of microorganisms. Metabolic inhibition (MI) and growth inhibition (GI) tests, both of which presumably involve surface antigens, have been used to identify various species of mycoplasmas. These tests have also been utilized to study antigenic variation among isolates of M. ovipneumoniae (Jones et al., 1976). Analysis of genome size, base composition, and nucleotide sequencing have become indispensable tools in the modern classification of bacteria into species, subspecies and varieties. A difference in the G + C contents of greater than 1.5 - 2.0 mol % between DNA of two bacteria is sufficient to rule out their inclusion in the same species (Johnson, 1984) . Hydrolysis of DNA by a series of restriction enzymes followed by electrophoretic separation in polyacrylamide gel provided a 44 promising approach to identify homology in strain specific patterns of nucleotides. As in other prokaryotes, some of the adenine and cytosine residues in mycoplasma genomes are methylated. Knowledge of the extent and sequence of methylation could be used as taxonomic markers for identification of strains within species. This can be done by pairs of restriction endonucleases recognizing the same site, one methylated and other non methylated adenosine or cytosine (Chan and Ross, 1984). DNA-DNA hybridization is a valuable technique for the determination of genetic relatedness among bacterial strains, but its use for mycoplasmas is restricted (Razin, 1992). Examination of DNA fragments following digestion with restriction endonucleases and separation by electrophoresis in agarose gel is an established method that provided valuable information on the type and number of specific nucleotide sequences in the genomes that could be considered genomic fingerprints, called restriction fragment length polymorphisms (Razin and Rottem, 1967; Dybvig et al., 1982; Razin et al., 1983a; Christiansen, 1987). This approach owes much to the work of Razin, who suggested that mycoplasmas of strict host specificity show homogeneous restriction endonuclease analysis (REA). The use of REA patterns to distinguish isolates implies that the patterns should be stable following long term propagation in vitro and that differences were not due to the 45 presence of plasmids. Further advancement in this regard was achieved by subjecting the DNA cleavage fragments, separated on an agarose gel, to Southern blot hybridization, with cloned conserved genes as probes. The rRNA genes of M. capricolum cloned in a plasmid were the most popular probes in use, in demonstrating genotypic heterogeneity among strains of various mycoplasma species (Amikam et al., 1982; Razin and Yogev, 1986; Yogev et al., 1988; Christiansen and Erno, 1990). Variability of surface antigens was another basis for genotypic variation of strains within mycoplasma species. Identification of an immunodominant surface antigen (V-l) was first reported to denote variability in different isolates of M. pulmonis (Watson et al., 1989). In addition, the variability due to V-l antigen was also reflected in surface properties and the virulence of that mycoplasma (Dybvig et al., 1989). This high degree of variability in surface protein antigens is quite common in mycoplasmas, which provide a versatile interaction of mycoplasmas with their environment, and may be related to their survival for a long time in immunocompetent hosts. Earlier reports indicated that different isolates of M. ovipneumoniae were found to be heterogenous by using metabolic inhibition and growth inhibition tests (Jones et al., 1976). Recently, marked heterogeneity was identified among different isolates of M. ovipneumoniae (Mew et al., 1985). A study on 46 sixteen isolates recovered from either the nasal tract or lungs of sheep from different flocks in New Zealand, were analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and REA by using EcoRI endonuclease. They gave unique banding patterns entirely different from one another and those patterns were consistent even over 20 passages. These observations were supported by lonas et al. (1991b) who studied 60 isolates of M. ovipneumoniae obtained from 20 different farms and found differences in their REA patterns, except in 3 isolates recovered from one farm which gave identical REA patterns. In addition, those REA patterns of individual isolates were highly reproducible and remained unchanged following long term passage in vitro. These differences in REA patterns were real and not because of genetic interchange or the presence of plasmids. Neither any plasmids in 12 M. ovipneumoniae isolates examined nor any genetic interchange between two isolates were detected by in vitro assays. It appeared that the differences in REA patterns observed in those isolates of M. ovipneumoniae organisms were a consequence of the existence of many strains with different and stable REA patterns that coexisted in sheep population. Moreover, those differences could not be accounted for by changes following the initial isolation. The heterogeneity of this mycoplasma in REA patterns together with their stability, allowed individual 47 isolates to be identified. It appeared to be difficult to examine multiple isolates from both nasal passages, trachea and lungs from sheep within a flock to find how many strains of M. ovipneumoniae existed within that flock, and to investigate the proportion of those stains which were capable of colonizing the lungs. The heterogeneity among isolates of M. ovipneumoniae as observed with REA was also reflected in their surface proteins when examined by SDS-PAGE (Mew et al., 1985; lonas et al. 1991a; Thirkell et al., 1990a; and lonas et al., 1991b). Among different isolates examined with SDS-PAGE, most of the protein bands were found to be common to all isolates but each isolate was unique in the sense that they differed from one another in one or more protein bands. The similarity was most evident in the lower molecular weight ranges (Mew et al., 1985). It was also noticed that those isolates which gave identical REA pattern also gave identical SDS-PAGE patterns. The reproducibility of SDS-PAGE patterns was also persistent with any one isolate even when prepared from replicate cultures, indicating that the differences seen represent genuine differences in isolates and were not due to lack of reproducibility of results (lonas et al., 1991b). Analysis of proteins by SDS-PAGE supported the conclusion drawn by examining REA patterns that a large number of M. ovipneumoniae strains exist in the sheep population. 48 Isolates of M. ovipneumoniae could be divided into two groups on the basis of antigenic differences in surface proteins, but these differences have limited value in characterization of mycoplasmas. A comparison of the polypeptide patterns of 22 isolates of M. ovipneumoniae by SDS-PAGE identified a marked degree of heterogeneity with only limited groupings identifiable (Thirkell et al., 1990b). Among 50 major polypeptides identified in one strain (956/2) of M. ovipneumoniae, 35 were shown to be antigenic using immunoblotting with a homologous polyclonal serum. Analysis with radioimmune-precipitation of ^^®I-surface-labeled proteins and phase partition using Triton X-114 detergent indicated that those peptides were membrane associated. Cross reactivity among M. ovipneumoniae isolates was examined by immunoblotting using one polyclonal serum and four monoclonal antibodies (MO/l to MO/4, recognizing 40, 56, 26, 105 kDa antigens respectively) raised against strain 956/2. The polyclonal serum revealed considerable antigenic heterogeneity, but at least 9 major antigens were conserved across all isolates. Two MAbs cross reacted with all 22 isolates but the other two allowed some differentiation of strains. One MAb (MO/3) divided the isolates into two groups of 16 and 6 isolates based on the presence or absence of a 26 kDa antigen. However, M. ovipneumoniae isolated from pulmonary adenomatosis did not possess the 26 kDa antigen (Thirkell et al., 1990a). This 49 polypeptide and antigenic variations among different isolates of M. ovipneumoniae might have some significance in respect to their potential to produce disease, although the basis for this has yet to be established. The literature indicates that great heterogeneity exists among the isolates of M. ovipneumoniae, not only from different flocks but also from a single farm. Multiple strains of M. ovipneumoniae have also been isolated from individual lungs of sheep suffering from chronic nonprogressive pneumonia, as detected by REA using EcoRl and SDS-PAGE. From each of those lungs having relatively severe lesions, three to four different strains of M. ovipneumoniae were detected, compared to only one or two from lungs with less severe lesions (lonas et al., 1991a). The presence of multiple isolates from individual lungs suggested the possibility that the lungs of sheep with severe chronic nonprogressive pneumonia might be simultaneously colonized by more than one strain of M. ovipneumoniae. If that is true then it could be speculated that mixed infection with different strains of one mycoplasma species might help to initiate more severe lesions. Similar differences in REA patterns as seen by using EcoRl were also observed with 12 other restriction endonucleases. However, some isolates gave partial digestion of DNA with Xhol and Haelll which recognize cytosine-rich sequences. Some isolates remained undigested (lonas et al.. 50 1991b). The presence of multiple strains of one species of mycoplasma (M. ovipneumoniae) is an unusual concept and is not limited to one mycoplasma of one host or disease. Multiple strains of species of mycoplasmas, ureaplasmas and acholeplasmas have been identified, using analysis of DNA cleavage patterns following digestion with a range of restriction enzymes. The patterns obtained with A. axanthum isolates differed markedly from one another and this observation is similar to that found with M. ovipneumoniae (Razin et al., 1983a,b). Multiple serovars (I, J, K, N, Q, and R) of M. iowae, a turkey pathogen, were recognized as strains of M. iowae. In addition, there is also variability in pathogenic potential among those strains (Bradbury and Ideris, 1982) . Comparative studies on different isolates M. iowae indicated that a few of these strains were homologous in some characteristics and heterogenous in others. The use of biochemical tests, immunofluorescence, and protein profiling by SDS-PAGE were considered as group specific tests for this mycoplasma. However, some minor differences in protein patterns were observed among different strains. The growth inhibition test tended to be strain specific although hemagglutination titers were very low and unstable for majority of strains. Restriction endonuclease DNA analysis by using either EcoRI or HindiII enzymes was the method of choice 51 for showing variation among different strains of M. lowae. The results of REA of 6 reference strains (I,J,K,N,Q, and R) and 2 field isolates of M. iowae, digested by EcoRI suggested that the heterogeneity of DNA patterns among them, was above the 6.6 kb level, and all shared many common bands below 6.6 kb levels. Similar to the EcoRI digests. Hind-III- digested DNA's of all reference strains were also different from each other (Zhao and Yamamoto, 1989). Studies on antigenic variation has also been reported for M. pulmonis (Watson et al., 1988), M. hominis (Hollingdale and Lemcke, 1970; Rosengarten and Wise, 1990), and M. gallisepticum (Khan et al., 1987; Panangala et al., 1992). Monoclonal antibodies have served as powerful probes for precise identification and characterization of specific immunodeterminants on the surface membranes of mycoplasmas (Watson et al., 1988). A panel of MAb against M. gallisepticum strain PG31 was used to probe the antigenic profile of 5 recognized strains and 6 field isolates. Monoclonal antibody (G9) reacted with all strains and isolates, but the molecular mass position of the antigens varied from 90-98 Kd among some isolates of this mycoplasma. Monoclonal antibody (G12) reacted with all strains and isolates and had identical banding patterns. However, MAb (GIO and Gil) reacted selectively only with a limited number of strains and/or isolates (Panangala et al., 1992). From these observations the workers speculated 52 that alterations in phenotypic structural or antigenic characteristics of mycoplasmas were likely to influence the host-parasite interactions, the immune response, and the outcome of the disease. Antigenic variation has been predominantly recognized as a mechanism for evasion of the host immune response. Among the prokaryotes, the most notable examples of immune evasion were observed with Neisseria gonorrhoeae (Meyer and van Putten, 1989) and Borrelia hermsii (Barbour, 1990), both of which cause recurrent or persistent diseases. A high rate of variation in major surface protein antigens of AT. pulmonis, M. gallisepticum, M. ovipneumoniae, and M. iowae led to belief that this phenomenon might be a contributing factor for persistent infection and chronicity of the mycoplasmal diseases (Watson et al., 1989). Another important phenomenon observed with M. ovipneumoniae and some other mycoplasmas was the presence of cross reacting antigens. Serological techniques, particularly the agar gel double diffusion (AGDD) and counter Immunoelectrophoresis (CIE) tests, tended to reveal antigenic relationships rather than differences between species of mycoplasma, since they identify cytoplasmic rather than membranous antigens (Erno and Jurmanova, 1973; Kenny, 1975). The AGDD and CIE tests have been used to identify cross- reacting antigens between M. dispar, M. hyopneumoniae, M. 53 hyorhinxB, and M. ovipneumoniae (Ball and Todd, 1978). Further studies using indirect hemagglutination, complement fixation, two dimensional Immunoelectrophoresis, and enzyme linked immunosorbant assay (ELISA) also confirmed the findings of the presence of common antigens among these mycoplasmas. Cross reacting antigens with apparent molecular weights of 63, 44, and 32 kDa were found in all 4 mycoplasma species mentioned earlier. An antigen of 184 kDa common to M. dispar, M. flocculare, and M. hyopnexmoniae was also reported (Priis and Jensen, 1984; Mori et al., 1988; and Thirkell et al., 1991). Monoclonal antibodies (MO/l to MO/4) against M. ovipneumoniae strain 956/2 were found to be species specific as they recognized only M. ovipneumoniae, but did not recognize any antigen on M. dispar, M. flocculare, or M. hyopneumoniae (Thirkell et al., 1991). Cross reactivity could not be detected by MI or GI tests which only detected surface antigens (Erno and Jurmanova, 1973). This cross reactivity among antigens of various mycoplasma species would reduce the specificity of diagnostic tests, particularly ELISA. 54

PAPER I: DETECTION OF MYCOPLASMA OVIPNEVMONIAE IN LAMBS WITH

A COUGHING SYNDROME 55

DETECTION OF MYCOPLASMA OVIPNEUMONIAE IN LAMBS WITH A COUGHING SYNDROME

Mumtaz A. Khan, M.Sc. (CMS), M,Sc. (TVM). Ricardo F. Rosenbusch, Ph.D.* John J. Andrews, D.V.M., Ph.D.** Mammadou Niang, M.Sc. Merlin L. Kaeberle, D.V.M., Ph.D.

* From the Veterinary Medical Research Institute, Iowa State University, Ames, lA 50011 ** From Veterinary Diagnostic Laboratory, Iowa State University, Ames, lA 50011 56

SUMMARY

Pneumonia is one the most common diseases of lambs world wide. The etiology of this disease is multifactorial and comprises both infectious and noninfectious agents. Paateurella sp. and M. ovipneumoniae are among the commonly isolated organisms from pneumonic lambs. The objective of this experimentation was to correlate infection of lambs with M. ovipneumoniae with outbreaks of a coughing syndrome. The clinical disease was characterized by paroxysmal cough, nasal discharge, exercise intolerance, and occasionally rectal prolapses. Mycoplaama ovipneumoniae was the most common organism isolated from 105 lambs investigated. Thirty-nine lung samples from pneumonic and healthy lambs were included for observation of gross and histologic lesions and standardization of an immunohistochemical staining technique for in aitu detection of mycoplasma antigens in formalin-fixed lung tissues. Gross lesions in lungs infected only with M. ovipneumoniae were classical in the form of focal consolidation. However, diffuse consolidation and congestion were observed in lungs having mixed infection of mycoplasma and other bacteria. The histological lesions associated with M. ovipneumoniae were in the form of peribronchiolar and perivascular lymphocytic cuffing, lymphoid hyperplasia, loss of cilia, and interstitial thickening. Indirect 57 immunohistochemical staining using specific M. ovipneumoniae antiserum as the primary antibody proved to be a sensitive and specific technique in detection of antigen in tissues. 58

INTRODUCTION

Pneumonia of lambs is one of the most common disease problems worldwide. The cause of this disease is multifactorial and comprises of both infectious agents and noninfectious factors. Paateurella sp. and Mycoplasmas are the most common infectious agents associated with this disease (Hamdy, 1959; Clarke et al., 1974; Alley et al., 1975; Jones et al., 1979; Bakke, 1982; Davies, 1985). The presence of these organisms in apparently normal animals and challenge experiments have indicated that these organisms individually have variable ability in producing disease and do require predisposing factors. Malnutrition, parasites, inclement weather, certain viruses and Chlamydia infections are examples (Foggie et al., 1976; Davies et al., 1981; Buddie et al., 1984). Mycoplasma ovipnexmoniae is the most common mycoplasma associated with lamb pneumonia (Carmichael et al., 1972; Jones et al., 1976; Alley and Clarke, 1979; Gilmour, 1980; Bakke, 1982; Davies, 1985; Malone et al., 1988). The occasional presence of this mycoplasma in apparently healthy lambs is also good evidence of its saprophytic nature (Davies, 1985; Brogden et al., 1988). Study of this agent and its association with respiratory disease in lambs in the United States of America is extremely limited. However, there is evidence of 59 its prevalence and association with an outbreak of a "coughing syndrome" in lambs (Kaeberle and Eness, 1988; Eness et al., 1990). The prevalence of this disease is widespread in the state of Iowa and primarily affects feeder lambs, 5-12 weeks of age. The clinical disease is characterized by chronic paroxysmal cough, mucoid nasal discharge, mild fever, and severe intolerance to exercise. The coughing sometimes leads to a high incidence of rectal prolapse in some flocks and secondary bacterial pneumonia. The mortality rate for this disease is low, but poor rate of growth, reduced feed conversion, delayed marketing, and secondary complications cause heavy economic losses to the sheep industry. While M. ovipneumoniae is the most common mycoplasma associated with ovine pneumonia, it has not received extensive study in the past. Current knowledge of the nature of this organism and its pathogenic potential in causing respiratory disease in lambs is extremely limited. The purposes of the present study were: 1) To isolate and characterize mycoplasmas from apparently healthy lambs and animals suffering from the coughing syndrome, 2) To study the pathological changes in lungs of infected lambs, 3) To standardize a technique for the demonstration of M. ovipneumoniae in formalin-fixed lung sections. 60 MATERIALS AND METHODS

Experimental Animals The experiment was conducted as an attempt to relate infection of M. ovipneumoniae to a coughing syndrome. This required the procurement of infected animals or samples for mycoplasma isolation from flocks with or without clinical disease. One hundred and five lambs utilized for isolation of mycoplasma were included in this study. For histopathology and immunohistochemical tests, a total of 3 9 lung samples from apparently healthy and naturally-infected lambs with the coughing syndrome were included. This goal was achieved through the cooperation of Iowa sheep producers and field veterinarians.

Mycoplasma Specimens Nasal swabs and bronchial washes from live animals, and bronchial swabs or homogenates of lungs were the source material for isolation of mycoplasmas. For bronchial washes, approximately a one cm long skin incision was made with a surgical scalpel over the midpoint of the trachea under local anesthesia with 2% Xylocaine with epinephrine (Astra, Worcester, Mass). A 16 gauge hypodermic needle was inserted into the tracheal lumen. A catheter equipped with a two way Luer connect valve was then inserted through the needle up to 61 bronchial bifurcation. Approximately 100 ml of Hank's balanced salt solution (Gibco Laboratories, Grand Island, NY) was injected through the catheter and then immediately aspirated with a syringe. From lungs, samples were taken either from bronchioles of each lobe of cut lung sections with microtipped swabs (Calgiswab, type 4, Spectrum Diagnostics, Inc., Glenwood, IL) or from lung homogenate, by blending lung tissues with IX Hank's balanced salt solution, followed by removal of tissue debris by low speed centrifugation. These samples were processed for subsequent growth and cultural examination of mycoplasmas.

Cultivation of Mycoplasmas Representative samples of mycoplasmas were grown on modified Friis broth medium (MFBM) and/or Friis agar (FA) (Friis, 1974; Knudtson et al., 1986). Briefly, a 2X Friis stock solution (FSS) was first made from brain heart infusion medium (Difco Laboratories, Detroit, MI, 49.29 g), mycoplasma broth base (Becton Dickinson Microbiology System, Cockeysville, MI, 52.29 g), lOX Hank's balanced salt solution (Gibco Laboratories, 300 ml), 1% deoxyribonucleic acid solution (Herring sperm DNA, Sigma Chemical Co., St. Louis, MO, 16 ml), L-arginine hydrochloride (Sigma Chemical Co., 0.45 g), L-glutamine (Sigma Chemical Co., 0.675 g) , 0.5% phenol red solution (Sigma Chemical Co., 5 ml), deionized water (3734 62 ml), 1% cysteine (Sigma Chemical Co., 15 ml), and nicotinamide adenine dinucleotide (Sigma Chemical Co., 0.75 g), and the mixture was filter sterilized. A working MFBM was made by mixing 2X FSS (100 ml), newborn calf serum (NBS, Gibco Laboratories Inc., 20 ml), Hye yeast extract (HYE, Gibco Laboratories Inc., 2 ml), lOOX bacitracin and lOOX thallium acetate (Sigma Chemical Co., made by mixing 2 g of bacitracin in 100 ml distilled water, and 1 g of thallium acetate in 100 ml distilled water, with sterilization by filtration, 1 ml each), distilled water (25 ml), and adjusting the pH to 7.5 with 1 N sodium hydroxide. The FA was made from: (1) a mixture of Noble agar (Difco Laboratories, 6 g), DEAE dextran (Sigma Chemical Co., 0.05 g), and distilled water (125 ml), autoclaved for 15 minutes and then cooled to 56 °C in a water bath, and (2) a mixture of 2X FSS (250 ml), FCS (100 ml), HYE (10 ml), lOOX bacitracin (5 ml), lOOX thallium acetate (5 ml), and 7.5% solution of sodium bicarbonate (5 ml), adjusted to pH 7.5 with 1 N sodium hydroxide and warmed to 45 °C. The ingredients in step 1 and 2 were mixed together and poured into agar plates. Ten-fold serial dilutions of each mycoplasma sample were made in MFBM in polystyrene round bottom tubes with caps (12 x 75 mm, Becton Dickinson Labware, Lincoln Park, NJ) and incubated at 37 °C in a 5% COg and 95% air atmosphere. Samples were also grown on FA directly or from broth at first observation of growth in the form of a color change. 63 Representative colonies of mycoplasmas were cloned at least three times on MFBM and FA as described by Tully (1983), and identified.

Indirect Xminunofluorescent Technique The indirect immunofluorescent technique (IIP) of Barile et al. (1962) and Del Guidice et al. (1967) was used with some modifications for identification of isolates of different mycoplasmas. Briefly, the mycoplasmas were plated on FA and early growth of mycoplasma colonies was observed under a dissecting microscope. One cm square agar blocks were cut with a sterilized spatula, and placed on a glass slide. Colony impression smears were obtained on a second glass slide by gentle touching to the agar block. The impression smear slides were then dried in air and fixed with acetone for 7 minutes. Each mycoplasma colony impression smear on glass slides were encircled with a marker (Mark-Tex Tech-Pen, Mark-Tex, Englewood, NJ) for boundary demarcation before IIF staining.

Approximately one to two drops of diluted (1:100) rabbit antiserum (against M. bovigenitalium (PGll), M. californicum (ST6), M. alkaleacena (PG51), M. putrefaciena (KSl), M. fermentans (PG18), M. arginini (G230), M. ovipnevmoniae (Y98), M. canadense (C275), M. conjunctivae (HRC581), M. capricolum (California Kid), A. axanthum (B107PA), A. oculi (19L), and A. modicum (PG49), Institute of Medical Microbiology, University 64 of Aarhus, Denmark) were added to the circle of each demarcated mycoplasma colony impression smear, as the primary antibody. The slides were incubated at 37 °C for 30 minutes in a moist chamber, washed twice for 5 minutes in phosphate buffered saline solution (PBS, Dulbecco's PBS, without calcium chloride and magnesium chloride, pH 7.4, Sigma Chemical Co), rinsed once in distilled water, and dried in air. One to two drops of diluted (1:40) fluorescein-labeled (goat) anti-rabbit IgG (H+L chain specific, Kirkegaard & Perry Laboratories Inc. Gaithersburg, MD) was added on all mycoplasmas impression smears as the secondary antibody. After an incubation for 30 minutes slides were washed as described previously and dried in air. The slides were mounted in glycerinated PBS (9 parts of glycerol and 1 part of PBS) and observed under a fluorescence microscope. Known cultures of AT. ovipnenmoniae, M. arginini, M. bovia, and M. conjunctivae (courtesy Dr. R. F. Rosenbusch, Veterinary Medical Research Institute, Ames, lA) were included as (positive) controls. A drop of PBS was used on negative control slides instead of the primary antibodies. All these specimens were also plated on 5% bovine blood and MacConkey's agar plates for isolation and identification of residual bacteria of pathogenic significance. 65

Hyperimmune Anti Mycoplaama ovipnevmoniae Serum Hyperimmune anti M. ovipneumoniae (Lomax strain, courtesy Dr. R. F. Rosenbusch, VMRI, Ames, lA) serum was produced in a horse by the procedure outlined by Stalheim et al. (1975) with modification. Briefly, M. ovipneumoniae (Lomax strain) was first adapted to grow in MFBM containing 20% horse serum (Gibco Laboratories) and then on dialysate medium (DM) as described by Kenny and Cartwright (1973) with 20% horse serum. A stock culture was prepared by growing the organism on DM for 36 hours, and harvested by centrifugation at 14000 rpm for 45 minutes to pellet the organisms. The pellet was washed thrice with PBS and stored in aliquots at -70 °C for further use. A formalin-treated M. ovipneumoniae antigen was prepared by treating the organisms with 1% formalin in sterile saline solution overnight at 4 °C, followed by centrifugation. The viability of the organisms was checked by growing them on MFBM and FA. Mycoplasma antigen was prepared by thorough mixing of 2 mg/ ml (in PBS) of mycoplasma protein-based antigen per injection dose with an equal volume of Freund's incomplete adjuvant. The antigen was injected intramuscularly at 4 different sites in the neck region. The booster injections were repeated at 2 week intervals. The horse was tested for production of antibodies by the agar gel double immunodiffusion test (Lemcke, 1965) on serum obtained before each booster injection and was finally bled for harvest of 66 reagent antiserum. The antiserum was stored in aliquots at -70 °C for use in immunofluorescence (as required) and immunohistochemical staining.

Gross and Histopathology General postmortem and histopathological examinations were conducted on two types of lambs: (1) Lambs that died of respiratory disease or those suffering from clinical respiratory disease that were euthanized with Beuthanasia-D Special (a euthanasia solution containing 350 mg pentobarbital sodium and 50 mg phenytoin sodium per ml, Schering-Plough Animal Health Corp., Kenilworth, NJ), at a dose of 10 ml/ animal by intravenous administration, and (2) Apparently normal animals. For histopathological (and immunohistochemical) examination tissue samples were included from each lobe of the lung and trachea from all 39 animals included in this study. Representative samples were fixed in 10% neutral buffered formalin for 24 to 48 hours and then processed by a routine paraffin embedding technique, microsectioned and stained with hematoxylin and eosin.

Immunohistocheinical Staining The indirect immunohistochemical (IH) staining procedure of Nakane and Pierce (1967) with some modification was utilized for detection of M. ovlpneumoniae antigen in situ. 67 Representative formalin-fixed lung sections were first embedded in paraffin wax at 56 °C, cut into sections, and dried to use for staining with this technique. The protocol of the IH technique was as follows: 1) Deparaffinize the slides through 2 changes of Pro-Par Clearant (Propylene based glycol ether with paraffin solvents, Anatech Ltd., Battle Creek, MI) for 15 minutes each and réhydraté by sequential immersion in graded concentrations of ethanol, each for 10 dips, and finally in tap water; 2) Inactivate the endogenous tissue peroxidase by immersion of the slides in a solution of 0.3% HgOg (Fisher Scientific, Fairlawn, NJ) in distilled water for 30 min at room temperature, and wash with wash buffer [Cadenza buffer, Shandon, Pittsburgh, PA., containing Tris HCl (7.44 g) , NaCl (5.1 g), and Brij-35 (1.0 g), in 1 liter of distilled water]. Shandon Sequenza^ reagent trays with cover plates (Shandon) were used for holding slides during the staining procedure; 3) Block nonspecific adherence of proteins to tissue sections by treating with a 2% bovine serum solution in PBS and incubate for 1 hour at 37 °C. Wash with wash buffer for 5 minutes; 4) Treatment with horse anti M. ovipneumoniae (Lomax strain) serum as primary antibodies, at a 1:10,000 dilution and incubate overnight at 4 °C. Wash with wash buffer for 5 minutes; 5) Treatment with biotinylated anti-horse IgG (H&L, affinity purified, made in goat. Vector Laboratories Inc. 68 Burlingame, CA), as secondary antibody at 1:200 dilution and incubate at room temperature for 30 minutes. Wash with wash buffer for 5 minutes; 6) Treatment with horseradish peroxidase-streptavidin (Vector Laboratories Inc) at 1:100 dilution and incubate for 30 minutes at room temperature. Wash with wash buffer for 5 minutes; 7) Treatment with substrate (3'3'-Diaminobenzidine tetrahydrochloride substrate kit, Zymed Laboratories Inc. San Francisco, CA), at manufacturer's direction and wash with distilled water; 8) Counter-stain with hematoxylin and eosin stain for 10 minutes and wash with tap water; and 9) Dehydrate with ethanol. Pro-par clearant, and mount with 36% xylene substitute mountant (Methacrylate polymer, Shandon) and examine under light microscope. Control slides were treated with normal horse serum or PBS in place of specific antiserum. 69 RESULTS

Isolation of Mycoplasmas Table 1 represents the species and number of microorganisms isolated from lambs or their lungs during this study. Mycoplasmas were isolated from both healthy and diseased lambs or their lungs. A total of 78 animals and 27 lung samples were found to be positive for having single or mixed types of mycoplasmas or other bacterial infection. Eighty-two percent of the isolates of mycoplasma were from lambs or lung samples having some evidence of respiratory disease, and the remaining isolates (18%) were from apparently healthy animals. Mycoplasma ovipneuwoniae was the most common microorganism isolated. It was isolated at a higher percentage from diseased animals (77%) compared to healthy ones (23%). The majority of cases (58 out of 86) with evidence of pneumonia had mixed mycoplasmas and/or other bacterial infection. All isolates of M. ovipneumoniae had "typical" colony morphology, i.e., centerless and lacy type colonies when grown on FA, while all other mycoplasmas isolated had "fried egg" type colony morphology (Figs 1 and 2). All mycoplasmas isolated were identified by the indirect immunofluorescent technique, and no cross reactivity was found among isolates of different mycoplasmas (Figs 3, 4, and 5). 70 Table 1. List of microorganisms isolated from 105 diseased or clinically normal lambs that were included in the study. Samples for culture included nasal swabs, bronchial washes, lung swabs, or lung homogenates. Mycoplasmas were identified by indirect immunofluorescent technique.

Lambs Microorganisms Total number® Percentage

M. ovipneumoniae 74 70 M. arginini 41 39 M. conjunctivae 3 3 M. capricolum 1 1 Unidentified mycoplasma 2 2 Ureaplasma sp 5" 5 Pasteurella sp gb 9 Staphylococcus sp 4b 4 Streptococcus sp 7b 7 E. coli/ Salmonella sp 13^ 12 Gram-negative hemolytic 24b 23 coccobacillus Bacillus sp 3b 3

® Total number of lambs from which agent(s) was isolated. ^ Isolates identified by morphological and biochemical tests. Fig 1. Centerless and lacy type colony morphology of M. ovipneumoniae at day 5 of growth on Friis agar.

Fig 2. Fried-egg-type colonies of Mycoplasma arginini at day 5 of growth on Friis agar. #4?#" Fig 3. Colony impression smear of M. ovipneumoniae stained by the indirect immunofluorescent technique which scored ++++.

Fig 4. Co]ony impression smear of M. ovipneumoniae stained by the indirect immunofluorescent technique which scored ++.

Fig 5. Colony impression smear of M. arginini stained by the indirect immunofluorescent technique. 76 77 The shape of the colonies of M. ovipneumoniae isolates was similar but minor differences were observed in sizes on PA. A difference in intensity of fluorescence color was also observed with the indirect immunofluorescent technique among different isolates of M. ovipneumoniae. When scored on the basis of intensity of fluorescence approximately 24% of isolates of M. ovipneumoniae were given an arbitrary score of ++++, 57% +++ and 19% ++ (Fig 3 and 4). A summary of results of isolation of other common bacteria from nasal swabs, bronchoalveolar washes or bronchial swabs are also presented in Table 1.

Gross Examination Gross lesions consisting of fibrinous adhesions of the lung to adjacent pleura, consolidation, and congestion were observed in both apparently healthy and clinically diseased lambs (Figs 6, 7, and 8). However, approximately 60% of the lungs from lambs with clinical respiratory disease were apparently normal on gross examination (Table 2). The lungs with severe consolidation and/or fibrinous adhesions involving multiple lobes usually had mixed M. ovipneumoniae and other bacterial infection. However, with only M. ovipneumoniae infection these lesions were mild in the form of focal consolidation. Gross lesions were not observed with pure M. arginini infection. Fig 6. Photograph of a normal lung of a lamb.

Fig 7. Photograph of the lung with congestion mostly in cranial and ventral half of lungs. TVyTT

tiwnM

#9 Fig 8. Photograph of a lung with fibrinous pleuritis and consolidation especially in the cranial lobes. 81 82 Table 2. Relationship between bacterial isolation and gross

lesions observed in lungs of lambs. Data from 39 specimens was included in the study.

Microorganisms No. of lungs with gross lesions Isolation Congestion Consolidation Adhesion

M. ovi.® 2" 3 1

M. ovi. & M. arg.^ - -

M. ovi. & other 6 6 3 bacteria®

M. ovi., M. arg. 1 1 - & other bacteria

M. arg. & bacteria 1 - - Bacteria only 2 1 1

No bacteria or Mp*^ 1 1 -

® Mycoplasma ovlpneumoniae, ^ Mycoplasmas arginini, ° mainly Gram-negative hemolytic coccobacillus and Pasteurella sp., ^ Mycoplasmas, ® Number of cases having lesions, ^ No gross lesions. 83 However, the lesions were present in a lung having mixed infection of this mycoplasma and other bacteria (Table 3). In lungs with consolidation, the bronchi and bronchioles were markedly dilated and more difficult to cut compared to other parts of the lung lacking such lesions.

Histopathology Typical histopathological lesions in the form of peribronchiolar and perivascular lymphocytic cuffing, lymphoid hyperplasia, and denudation of ciliated epithelium were observed in all lungs of lambs from which M. ovipneumoniae was isolated. These lesions were scored as mild (+), moderate (++), severe (+++), or very severe (++++), as indicated in Figs 10, 11, 12 and 13 respectively. The data representing severity of lesions and areas of lungs involved is presented in Table 4. Histopathological lesions were also observed in sections of lungs from apparently normal animals from which M. ovipneumoniae was not isolated. Mild to moderate microscopic lesions typical of mycoplasma infection were also observed in one out of four lung sections where M. arginini was the only organism isolated. This distinction could not be made with other mycoplasmas as they were isolated from mixed infection with M. ovipneumoniae and/or other bacteria. 84 Table 3. Isolation of microorganisms and pathological lesions in lungs of 39 lambs.

Microorganisms Number Gross Microscopic isolated of lungs lesions lesions* of Mp

M. ovi.^ 7 43 % 100 %

M. ovi. & M. arg.c 3 - 100 % M. ovi. & other 8 75 % 87 % bacterial M. ovi., M. arg. 1 100 % 100 % & other bacteria M. arg. & bacteria 4 25 % 50 %

Bacteria only 3 66 % - No bacteria or Mp® 13 22 % 33 %

® Peribronchiolar, perivascular lymphocytic cuffing, lymphoid hyperplasia, and loss of ciliated epithelium, ^ Mycoplasma ovipneumoniae, ° M. arginini, Includes Pasteurella sp.. Gram negative hemolytic coccobacillus, E. coli, Staphylococcus sp., ® Mycoplasmas. Fig 9. Photograph of a histological section of a normal lung stained with H & E stain.

Fig 10. Photograph of a histopathological section of the lung of a lamb. Histological changes indicated mild and focal areas of peribronchiolar and perivascular lymphocytic aggregation and scored +. A4: \' if A ?;1. ^Z.'iJ;, Fig 11. Photograph of a histopathological section of the lung of a lamb. Histological changes included multifocal areas of peribronchiolar and perivascular lymphocytic cuffing, lymphoid hyperplasia, and denudation of cilia, which scored ++. The majority of infiltrating cells were lymphocytes, plasma cells and macrophages. This type of reaction was common in lungs where M. ovipneumoniae was isolated.

Fig 12. Photograph of a histological section of the lung of a lamb. Histological changes included multifocal to diffuse areas of purulent exudation, atelectasis, occasionally with bacterial colonies, and other changes as in Fig 11 and which scored +++. This type of reaction was common in lungs having mixed M. ovipneumoniae and other bacterial infection. 88

«A*'

VSV'' Fig 13. Photograph of a histological section of the lung of a lamb. Histological changes included diffuse areas of loss of detail, necrosis, marked fibrosis around bronchioles, atelectasis, and obliteration of bronchioles and scored ++++. This type of response was observed near areas of consolidation. 90

• 91 Table 4. Histological lesions and bacterial isolation from lungs of 39 lambs.

RM° M03 Taa No EM Ldf RÇ! LC^ Il Bac^

1. s-ij + ++ + + + + - - -

2 . No Tag] ++ + + + + + + Y Y -

3 . S-14 ------

4. 10-green + + + + + - - Y Y

5. S-20 ------

6 . 21-yellow ------

7. 24-orange ++ + + + - - - - -

8. S-23 ------

9. S-24 +++ + + - - - Y Y -

10. S-25 + - + ------

11. S-26^ ------

12. 1053 +++ + ++ + + + Y - Y

13 . 1618 ------

14. IDE-lj ++ + + + + + Y - Y

15. IDE-144^ ++ + ++ + + + Y - Y

16. 351 ++ + ++ + + + Y Y Y

17. 349-red^ ++ ++ ++ + ++ ++ Y - Y

18. 927-blue ++ + + + ++ + Y - Y

19. 309McNayj + + + + ++ + + Y - -

20 . 327McNayj ++ + + - - - - - Y

21. 402McNay ++ + + + ++ + + - Y Y 92 Table 4 (continued)

22 . 362-McNay +++ ++ ++ ++ + + Y - Y

23 . Eness-1 + + + + ++ + Y - -

24 . BL-14 ++ + - ++ + + - - -

25. BL-13 +++ + ++ + ++ + - Y Y

26. 118-4^ ++ + ++ + + + Y - -

27. 117-53 +++ + ++ + ++ + Y - -

28 . 714-144^ ++ + + + + + Y - -

29. 126^ +++ ++ +++ ++ ++ ++ Y - Y

30. ML-1 + + ++ ++ + + Y - -

31. ML-2 + + + + + + - Y -

32. 265 ++ ++ + + + + - Y Y

33 . NT-Don C + + + + + + - - Y

34 . 221-redj + + + + + + Y - Y

35. 206-red^ +++ ++ ++ ++ + + Y Y -

36. K-lj + + + + + + - - -

37. 2350-Kj + + + + + - - - -

38. Eness-5 ++ + + + - - - - Y

39. 58-C ++ + + + + + Y - -

® right anterior, ^ left anterior, ° right middle, ^ left middle, ® right caudal, ^ left caudal lobes. ^ M. ovipneimoniae, ^ Mycoplasma arginini, ^ other bacteria. ^ coughing syndrome, + mild, ++ moderate or +++ severe lesions typical of mycoplasmal infection. Y isolation positive. 93

Immunohls bochemis try The indirect immunohistochemical staining procedure used in this study allowed the detection of M. ovipneumoniae antigen in situ. Out of 39 lung sections examined by this technique using a light microscope with a blue filter, 20 were identified as being positive for having M. ovipneumoniae antigens. The positive reaction was observed in the form of dark brown staining with a blue background only on intact ciliated epithelium. In other tissues or areas of the bronchiolar epithelium in which cilia were absent no staining reaction was observed. Macrophages or other phagocytic cells were not stained and background staining was minimal. A positive reaction was scored when at least one or more lobe sections from lungs were found stained with this technique. The mycoplasma antigens were observed as tiny small brown pleomorphic granules attached to ciliated epithelium. Control slides showed no staining (Figs 14, 15 and 16). A total of 16/20 IH positive cases were confirmed by isolation and identification methods and 4/20 by histopathology. However, 3 cases could not be detected by IH technique while M. ovipneumoniae was isolated. Lung sections of lambs infected with other mycoplasmas were not stained using specific M. ovipneumoniae antisera at optimum dilution. dumpiness and varying loss of cilia were observed in the bronchi or bronchioles of almost all lung sections infected Fig 14. Photograph of a histologic section of lung with negative control indirect immunohistochemical stain in which either normal horse serum or PBS was used instead of specific M. ovipneumoniae horse serum as primary antibody.

Fig 15. Photograph of a histologic section of lung with indirect immunohistochemical stain. Dark brown staining bodies sticking to ciliated epithelium is indicative of IH positive for M. ovipneumoniae. 95 Fig 16. Photograph of a histologic section of lung with indirect immunohistochemical positive stain as in fig 15. dumpiness and patchy loss of cilia in M. ovipneumoniae infected bronchioles is apparent. 97 98 with M. ovipneumoniae. However, these abnormalities were not observed in tissues from sole M. arginini positive lungs. 99

DISCUSSION

The M. ovipneumoniae associated coughing syndrome reported was characterized by acute paroxysmal cough, mucoid nasal and ocular discharges, mild fever, severe exercise intolerance, restricted growth, and associated rectal prolapses. These signs were acute in feeder lambs between the age of 5-12 weeks and decreased in severity as they grew older. These features of a coughing syndrome of lambs have not been reported in the previous literature. However, a chronic non-progressive interstitial to exudative type pneumonia had been reported (Sullivan, 1973; Alley et al., 1975; Davies et al., 1981; Malone et al., 1988). Mycoplasma ovipneumoniae was the predominant species of Mycoplasma and was isolated from 70% of lambs investigated. However, it was isolated from both clinical cases and apparently healthy lambs. Twenty-three percent of M. ovipneumoniae isolates were from clinically normal animals. These isolates of M. ovipneumoniae were recovered as a sole organism or as a mixed infection with other mycoplasmas and/or bacteria from both diseased and normal animals or their lungs. Mycoplasma ovipneumoniae were generally isolated together from the nasal mucosa, bronchial washes, or lungs at the same time but their apparent absence in the nasal mucosa did not rule out the infection. Bronchial washes of live animals and lung 100 swabs were more successful recovery methods for isolation of mycoplasma from positive animals. Mycoplaawa arginini was the second most common mycoplasma isolated from these animals. However, it was mainly found as a mixed infection with M. ovipneumoniae and/or other bacteria, and could not be isolated as a sole organism from diseased lambs or their lungs. This presence of mixed infection correlated with the previous reports of studies on exudative type of pneumonia or nonprogressive chronic interstitial type of pneumonia (Sullivan et al., 1973; Alley et al., 1975; Alley and Clarke, 1977; Davies et al., 1981; Bakke, 1982; Babu et al., 1982; Pfeffer et al., 1983; St. George and Carmichael, 1975; Malone et al., 1988). However, the exudative type pneumonia associated with M. ovipneumoniae was not observed in our study as described previously (Pfeffer et al., 1983; Babu et al., 1982) . Mycoplasma conjunctivae and ureaplaamaa were isolated from 3 and 5 lambs respectively (Table 1), in association with other mycoplasma or bacteria. Therefore, they could not be directly associated with this disease or pathology. Mycoplasma conjunctivae is usually associated with keratoconjunctivitis in lambs (Barile et al., 1972), but its role in pulmonary infection has not been documented. Mycoplasma capricolum was isolated from nasal mucosa of a healthy lamb as mixed infection with M. arginini. Previous studies has also 101 indicated the presence of this mycoplasma in lambs without any evidence of pneumonia (Banergee et al., 1979). Pasteurella sp. are reported to be the most common cause of pulmonary infection in lambs and are associated with an acute exudative type pneumonia with pleurisy, and commonly with a fatal outcome (Stamp and Nisbet, 1963; Gilmour, 1978; Gilmour, 1980) . Various species of bacteria were isolated from 9 cases and 7 of them had mixed M. ovipneumoniae infection. We could not isolate P. haemolytica as frequently as reported earlier, even from a group of diseased lambs that had not been treated with antibiotics (McGrown et al., 1957; Stevenson, 1969; Davies et al., 1981; Jones et al., 1982; Ellis et al., 1984; Foder et al., 1984). The pathologie findings (described elsewhere), associated with these organisms alone or in association with M. ovipneumoniae were more severe compared to either of these infections alone. These findings support the observations previously reported (Jones et al., 1978; Jones et al., 1982). A number of other bacterial isolates from the respiratory tract of sheep have been reported but most of them are considered opportunistic (Stevenson, 1977). The isolation of a Gram-negative hemolytic coccobacillus mostly from the upper respiratory tract of lambs and occasionally from lungs in this study was a unique observation and their identification as to type could not be established. These organisms were isolated 102 from lambs with M. ovipneumoniae associated coughing syndrome of lambs as well as from apparently normal animals. Isolation of a similar type of bacteria from cattle has been reported previously (Bosworth and Lovell, 1944), but the etiological significance of these organisms was not clear. The colony morphology and growth characteristics of M. ovipneumoniae in our study were similar to those observed by others (Jones et al., 1979; Cottew, 1983; Erno et al., 1978; Goltz et al., 1986; Brogden et al., 1988). Mycoplasma ovipneumoniae required 5-7 days to develop colony size identifiable morphologically under a dissecting microscope, compared to 3-4 days for M. arginini and other mycoplasmas. Different isolates of M. ovipneumoniae did show minor differences in their growth and morphological characteristics. It may be due to some genetic differences in each of the isolates, as the growth environments were similar for all isolates. The indirect immunofluorescent technique utilized in this study enabled easy identification of different isolates of mycoplasmas especially M. ovipneumoniae. The protocol utilizing colony impression smears on glass slides rather staining directly on agar surface may also be utilized for other mycoplasmas with centerless colonies as they easily clean off from agar surfaces during washing steps. Various isolates M. ovipneumoniae behaved differently in picking up 103 fluorescent stain as reported in the text. Whether this represents a specific characteristic of different isolates or was due to some variations in passages or growth microenvironment. The development of gross lesions was observed in 43% of the M. ovipneumoniae infected lungs. However, these lesions were mild and focal in character appearing primarily in the anterior lobes. Infection with Pasteurella sp. or other bacteria (mainly Gram-negative coccobacillus) either alone or associated with M. ovipneumoniae produced comparatively diffuse type lesions, consisting of congestion, consolidation, and/or adhesions. Mycoplasma ovipneumoniae associated gross lesions described in the text could not be correlated with previous reports in the literature. However, gross lesions associated with mixed infections were similar to previous reports (Foggie et al., 1976; Jones et al., 1982; Davies, 1985; Malone et al., 1988). Although a Gram-negative hemolytic coccobacillus type bacteria represented approximately 40% of total bacterial isolates from both normal and clinically diseased lambs, its role in contributing to pneumonia or related pathology could not be determined. The histological findings associated with M. ovipneumoniae were very typical and consistently present in all lungs obtained from diseased lambs. The histological lesions were severe in anterior lobes and with decreasing 104 intensity toward the caudal lobes (Table 4). The prominent features observed were peribronchiolar and perivascular lymphocytic cuffing, lymphoid hyperplasia, interstitial thickening and loss of ciliated epithelium in major bronchioles or bronchi of infected lungs. The majority of cells of infiltration were lymphocytes, plasma cells and macrophages. The lesions were severe and of proliferative and exudative types in lungs where other bacteria were also involved. These findings were similar to those reported earlier (Foggie et al., 1976; Davies, 1985). However, we did not observe lesions such as hyaline scars in lung sections as described previously by Gilmour et al. (1982). The detection of antigen in formalin-fixed paraffin- embedded lung tissue sections by the enzyme-linked immunohistochemical staining appeared to be a reliable method as described earlier for use in veterinary pathology (Imada et al., 1987; Doster and Lin, 1988; Deborah and Chelack, 1991). We successfully used this technique for the detection of M. ovipneumoniae antigen in formalin-fixed lung sections. The M. ovipnexiiaoniae antigens were found located on the ciliated epithelium of bronchi, bronchioles and trachea in the form of dark brown stain. Similar observations have been reported for detection of M. hyopneumoniae, M. galliaepticum, M. aynoviae, and ureaplasmaa antigens in other animals (Hsu et al., 1981; Quinn et al., 1981; Imada et al., 1982; Doster and Lin, 1988). 105 The cellular architecture was not altered with this technique as has been reported for frozen lung tissues. This provided the opportunity to study the relationship of M. ovipneumoniae to the cells in the tissues. In addition IH stained sections were permanent and could be stored for re- evaluation at a later date (Chasey and Wood, 1984). Indirect immunohistochemical staining proved to be a more sensitive method in detection of M. ovipneumoniae antigen in tissue sections as compared to isolation and cultivation or IF techniques. We were able to detect 20% more IH positive cases where isolation attempts failed to identify the presence of M. ovipneumoniae, but histological changes in lungs did indicate their presence. The observation of IH stain required a medium to large airway with intact ciliated epithelium in the tissue section to detect mycoplasma antigen in situ. Only 16% the cases were found negative by IH technique where isolation and/or histological changes confirmed the presence of M. ovipneumoniae infection. This may have been due to the absence of larger bronchioles bearing ciliated epithelium or complete loss of cilia as observed in some lung sections. One may also suspect immune clearance of mycoplasma from those lungs. The protocol adopted in our study appeared to be species specific as antigens of other mycoplasmas could not be detected from lungs having other evidences of their presence. The results of our study suggested that M. ovipneumoniae 106 was the main organism isolated from a coughing syndrome of lambs. However, the mere isolation from respiratory tract did not indicate the clinical disease. The development of gross lesions in M. ovipnewnoniae infected lungs, although present only in a few percent of lungs, were characteristic in the form of focal areas of consolidation. Lesions due to mixed mycoplasmal and other bacterial infections were in the form of congestive, consolidative and/or adhesive types. The histologic features in M. ovipneumoniae infected lungs were classical and significantly present in all M. ovipneumoniae infected lungs. Indirect immunofluorescent and indirect immunohistochemical staining were sensitive and specific tests for detection of this mycoplasma. These tests may be used for identification of mycoplasma isolates in culture or their detection at postmortem in lung tissues, respectively. Immunohistochemical staining has the advantages to study the relationship of antigen to the cells in the tissues and detection of even small quantities of M. ovipneumoniae antigens in lungs where isolation attempts usually fail. In addition the difficulties of nonspecific staining in tissue sections as seen with IF method were minimal with IH technique. 107

REFERENCES

Alley, M. R. and Clarke, J. K. 1977. The influence of microorganisms on the severity of lesions in chronic ovine pneumonia. N. Z. Vet. J. 27:217-220. Alley, M. R. and Clarke, J. K. 1979. The experimental transmission of ovine chronic non-progressive pneumonia. N. Z. Vet. J. 27:217-220. Alley, M. R., Quinlan, J. R., Clarke, J. K. 1975. A survey of the prevalence of Mycoplasma ovipneumoniae and M. arginini in the respiratory tract of sheep in New Zealand. N. Z. Vet. J. 23:137-141. Babu, Y. H., Murthy, K. G., and Krishnaswamy, S. 1982. Isolation and identification of mycoplasmas from respiratory tract of sheep. Kerala J. Vet. Sci. 13:79-88. Bakke, T. 1982. The occurrence of mycoplasmas and bacteria in lungs from sheep in southern Norway. Acta. Vet. Scand. 23:235-247. Banerjee, M., Singh, N., and Gupta, P. P. 1979. Isolation of Mycoplasmas and Acholeplasmas from pneumonic lesions in sheep and goats in India. Zbl. Veterinar. Med. B. 26:689- 6995. Barile, M. F., Del Guidice, R. A., Tully, G. 1972. Isolation and characterization of Mycoplasma conjunctivae sp. n. from sheep and goats with keratoconjunctivitis. Infect. Immun. 5:70-76. Barile, M. F., Malizia, W. F., and Riggs, D. B. 1962. Incidence and detection of pleuro-pneumonia-like organisms in cell cultures by fluorescent antibody and culture procedures. J. Bacterid. 84:130-136. Bosworth, T. J. and Lowell, R. 1944. The occurrence of hemolytic coccobacilli in the nose of normal sheep and cattle. J. Comp. Pathol. Ther. 54:168-171. Brogden, A., Rose, D., Cutlip, R. C., lehmkuhl, H. D., and Tully, J. G. 1988. Isolation and identification of mycoplasmas from the nasal cavity of sheep. Am. J. vet. Res. 49:1669-1672. 108 Buddie, B. M., Herceg, M., and Davies, D. H. 1984. Experimental infection of sheep with Mycoplasma ovipneumoniae and Pasteurella haemolytica. Vet. Microbiol. 9:543-558. Carmichael, L. E., St.George, T. D., Sullivan, N. D., and Horsefall, N. 1972. Isolation, propagation, and characterization studies of an ovine mycoplasma responsible for proliferative interstitial pneumonia. Cornell Vet. 62:654 Chasey, D. and Wood, S. B. 1984. Detection of immunoperoxidase labelled mycoplasmas in cell culture by light microscopy and electron microscopy. J. Med. Microbiol. 17:23-30. Clarke, J. K., Brown, V. G., and Alley, M. R. 1974. Isolation and identification of mycoplasmas from the respiratory tract of sheep in New Zealand. N. Z. Vet. J. 22:117-121. Cottew, G. S. 1983. Recovery and identification of caprine and ovine mycoplasmas. In: Tully, J. G. and Razin, S. (eds.). Methods in Mycoplasmology. Vol. II. Academic Press, New York. pp. 91-104. Davies, D. H. 1985. Aetiology of pneumonias of young sheep. Prog. Vet. Microbiol. Immun. 1:229-248. Davies, D. H., Jones, B. A. H., and Thurley, D. C. 1981. Infection of specific pathogen free lambs with parainfluenza virus type-3, Pasteurella haemolytica and Mycoplasma ovipneumoniae. Vet. Microbiol. 6:295-308. Deborah, M. H. and Chelack, B. J. 1991. Technical consideration for developing immunohistochemical staining procedures on formalin-fixed paraffin-embedded tissues for diagnostic pathology. J. Vet. Diagn. Invest. 3:101- 112. DelGuidice, R. A., Robollard, N. F., and Carski, T. R. 1967. Immunofluorescence identification of mycoplasma on agar by use of incidence illumination. J. Bacterid. 93:1205. Doster, R. and Lin, B. C. 1988. Identification of Mycoplasma hyopneumoniae in formalin-fixed porcine lung, using an indirect immunoperoxidase method. Am. J. Vet. Res. 49:1719-1721. Ellis, J. 1984. Pasteurella haemolytica infection in sheep. Comp. Community Ed. 6:5360-5366. 109 Eneas, G. P., Kaeberle, M. L., and Hummel, D. 1990. Association of Mycoplaama ovipneumoniae and respiratory- disease in feeder lambs. Beef-Sheep Research Report. Iowa State University, Ames, Iowa. pp. 44-45. Erno, H., Al-Aubaidi, J. M., Ojo, M. 0., Minga, U. M., and Sikdar, A. 1978. Classification and identification of ovine and caprine mycoplasmas. Acta Vet. Scand. 19:392- 406. Fodor, L., Varga, J., Hajtos, I., and Szemeredi, G. 1984. Serotypes of Paateurella haemolytica isolated from sheep, goats and calves. Zbl. Vet. Med. B. 31:466-469. Foggie, A. and Angus, K. W. 1972. Observation on the distribution of Mycoplasma arginini as a respiratory tract infection in sheep and its pathogenicity for specific pathogen free lambs. Vet. Rec. 90:312-313. Foggie, A., Jones, G. E., and Buxton, D. 1976. The experimental infection of specific pathogen free lambs with Mycoplasma ovipneumoniae. Res. Vet. Sci. 21:28-35. Friis, N. F. 1974. Mycoplasmas in pigs with special regard to the respiratory tract. Commissioned by: Forlag, D.S.R. Royal Veterinary and Agriculture University, Copenhagen, Denmark. Gilmour, N. J. L. 1978. Pasteurellosis in sheep. Vet. Rec. 102:100-102. Gilmour, N. J. L. 1980. Pasteurella haemolytica infection in sheep. Vet. Quart. 2:191-198. Gilmour, J. S., Jones, G. E., Keir, W. A., and Rae, A. G. 1982. Long term pathological and microbiological progress in sheep of experimental disease resembling atypical pneumonia. J. Comp. Pathol. 92:229-238. Goltz, J. P., Rosendal, S., McCrew, B. M., and Ruhnke, H. L. 1986. Experimental studies on the pathogenicity of Mycoplasma ovipneumoniae and M. arginini for the respiratory tract of goats. Am. J. Vet. Res. 50:59-67. Gourlay, R. N. and Haward, C. J. 1983. Recovery and identification of bovine mycoplasmas. In: Tully, J.G. and Razin, S. (eds.). Methods in Mycoplasmology. Vol. II. Academic Press. New York. pp. 81-90. 110 Hamdy, A. H. and Pounden, W. D. 1959. Experimental production of pneumonia in lambs. Am. J. Vet. Res. 20:78-83. Hsu, S. M., Raine, L., and Fanger, H. 1981. The use of avidin- biotin-complex in immunoperoxidase techniques. Am. J. Clin. Pathol. 75:816-821. Imada, Y., Nonomura, I., and Furuta, K. 1982. Indirect immunoperoxidase technique for the assay of antibodies against Mycoplasma galliaepticum and AT. synoviae in chicken serum. Natl. Inst. Anim. Health.' Q. 22:16-22. Imada, Y., Uchida, I., and Hashimoto, K. 1987. Rapid identification of mycoplasmas by indirect Immunoperoxidase test using small square filter paper. J. Clin. Microbiol. 25:17-21. Jones, G. E., Buxton, D., and Harker, D.B. 1979. Respiratory infections in housed sheep, with particular reference to mycoplasmas. Vet. Microbiol. 4:47-59. Jones, G. E., Foggie, A., Mould, D. L., and Livitt, S. 1976. The comparison and characterization of glycolytic mycoplasmas isolated from the respiratory tract of sheep. J. Med. Microbiol. 9:39-52. Jones, G. E., Gilmour, J. S., and Rae, A. G. 1978. Endobronchial inoculation of sheep with pneumonic lung- tissue suspensions and with the bacteria and mycoplasmas isolated from them. J. Comp. Path. 88:85-96. Jones, G. E., Gilmour, J. S., and Rae, A.G. 1982. The effects of different strains of Mycoplasma ovipneumoniae on specific pathogen-free and conventionally-reared lambs. J. Comp. Pathol. 92:267-272. Kaeberle, M. L. and Eness, G. P. 1988. Serological analysis of pneumonia in feeder lambs. Beef-Sheep Research Report. Iowa State University, pp. 54-55. Kenny, G. E. and Cartwright, F. D. 1973. Effect of urea concentration on growth of Ureaplasma urealyticum (T- strain mycoplasma). J. Bacterid. 132:144-150. Knudtson, W. U., Reed, D. E., and Daniel, G. 1986. Identification of Mycoplasmatales in pneumonic calf lungs. Vet. Microbiol. 11:79-91. Ill Lemcke, R. 1965. A serological comparison of various species of Mycoplasma by an agar gel double-diffusion technique. J. Gen. Microbiol. 38:91-100. McGowan, B., Moulton, J. E., and Shultz, G. 1957. Pneumonia in California lambs. J. Am. Vet. Med. Ass. 131:318-323. Mew, A. J., lonas, G., Clarke, J. K., Robinson, A. J., and Marshall, R. B. 1985. Comparison of Mycoplasma ovipneumoniae isolates using bacterial restriction endonuclease DNA analysis and SDS-PAGE. Vet. Microbiol. 10:541-548. Nakane, P. K. and Pierce, G. B. Jr. 1967. Enzyme labelled antibodies: preparation and application for the localization of antigens. J. Histochem. Cytochem. 14:929- 931. Pfeffer, A., Thurley, D. C., Boyes, B. W. et al. 1983. The prevalence and microbiology of pneumonia in a flock of lambs. N. Z. Vet. J. 31:196-202. Quinn, P. A., Arshoff, L. U., and Li, H. C. S. 1981. Serotyping of Ureaplaama urealyticum by immunoperoxidase assay. J. Clin. Microbiol. 13:670-676. Rosendal, S. and Black, F. T. 1972. Direct and indirect immunofluorescence of unfixed and fixed mycoplasma colonies. Acta. Pathol. Microbiol. Scand. Sect. B. 80:615-622. St.George, T. D. and Carmichael, L. E. 1975. Isolation of Mycoplasma ovipneumoniae from sheep with chronic pneumonia. Vet. Rec. 97:205-206. Stalheim, O. H., Foley, J. W., Stone, S. S., and Rhoades, K. R. 1975. Production of antiserums in horses to mycoplasmas of bovine origin and their use in growth inhibition and fluorescent antibody tests, In: Proceedings, 18th Annu. Meet. Am. Assoc. Vet. Lab. Diagnosticians, pp. 207-219. Stamp, J. T. and Nisbet, D. I. 1963. Pneumonia in sheep. J. Comp. Pathol. 73:319-328. Stevenson, R. G. 1969. Respiratory diseases of sheep. Vet. Bull. 39:747-755. Stevenson, R. G. 1977. Proliferative interstitial pneumonia in lambs. Can. Vet. J. 18:313-317. 112 Sullivan, N. D., St.George, T. D., and Horsefall, N. 1973. A proliferative interstitial pneumonia of sheep associated with mycoplasma infection. I. Natural history of the disease in a flock. Aust. Vet. J. 49:57-62. Tully, J. G. 1983. Cloning and filteration techinques for mycoplasmas. In: Razin, S., and Tully, J. G. (eds.). Methods in Mycoplasmology. Vol. I. Academic Press, New York. pp. 173-177. 113 ACKNOWLEDGMENTS

The author thank Dr. Paul G. Eness for his constant help in obtaining field samples for isolation of microorganisms, David L. Cavanaugh (VDL) for technical assistance with immunohistochemical staining, Bob Ross (VMRI) for tissue sectioning, and Dr. John J. Andrews, for his guidance in interpretation of microscopy. 114

PAPER II. MITOGENIC ACTIVITY OF MYCOPLASMA OVIPNEVMONIAE ANTIGENS FOR OVINE LYMPHOCYTES 115

MITOGENIC ACTIVITY OF MYCOPLASMA OVIPNEUMONIAE ANTIGENS FOR OVINE LYMPHOCYTES

Mumtaz A. Khan, D.V.M., M.Sc. (CMS), M.Sc. (TVM) Merlin L. Kaeberle, D.V.M., Ph.D. .116

SUMMARY

Mycoplasmas are ubiquitous pathogens which cause inflammatory diseases of the respiratory tract and joints. The pathogenic mechanisms through which they cause disease remains an enigma. A characteristic feature of respiratory mycoplasmas is hyperplasia of bronchus-associated lymphoid tissue. This may be related to a cellular immune response to an antigen. In vitro lymphocyte blast transformation with specific antigens or nonspecific mitogens is one of the routine assays for evaluating potential cellular immunity of an individual. Some mycoplasma antigens have been reported to stimulate lymphocytes of different hosts nonspecifically while others have suppression effects. Mycoplasma ovipneumoniae is commonly associated with ovine pneumonia. Three different antigen preparations from M. ovipneumoniae were evaluated for their effect on in vitro transformation of ovine peripheral blood lymphocytes. The lambs under study were: (1) lambs with a clinically apparent M. ovipneumoniae associated coughing syndrome, (2) lambs experimentally infected with M. ovipneumoniae, and (3) disease-free lambs. The three M. ovipneumoniae antigen preparations appeared to have nonspecific mitogenic activity, although the response was mild in term of stimulation indexes. The mycoplasma membrane fraction appeared to have more 117 mitogenic activity than heat-killed or SDS treated mycoplasma antigens. The blast transformation response was considerably higher with nonspecific mitogens (PHA and Con-A). 118

INTRODUCTION

Mycoplasmas are common parasites of man, animals and poultry, and cause a variety of diseases, both acute and chronic. The pathogenic potential of an organism is dependent upon its specific ability to cause tissue damage and/or its interaction with the host defense mechanisms, primarily those related to immune system. The pathogenic mechanisms through which mycoplasmas establish a disease is still a mystery. However, reports have indicated their role for immuno- pathological reactions, especially in causing lung damage and arthritis (Aldridge et al., 1977; Biberfeld and Gronowicz, 1978; Cassell et al., 1980; Cole and Atkin, 1991; Cole et al., 1992). A characteristic feature of most respiratory mycoplasma infections is hyperplasia of the bronchus- associated lymphoid tissue. This may be due to proliferation of lymphocytes and/or their recruitment from the circulation. This response can be related to delayed type hypersensitivity and is considered a part of the specific immune reaction to the causative organisms. The in vitro cell mediated immune response to infection has been studied by various research workers by examining the development of lymphocyte blast- transformation, delayed type hypersensitivity skin reaction, and macrophage migration inhibition by using different mycoplasmas or their antigens (Ginsburg and Nicolet, 1973; 119 Biberfeld, 1977; Naot et al., 1979; Naot, 1982; Kishima et al., 1983; Howard and Taylor, 1985; Ross et al., 1992). Various agents are known to be capable of inducing the transformation of lymphocytes into blast cells and are referred to as mitogens (Moler, 1972). Mitogen driven blast transformation of both sensitized or nonsensitized lymphocytes is a common assay for evaluation of immunopotency of both T and B lymphocytes (Barile and Razin, 1979; Kristensen et al., 1982). Phytohaemagglutinin (PHA), concanavalin-A (Con-A), and pokeweed mitogen (PWM) are some natural lectins having mitogenic capability to cause blast transformation of various sub-population of lymphocytes. This activity has also been observed with lipopolysaccharides (LPS) of Gram-negative bacteria, the Fc fragment of immunoglobulins, and BCG vaccine (Naot et al., 1977). Many members of Mycoplasmatales have been reported to behave as specific or nonspecific mitogens and stimulate lymphocytes of different hosts to undergo blast transformation (Ginsburg and Nicolet, 1973; Cole et al., 1975; Naot et al., 1977; Naot, 1982). Other workers suggested that some mycoplasmal components suppress the blast transformation response of lymphocytes (Naot et al., 1977; Sabato et al., 1981). Previous studies have indicated that the pathologic lesions associated with mycoplasmal diseases were immune injuries (Barile and Tully, 1979). In vivo development of 120 lymphocytic hyperplasia and interstitial pneumonia induced by mycoplasma membranes antigen may also be an association of this mechanism (Cole et al., 1985; Messier et al., 1990). Mitogenic activity of mycoplasmas is mainly associated with membrane proteins, to some extent carbohydrates, but not with lipid moieties (Naot et al., 1979). The blastogenic response could be increased by using pre-sensitized lymphocytes from diseased or vaccinated animals (Salih et al., 1987). The activated lymphocytes may elaborate a variety of lymphokines, interferons, chemotactic substances, and other secretory products which could influence the immune response or may trigger inflammation (Liggit, 1985). This in vitro ability of mycoplasmas to interact with host lymphocytes led to the hypothesis that this activity may contribute to the pathogenesis of pneumonia. Mycoplasma-lymphocyte interaction could provide an important model to understand some aspects in the pathogenesis of diseases. Studies on mitogenic potential of M. ovipneumoniae are not available in the literature. However, lymphocytic aggregation and lymphoid tissue hyperplasia with natural disease has led us to hypothesize that the immune- related response may be the main cause of disease with this mycoplasma. To test this hypothesis, we studied the in vitro interaction of M. ovipneumoniae antigens with lymphocytes of sheep. We believe that the findings of this study will 121 contribute to our understanding of the pathogenesis and disease development of M. ovipneumoniae infection. 122

MATERIALS AMD METHODS

Experimental Animals Lymphocyte blastogenic assay was conducted to evaluate the mitogenic potential of three different preparations of M. ovipneumoniae on peripheral blood lymphocytes from three groups of lambs between the age of 4-6 months, each group composed of 8 animals. Group A. Lambs in this group were culturally negative for any mycoplasmas at the time of experiment. They were kept in 4 separate pens (# 42, 43, 49, and 56) in the isolation facility of the Veterinary Medical Research Institute, with 2 animals in each pen. The lambs with Tag numbers 59, 60, 61, 62, 63, and 64 were experimentally infected with M. ovipneumoniae (P- 21, a field strain), at a dose rate of 5 X 10® colony forming units/ ml/ animal, intratracheally. Animals in pen # 56 (Tag Nos. 57 and 58) were kept as untreated controls. Group B. Lambs (Numbered 358, 359, 385, 399, 406, 411, 412, 415) were randomly selected from a flock naturally- infected with M. ovipneumoniae and had typical clinical features of a "coughing syndrome" described elsewhere. Group C. Lambs (Numbered 1-8) were culturally negative for AT. ovipneumoniae infection, and were housed together in an open shed in isolation from other sheep. 123

Blood Samples For lymphocyte blastogenic assays blood samples were collected from each animal in group A before challenge with M. ovipneumoniae and then afterward from all animals of the three groups at two-week intervals. Ten ml of blood was collected from the jugular vein of each animal in a 50 ml siliconized sterile tube containing 1.5 ml of 2X ACD [Citric acid (8 g), Trisodium citrate (22 g), Dextrose (25 g), and distilled water 500 ml)]. Each blood sample was diluted with 10 ml of prewarmed PBS (Dulbecco's PBS, Sigma Chemical Co., St.Louis, MO).

Mitogens Two nonspecific mitogens and three M. ovipneumoniae antigen preparations were used in this study. These were: (1) Phytohemagglutinin (PHA-P, Sigma) at a concentration of 100 ug/ ml, (2) Concanavalin-A (Con-A, Sigma) at concentration of 50 ug/ ml, and (3) Three different M. ovipneumoniae (Lomax strain) antigen preparations from a stock culture of AT. ovipneumoniae. The M. ovipneumoniae antigens were: (1) M. ovipneumoniae membrane fraction (MMF). Mycoplasma ovipneumoniae was grown in 1000 ml of MFBM as described previously in Paper 1. The MMF was prepared by digitonin induced osmotic lysis, by the method of Razin (1983). Briefly, a 36-hour broth culture was 124 centrifuged at 1200 rpm for 30 minutes and the pellet was harvested, washed twice with 0.25 M NaCl and resuspended in 100 ml solution of 0.25 M NaCl, 2 N glycerol, and 0.25 mg digitonin. After incubation for 30 minutes at 37 °C, the suspension was poured into 100 ml of pre-warmed triple distilled water. Following an additional 30 minutes incubation, the lysed material was harvested by centrifugation at 34,000 rpm for 30 minutes. The pellet was resuspended in beta buffer (0.15 M NaCl, 0.05 Tris aminomethane, and 0.01 M beta mercaptoethanol, pH. 7.4, diluted to 1:20 with deionized water). This membrane material was free of any viable mycoplasma as attested by plating samples on both MFBM and FA. The protein content was determined by the Pierce BCA Protein Assay (Pierce Chemical Co., Rockford, 111) according to manufacturer's directions and used as MMF antigen containing 400 ug of protein base/ ml of suspension. The aliquots were stored at -20 °C before use. (2) Mycoplasma ovipneumoniae heat killed antigen (HKA). A stock culture of M. ovipneumoniae was prepared as described earlier. The pellet was washed twice with PBS and finally resuspended in 10 ml of PBS. The suspension was then heated in a water bath at 56 °C for 30 minutes. The viability of organisms was attested by plating on agar and broth as described previously. The protein content was determined as before and the final dilution of mycoplasma HKA was made to 125 400 ug of protein base/ ml of suspension, divided into small aliquots and stored at -20 °C before use. (3) Sodium dodecyl sulfate M. ovipneumoniae antigen (SMA). A stock culture of M. ovipneumoniae was prepared as described earlier. The pellet was resuspended in PBS to give a final concentration of 1 mg of protein/ ml. It was mixed with 1% SDS and incubated for 30 minutes at 37 °C and dialyzed in 0.01 M Tris NaCl buffer pH 7.2 for 48 hours. The final dilution of SMA was made to 400 mg of protein base/ ml and stored in aliquots at -20 °C.

Lymphocyte Blast Transforming Assay The peripheral blood lymphocyte blast transforming assay was conducted by measuring the incorporation of [^H] thymidine into DNA. Lymphocytes were harvested from peripheral blood by the procedure of Boyum (1968). Briefly, 10 ml of diluted blood was gently layered on 6 ml of prewarmed Histopaque 1077 (Sigma) and centrifuged at 1400 rpm for 20 minutes at room temperature. The lymphocyte rich (opaque) interface was aspirated with a pipet and propipet and transferred to a tube containing 20 ml of Hank's balanced salt solution (HBSS, without calcium and magnesium, Gibco Laboratories), and centrifuged again at 750 rpm for 15 minutes. The supernatant was aspirated, the pellet resuspended in 10 ml of HBSS, and centrifuged at 750 rpm for 15 minutes. Finally, the pellet was 126 resuspended in 1 ml of HBSS and the mononuclear cell concentration determined with a Coulter Counter (Coulter Electronics Inc., Hialeah, FL). The viability of cells was determined with trypan blue stain. Those samples having 80% or more viable cells were used in the blastogenic assay. Each cell sample was diluted to a concentration of 50,000 cells/ 200 ul with diluting media containing 89% M-199 (Gibco Laboratories), 1% Antibiotics-Antimycotic 100 X (Penicillin-G, Streptomycin sulfate, and Amphotericin-B, Gibco Laboratories), and 10% fetal calf serum (Gibco Laboratories). A 200 ul aliquot of diluted cell suspension was dispensed in flat bottom 96 well cell culture plates, using 3 wells per mitogen/ animal, and 3 wells left as untreated controls for each animal. The mitogens were added at a dose rate of 25 ul/ mitogen/ well and the same volume of medium was added to cells of control wells. The plates were incubated for 96 hours at 37 °C in a COj incubator. After the required period of incubation, plates were labeled with [^H] thymidine (0.375 uCi/ well, Amersham International Pic., Amersham, UK). After an additional 18 hours of incubation, the cells were harvested on fiberglass filters (Filtermats, Skatron Inc., Sterling, VA) with a Titertek cell harvester (Flow Laboratories, Rockville, MD), and dried in an incubator. Dried filter paper discs were transferred to scintillation vials containing Scintiverse BD (Fisher Chemical, Fair Lawn, NJ), and radioactivity was 127 determined with a scintillation counter (Packard Instrument Co., Downers grove. 111). The stimulation index (SI) was calculated by the following formula: Stimulation index = mean count of 3 wells of mitogen stimulated lymphocyte cell culture/ mean count of cells of 3 control wells. Group average for each mitogen used was calculated and the results were analyzed by analysis of variance with a 95 % confidence level (P < 0.05). 128 RESULTS

The peripheral blood lymphocyte blastogenic response in the form of SI as measured by the uptake of [^H] thymidine by DNA of lymphocytes of lambs in all groups are presented in Tables 1, 2, 3, and 4. The overall mean values of all experiments for separate groups is presented in Pig 1. All lambs of groups A, B, and C gave significantly higher mean SI values (P < 0.01) by using nonspecific mitogens (Con-A and PHA), compared to their respective controls. However, when these mitogens were compared to each other and at different periods in each group, no significant differences among them were noted. Values of individual animals in each experiment, in all groups varied (data not shown). The three specific mycoplasma antigens (HKA, SMA, and MMF) used in this study stimulated lymphocytes in culture as identified by SI values, however, the response was significantly lower (P < 0.01) than nonspecific mitogens in all groups. There were differences in average SI values between each group at different time intervals when mycoplasma antigens used. All three antigen preparations were compared in each respective group of animals for their stimulating capabilities. Mycoplasma membrane fraction antigen gave higher SI (P < 0.05) compared to HKA and SMA in groups A (treated) and B. A similar pattern in stimulation indexes was observed 129 Table 1. Mitogen/ antigen induced stimulation indexes of lymphocytes of lambs of group A (treated).

Exp® PHA'' Con-A° HKA"^ SMA° MMF^ 0 o 0 1 124 ± 50* 137 + 76* 1,,8 ± .63 1.A ± 3 .9 ± .77 2 159 ± 66* 178 + 50* 1.,4 + .17 2 . 7 ± .98 2 .8 ± .78 3 194 ± 40* 273 ± 113* 1. 7 ± .16 1. 5 ± .18 1.8 + .42 4 150 ± 46* 167 ± 69* 1. 7 + .24 1.8 ± .34 2.2 ± .33 5 153 ± 21* 98 + 31* 1.4 ± .18 1.6 ± .29 1.8 ± .49

6 41 ± 13* 32 + 9* 1.7 ± to 2 . 0 ± .34 1.8 ± .26 7 65 ± 21* 54 ± 24* 1.4 ± .31 1.6 ± .20 3 .4 ± .56 8 72 ± 21* 102 + 33* 2.4 ± .53 2 . 1 ± .27 5 .4 ± 1.0 9 82 ± 27* 109 + 28* 1.6 ± .42 1.5 ± . 08 2.8 ± .80 10 46 ± 11* 75 ± 20* 1.6 ± .30 1. 5 ± .16 1.8 + .24

Peripheral blood lymphocytes were cultured with specific mitogens and M. ovipneumoniae antigens. After 96 hours the cultures were labeled with thymidine and harvested at 18 hour later. Stimulation indexes are average of 6 animals. * Each lymphocyte blast transformation experiment was conducted at 2 week intervals, ^ Phytohemagglutinin, ° Concanavalin A, Heat killed M. ovipneumoniae antigen, ® Sodium dodecyl sulfate treated M. ovipneumoniae antigen, ^ M. ovipneumoniae membrane fractions, + Standard error of mean, * P > 0.01 when nonspecific mitogens were compared to M. ovipneumoniae antigens. 130 Table 2. Mitogen/ antigen induced Stimulation Indexes of lymphocytes of lambs of group A (control).

Exp* PHA'' Con-A° HKA"^ SMA® MMF^

1 166 ± 103* 273 ± 95* 1.2 ± .22 1.1 ± .16 1.8 + .24 2 246 ± 183* 270 ± 206* 2 .9 ± 1.5 3 .8 ± 1.6 2 .4 + .56 3 240 ± 32* 282 ± 105* 1 .8 + .14 6 .7 ± 5.2 3 .7 ± 2.6 4 277 + 65* 278 ± 120* 1.2 ± .39 1.4 ± .29 1.5 ± .09 5 170 ± 15* 247 ± 41* 1.1 ± .11 1 .2 ± .22 1.8 ± .12 6 45 ± 8* 29 ± 6* 1.4 ± .23 1 .5 ± .31 1 .5 ± .22 7 117 ± 74* 95 ± 47* 1 .3 ± .12 1 .9 ± .48 2 .4 ± 1.2 8 25 ± 6* 36 ± 7* 7.0 ± .33 5 .4 ± 1.3 5 .5 ± .33 9 23 ± 11* 34 ± 14* 0 .9 ± .19 1 .8 ± .69 4 .6 ± .58 10 57 ± 41* 60 ± 31* 1 .8 ± .33 1 .7 ± .35 2 .2 ± .28

Peripheral blood lymphocytes were cultured with specific mitogens and M. ovipneumoniae antigens. After 96 hours the cultures were labeled with thymidine and harvested at 18 hour later. Stimulation indexes are average of 2 animals. ® Each lymphocyte blast transformation experiment was conducted at 2 week intervals, ^ Phytohemagglutinin, ° Concanavalin A, ^ Heat killed M. ovipneumoniae antigen), ® Sodium dodecyl sulfate treated M. ovipneumoniae antigen, ^ AT. ovipneumoniae membrane fractions, + Standard error of mean, * P > 0.01 when nonspecific mitogens were compared to M. ovipneumoniae antigens. 131 Table 3. Mitogen/ antigen induced stimulation Indexes of lymphocytes of lambs of group B.

Exp* PHA*' Con-A° HKA^ SMA® MMF^

1 51 ± 5* 66 ± 9* 1.8 ± .14 2.5 ± .24 2 .7 ± .16 2 42 + 4* 54 ± 7* 2.2 ± .24 2.5 ± .19 2 .8 ± .13 3 86 ± 20* 116 ± 21* 2.4 + .15 2.2 ± .14 3 .6 + .63 4 42 ± 7* 43 ± 13* 2.2 ± .29 2.2 ± .33 2 .9 ± .56 5 215 + 55* 277 ± 66* 2.4 ± .16 2.8 ± .63 3 .5 ± .77 6 197 + 9* 128 ± 29* 2.2 ± .9 3.8 ± .8 8 .8 ± 4.2

Peripheral blood lymphocytes were cultured with specific mitogens and AT. ovipneumoniae antigens. After 96 hours the cultures were labeled with thymidine and harvested at 18 hour later. Stimulation indexes are average of 8 animals. * Each lymphocyte blast transformation experiment was conducted at 2 week intervals. Phytohemagglutinin, ° Concanavalin A, Heat killed M. ovipneumoniae antigen, ® Sodium dodecyl sulfate treated M. ovipneumoniae antigen, ^ M. ovipneumoniae membrane fractions, ± Standard error of mean, * P > 0.01 when nonspecific mitogens were compared to M. ovipneumoniae antigens. 132 Table 4. Mitogen/ antigen induced stimulation indexes of lymphocytes of lambs of group C.

Exp° PHA*' Con-A° HKA*^ SMA® MMF^

1 36 ± 9* 43 ± 11* 1.20 ± .15 1.70 ± .30 1.5 ± .15 2 23 ± 7* 28 ± 8* 1.63 ± .63 1.64 ± .19 1.6 + .15 3 227 + 96* 87 ± 23* 1.87 ± .90 2 .40 ± .60 3.0 + .98 4 38 ± 8* 34 ± 9* 1.70 4" .19 1.70 ± .20 1.8 ± .11 5 31 ± 8* 37 ± 10* 1.60 ± .16 1.80 ± .19 1.8 + .42 6 23 ± 14* 49 ± 13* 1.50 + .16 1 .60 + .23 1.8 + .34

Peripheral blood lymphocytes were cultured with specific mitogens and M. ovipneumoniae antigens. After 96 hours the cultures were labeled with thymidine and harvested at 18 hour later. Stimulation indexes are average of 8 animals. * Each lymphocyte blast transformation experiment was conducted at 2 week intervals. ^ Phytohemagglutinin, ° Concanavalin A, Heat killed M. ovipneumoniae antigen, ® Sodium dodecyl sulfate treated M. ovipneumoniae antigen, ^ M. ovipneumoniae membrane components. + Standard error of mean, * P > 0.01 when nonspecific mitogens were compared to M. ovipneumoniae antigens. 133

g

A (Treated) (Control)

Groups of lambs

Pig 1. Stimulation indexes of peripheral blood lymphocytes cultured with mycoplasma antigens for 96 hours and labeled with thymidine. The SI values in group A (experimentally infected and control) are the mean of 10 experiments at 2 week interval comprising of 6 and 2 animals respectively. Values of groups B (naturally infected) and C (M. ovipneumoniae free) are of 6 experiments each comprising 8 animals. Error bars is S.E. of mean. HKA, SMA, and MMF are Mycoplasma antigens. * P > 0.05, when comparing mycoplasma antigens in respective groups. 134 in animals of group C and untreated control of group A. A comparative differences of SI among animals of all three groups could not be determined as blastogenic assays were performed on different days. However, a trend of higher SI was observed in animals from experimentally or naturally infected groups with MMF. These result indicated that M. ovipneumoniae antigens have nonspecific mitogenic effects for sheep lymphocytes. 135

DISCUSSION

Lymphocyte blastogenesis is a common method of evaluating the activity of both T and B lymphocytes. Mycoplasma-induced blastogenic response was originally reported by Ginsburg and Nicolet (1973) for lymphocytes of the rat with M. pulmonis. Since then it has been reported by several investigators that a wide variety of mycoplasmas share the potential of inducing nonspecific mitogenic activation of lymphocytes although some have inhibitory effects on blast transformation (Adegboye, 1978; Stanbridge et al., 1981). Lymphocyte blastogenesis induced by mycoplasma antigens differs relative to the mycoplasma species used and the origin and type of lymphocytes. Further, one mycoplasmal species can activate lymphocytes obtained from different species. Mycoplasma pneumoniae activates lymphocytes from mice, A. laidlawii stimulates only mouse B lymphocytes and human T lymphocytes (Naot, 1982). The results of our study showed that different antigen preparations of M. ovipneumoniae have the ability to induce blast transformation of sheep lymphocytes in vitro. This blast transformation was not as strong as reported previously with other mycoplasmas especially M. pulmonis (Naot et al., 1977). Of the three mycoplasma antigens compared for blast transforming potential, mycoplasma membrane protein fraction 136 (MMF) appeared to be a more potent mitogen for sheep lymphocytes as compared with HKA and SMA antigens. This correlates with previous studies indicating that mitogenicity of different mycoplasmas resides in their membranes. Purified membranes resulted in greater mitogenicity than observed with an equal protein dose of nonviable, lysed mycoplasma cells (Naot, 1982). A slightly lower mitogenicity for sheep lymphocytes was exhibited by the other two antigens (HKA and SMA). The restricted activity of HKA may be because of heating effects on mycoplasma antigens as reported with M. pulmonis (Ginsburg and Nicolet, 1973) . However, other mycoplasma (AT. arthritidis, A. laidlawii, and M. pneumoniae) maintained their mitogenic activity following heat treatment (Kirchner et al., 1977; Cole et al., 1977; Biberfeld and Gronowicz, 1978). We also observed that the mitogen preparations used in our study have similar and nonspecific blastogenic effects on lymphocytes from either disease free, naturally diseased, or experimentally infected lambs. These findings correlate with previous reports, indicating nonspecific mitogenic activation of lymphocytes of different species of animals by various other mycoplasmas (Biberfeld, 1977; Cole et al., 1977; Naot et al., 1977; Stanbridge and Weiss, 1978). Mycoplasma antigen behave as multivalent ligands, cap with receptors on the surface of lymphocytes and induced them to form blast cells. The presence of serum antibodies appeared 137 to have an inhibitory effect on lymphocyte blastogenesis by preventing mycoplasma antigen to cap with receptors on lymphocytes (Razin, 1983). Colostrum-deprived calves with no serum antibodies to M. bovoculi showed a much greater stimulation response than seropositive calves (Salih et al., 1987) . Such an inhibitory effect was also noticed when antibodies to M. pulmoniB were added to M. pulmonis membranes incubated with rat lymphocytes (Naot, 1982). The overall low blastogenic response by sheep lymphocytes to various antigenic preparations of M. ovipneumoniae in our studies may be due to the presence of low titers of serum antibodies in our experimental animals as identified by the ELISA method (unpublished data). This in vitro blast transformation of lymphocytes provides a model for examining the cell mediated response to mycoplasma. Previous reports suggest that there is a correlation of mitogenicity and development of disease. Naot et al. (1981) reported the induction of interstitial pneumonia and tracheitis in pathogen free rats by nonviable M. pulmonis cells or membrane components. In addition, both pathologic and mitogenic effects were significantly reduced by prior treatment of membranes with heat or proteolytic enzyme. The pneumonia, characterized by peribronchial, perivascular, and alveolar wall infiltration by lymphocytes, was indistinguishable from that produced by viable M. pulmonis. 138 Moreover, intranasal administration of concanavalin-A, a T- cell mitogen, also produced interstitial pneumonia. Similarly, the characteristic lesions produced by M. pneumoniae in the form of peribronchiolar and perivascular lymphocytic accumulations was due to constant presence of mycoplasma in ciliated epithelium (Fernald, 1979). Our data did indicate a nonspecific transformation of sheep lymphocytes by M. ovipneumoniae antigens. This may also be correlated with histopathologic lesions observed (Papers 1 and 4) in the form of peribronchial and perivascular lymphocytic cuffing, and lymphoid hyperplasia with the natural and experimental infections with M. ovipneumoniae. These observations suggests that M. ovipneumoniae may behave as a nonspecific mitogen and can stimulate lymphocytes to transform into blast cells. This nonspecific activation of lymphocytes is a major cause of the classical histological lesions resulting from natural infection. 139

REFERENCES

Adegboye, D. S. 1978. A review of mycoplasma-induced immunosuppression. Br. Vet. J. 134:556-560. Aldridge, K. E., Cole, B. C., and Ward, J. R. 1977. Mycoplasma dependent activation of normal lymphocytes: induction of a lymphocyte-mediated cytotoxicity for allogeneic and syngeneic mouse target cells. Infect. Immun. 18:377-385. Barile, M. F. and Razin, S. (eds.). 1979. The Mycoplasmas. Human and Animals Mycoplasmas. Vol. II. Academic Press, New York. p. 509. Biberfeld, G. 1977. activation of human lymphocyte sub- populations by Mycoplasma pneumoniae. Scand. J. Immunol. 6:1145-1150. Biberfeld, G. and Gronowicz, E. 1978. Mycoplasma pneumoniae is a polyclonal B-cell activator. Nature, 261:238-239. Boyum, A. 1968. Separation of lymphocytes from blood and bone marrow. Scand. J. Clin. Lab. Invest. 21 (S-97):77. Cassell, G. H., Lindsey, J. R., Baker, H. J., and Davis, J. K. 1980. Mycoplasmal and Rickettsial Diseases. In: Baker, H. J. et al., (eds.). The Laboratory Rat. Vol. I. Academic Press Inc., New York. pp. 244-259. Cole, B. C., Aldridge, K. E., and Ward, J. R. 1977. Mycoplasma-dependent activation of normal lymphocytes : Mitogenic potential of mycoplasmas for mouse lymphocytes. Infect. Immun. 18:393-399. Cole, B. C., Atkin, C. L. 1991. The Mycoplasma arthritidis T- cell mitogen, MAM: A model superantigen. Immunol. Today, 12:271-276. Cole, B. C., Naot, Y., Stanbridge, E. J., and Wise, K. S. 1985. Interaction of mycoplasmas and their products with lymphoid cells in vitro. In: Razin, S. and Barile, M. F. (eds.). The Mycoplasmas, Vol. IV. Academic Press, New York. pp. 203-257. Cole, B. C., Griffiths, M. M., Ahmed, E., Kamerth, C., and Atkin, C. L. 1992. Immunomodulation and autoimmunity induced by the M. arthritidis super-antigen. Abst. 9th International Congress on the International Organization for Mycoplasmology. Ames, lA. p. 17. 140 Ginsburg, H. and Nicolet, J. 1973. Extensive transformation of lymphocytes by a mycoplasma organism. Nature (London), New Biol. 246:143-146. Howard, C. J. and Taylor, G. 1985. Immune responses to mycoplasma infections of the respiratory tract. Vet. Immunol. Immunopathoi. 10:3-32. Kirchner, H., Brunner, H., and Ruhl, H. 1977. Effect of A. laidlawii on murine and human lymphocyte cultures. Clin. Exp. Immunol. 29:176-180. Kishima, M., Kuniyasu, C., and Nakagawa, M. 1983. Delayed hypersensitivity to non-viable Mycoplasma pulmonis in mice is enhanced by dextran sulfate. Infect. Immun. 39:823-829. Liggit, H. D. 1985. Defense mechanisms in bovine lung. Symposium Bov. Resp. Dis. 1:347-361. Messier, S., Ross, R. P., and Paul, P. S. 1990. Humoral and cellular immune responses of pigs inoculated with Mycoplasma hyopneumoniae. Am. J. Vet. Res. 51:52-58. Moler, G. 1972. Lymphocyte activation by mitogens. Transplant. Rev. 11:3-267. Naot, Y. 1982. In vitro studies on the mitogenic activity of Mycoplasma species toward lymphocytes. Rev. Infect. Dis. 4(S) :205-209. Naot, Y., Davidson, S., and Lindenbaum, E. S. 1981. Mitogenicity and pathogenicity of Mycoplasma pulmonis in rats. I. Atypical interstitial pneumonia induced by mitogenic mycoplasmal membranes. J. Infect. Dis. 143:55- 62 . Naot, Y., Siman-Tov, R., and Ginsburg, H. 1979. Mitogenic activity of Mycoplasma pulmonis. II. studies on the biochemical nature of the mitogenic factor. Eur. J. Immunol. 9:149-154. Naot, Y., Tully, J. G., and Ginsburg., H. 1977. Lymphocyte activation by various Mycoplasma strains and species. Infect. Immun. 18:310-317. Ross, S. E., Simecka, J. W., Gambill, G. P., Davis, J. K., and Cassell, G. H. 1992. Mycoplasma pulmonis possesses a novel chemoattractant for B lymphocytes. Infect. Immun. 60:669-674. 141 Razin, S. 1983. Cell lysis and isolation of membranes. In; Razin, S. and Tully, G. (eds.). Methods in Mycoplasmology. Vol. 1. Academic Press, New York. pp. 225-233 . Sabato, A. R., Cooper, D. M., and Thong, Y. H. 1981. Transitory depression of immune function following Mycoplasma pneumoniae infection in children. Pediatr. Res. 15:813-816. Salih, B. A., Ostle, A. G., and Rosenbusch, R. F. 1987. Vaccination of cattle with Mycoplasma bovoculi antigens Evidence for field immunity. Comp. Immun. Microbiol. Infect. Dis. 10:109-116. Stanbridge, E. J., Bretzius, K. A., and Good, R. F. 1981. Mycoplasma lymphocyte interaction, Ir gene control of mitogenesis and a paradoxical interaction with thy-1 bearing cells. Israel. J. Med. Sci. 17:628-632. Stanbridge, E. J. and Weiss, R. L. 1978. Mycoplasma capping lymphocytes. Nature, 276:583-587. 142 ACKNOWLEDGEMENTS

The authors would like to thank Kevan Flaming for assistance in statistical analysis and Ervin Johnson for his early technical assistance in starting the experiments. 143 PAPER III: HETEROGENEITY OF ISOLATES OF MYCOPLASMA OVIPNEOMONIAE 144 HETEROGENEITY OF ISOLATES OF MYCOPLASMA OVIPNEUMONIAE

Mumtaz A. Khan, D.V.M., M.Sc. (CMS), M.Sc. (TVM) Ricardo F. Rosenbusch, Ph.D.* Chris F. Minion, Ph.D.* Eligius Tigges Merlin L. Kaeberle, D.V.M., Ph.D.

* From the Veterinary Medincal Research Institute, Iowa State University, Ames, lA 145

SUMMARY

Mycoplasma ovipneumoniae is one of the most common microorganisms isolated from the respiratory tract of normal as well as pneumonic lambs. Results of previous investigations with different isolates of M. ovipneumoniae in production of disease or their genetic relatedness are controversial. This study was designed to identify genetic relatedness among 30 isolates of M. ovipneumoniae. Twenty five of them were from lambs suffering from a coughing syndrome and 5 from normal lambs. Restriction endonuclease DNA analysis and a DNA hybridization technique were used to identify heterogeneity among different isolates of M. ovipneumoniae. Restriction endonuclease DNA analysis identified 8 isolates of M. ovipneumoniae as belonging to one strain cluster, with 100% homology by restriction pattern. Six of the 8 isolates were from either nasal mucosa or lungs of 3 different animals and the other 2 isolates were from lungs of different lambs. Outside the group of 8, two isolates obtained from nasal swabs gave similar restriction patterns to the isolates from lungs from the same animals. The other 18 isolates were heterogenic among each other in their restriction patterns. However, all these had many common bands especially at lower molecular weight (MW 6 kb) level. A radiolabeled rRNA probe identified 3 strain clusters of M. 146 ovipneumoniae isolates based on homology of restriction DNA bands. The latter approach has the potential to recognize multiple clusters of M. ovipneumoniae isolates. 147 INTRODUCTION

Mycoplaama ovipneumoniae is the most common mycoplasma isolated from the ovine respiratory tract. It can be isolated from sheep with a coughing syndrome, chronic nonprogressive pneumonia, pneumonia associated with other organisms and from the respiratory tract of normal sheep (see literature review). Challenge experiments conducted with specific pathogen free or conventionally reared lambs using isolates of M. ovipneumoniae have given incongruous results. Experimental transmission of disease with isolates of M. ovipneumoniae resulted in mild lesions as compared to natural disease (Foggie et al., 1976; Alley and Clarke, 1980; Jones et al., 1982b). Early reports suggested a role for M. ovipneumoniae in association with P. haemolytica in the production of respiratory disease (Jones et al., 1978; Alley and Clarke, 1979). Challenge with mixed clones of M. ovipneumoniae followed by P. haemolytica infection produced major lesions of proliferative exudative pneumonia more frequently than did individual isolates of M. ovipneumoniae followed by P. haemolytica (Jones et al., 1978; Gilmour et al., 1979; Jones et al., 1982a). It was apparent that the presence of mixed infection with M. ovipneumoniae and P. haemolytica contributed to the production of severe disease. We have reported the association of M. ovipneumoniae in outbreaks of a coughing syndrome (Paper 1 of this thesis). 148 Recent studies have pointed to the presence of multiple strains of M. ovipneumoniae in sheep (Mew et al., 1985; lonas et al., 1991). Mycoplasma species and strains have been identified and differentiated by use of different biochemical and serological tests, by comparing polypeptide profiles using polyacrylamide gel electrophoresis, and by comparing DNA fragments following digestion with restriction endonucleases (Daniels and Meddins, 1973; Razin et al., 1983b; Andersen et al., 1987). Isolates of M. ovipneumoniae from New Zealand and Scotland have been studied for genetic relatedness among strains by comparing their DNA profiles and great heterogeneity was reported (Mew et al., 1985; Thirkell et al., 1990; lonas et al., 1991b). This heterogeneity among isolates of M. ovipneumoniae was not only noted among isolates from different flocks but also within a flock or even isolates from individual animals (Jonas et al., 1991a,b). These observations led to the hypothesis that there may be a prevalence of multiple strains of this mycoplasma with variability in their pathogenic potential. The experiment was designed to study the homogeneity, if any, among different isolates of M. ovipneumoniae obtained from field outbreaks of a coughing syndrome by using restriction endonuclease DNA analysis and hybridization of DNA with an rRNA probe. We believe that the results of this study will help to differentiate strains of M. ovipneumoniae. 149

MATERIALS AND METHODS

Isolates M. ovipnevmonlae Thirty isolates of M. ovipneumoniae from normal as well as lambs suffering from a coughing syndrome were included in this study. Twenty five isolates were from naturally infected lambs obtained either from nasal swabs, bronchial washes or bronchial swabs. The other 5 isolates were from nasal mucosa of normal animals. In addition, a laboratory adapted M. ovipneumoniae isolate (courtesy of Dr. R. F. Rosenbusch, VMRI) was also included. The isolates were identified by their morphological characteristics and by the indirect immunofluorescent technique of Rosendal and Black (1972) as M. ovipneumoniae and cloned by the method of Tully (1983) as described in Paper 1 of this manuscript. All isolates were stored at -70 °C in small aliquots for further use in restriction endonuclease DNA analysis and hybridization with labeled rRNA studies.

Preparation of DNA Mycoplasma ovipneumoniae DNA was isolated by the procedure initially used to isolate DNA of M. pulmonis as outlined by Mahairas et al. (1989) with some modifications. Briefly, each isolate of M. ovipneumoniae was propagated in 300 ml of MFBM (described in Paper 1) to the mid-exponential 150 Stage of growth. The cells were collected by centrifugation at 10,000 rpm for 30 minutes. Each mycoplasma cell pellet was suspended in 100 ul of TNE (0.01 M Tris, 0.15 M NaCl, 0.001 M EDTA, pH 8.0) and 100 ug of Proteinase K (Gibco BEL, Life Technologies Inc., Gaithersburg, MD). They were transferred to a 2 ml microfuge tube (Sarstedt, Princeton, NJ), incubated to equilibrate to 37 °C, and then 100 ul of 2% low-melting- temperature agarose (SeaPlaque, FMC Corp., Rockland, ME., in TNE, equilibrated to 48 °C) was added. The contents were thoroughly mixed, placed on ice to harden the agarose, and 100 ul of lysis buffer [2% Tween 20 (Sigma) in TE (0.01 M Tris, 0.001 M EDTA, pH 8.0)] was added. Following an overnight proteolytic digestion at 37 °C the plug containing DNA was dialyzed against a 1.5 ml volume of TE (at 4 °C) , with 8 changes of the dialysate at intervals of 1 hour and once in double distilled water, and then maintained in TE buffer at 4 °C.

Restriction Endonuclease Digestion of DNA Restriction endonuclease digestion of DNA was carried out with EcoRl (Promega Corp., Madison, WI) restriction enzyme. The agarose containing DNA samples were first heated to 65 °C for 10 minutes and then cooled to 40 °C. Eight microliters of each DNA sample was added to a mixture containing 2 ul of lOX restriction enzyme digestion buffer (Promega), 2 ul of 1 mg/ml 151 BSA buffer, 5 units of EcoRl, and distilled water to make a total volume of 20 ul. Following an incubation of 4 hours, the reaction was stopped by adding 2 ul of running buffer to each DNA sample before transferring into electrophoresis gel. A 1 kb molecular weight ladder (BRL) was used as size standards.

Gel Electrophoresis and Photography Electrophoresis of DNA was carried out in a 0.6% w/v agarose gel in IX TAE electrophoresis buffer (0.04 M Tris acetate, 0.001 M EDTA) and run at 1.6 V/cm nominal voltage overnight. The gel was stained in 100 ul ethidium bromide in 1 liter distilled water for 1 hour and destained in deionized water. The gel was photographed by placing the gel on an ultraviolet illuminator and exposure of Polaroid film Type 57 with a Wratten 22A filter for 4 seconds.

Transfer of DNA onto Nylon Membrane The transfer of fractionated DNA from the gel to a nylon membrane (Nytran, Schleicher and Schuell, Keene, NH) was accomplished by the downward capillary transfer method of Lichtenstein et al. (1990), modified by Chomczynski (1992). Briefly, each nylon membrane was soaked in deionized distilled water for 10 minutes, and placed on a 3-4 cm stack of S&S GB004 gel blot paper cut 2 cm larger than the gel, with a 2 inch stack of paper towels underneath. The DNA gel was placed 152 on the top of the membrane and then covered with 3 sheets of presoaked (in denaturing buffer, 3 M NaCl, 0.4 M NaOH) S&S GB002 gel blot paper cut to the size of the gel. A wick was formed with two sheets of GB002 gel blot paper presoaked in denaturing buffer (DB) placed on top of the stack and extended off into the DB reservoir. Transfer was allowed to continue for one hour and then DB was replaced with a low concentration transfer solution (3 M NaCl, 8 mM NaOH) and allowed to run overnight at room temperature. The membrane was neutralized prior to immobilization and hybridization by soaking for 10 minutes in 0.2 M sodium phosphate buffer (pH 6.7). Immobilization of DNA on the nylon membrane was accomplished by baking for 1 hour at 80 °C.

DNA Hybridization and Autoradiography Fragmented DNA on the nylon membrane was probed with the 16S rRNA gene (insert size 1.5 kb), obtained from Acholeplasma ISM 1499 (a laboratory isolate, courtesy of Dr. Chris F. Minion, VMRI, Ames, Iowa), labelled with by the direction of the manufacturer (Amersham International Pic., Amersham, UK). The hybridization of DNA on the nylon membrane was achieved by the procedure of Maniatis et al. (1989) modified by McCully and Brock, (1992) . Briefly, the hybridization membrane was placed in a special hybridization chamber cylinder and wetted with prehybridization buffer (Ausubel et 153 al., 1992) for 2 hours at 40 °C in a rotating hybridization chamber. After incubation the prehybridization buffer was removed and a hybridization solution (Ausubel et al., 1992) with radiolabeled rRNA probe was added and incubated overnight at 40 °C in a hybridization chamber with rotation. Following completion of hybridization, the membrane was washed first with 4X SSPE and 1% (w/v) SDS, next with 2X SSPE and 1% SDS, and then finally with IX SSPE with 1% SDS for 30 minutes each at 40 °C in the hybridization chamber with rotation. The membrane was blotted dry, attached to blot paper, sealed with plastic wrap, and placed on radiograph film for 48 hours at -70 °C. The radiograph was developed by routine methods. 154 RESULTS

Restriction Endonuclease DNA Analysis Thirty field isolates of M. ovipneumoniae from normal lambs as well as animals with the coughing syndrome were subjected to restriction endonuclease DNA analysis. The isolates and their source are presented in Table 1. Visual restriction bands from EcoRl digestion ranged from 1 to 16 kb and are illustrated in Figs 1 and 2. Eight isolates (#3, 6, 8, 12, 13, 24, 25, and 29) from 5 different lambs gave identical restriction patterns. Six of them (Nos. 6, 8, 12, 13, 24, and 25) were from 3 different animals isolated from nasal swabs and bronchial washes and the other (Nos. 3 and 29) were from different lambs. Outside the group of 8 isolates, four isolates (Nos. 10, 17, 22, and 23) obtained from nasal swabs and lungs of 2 different lambs had restriction patterns similar to corresponding isolates from the same animal. The remaining isolates including M. ovipneumoniae (laboratory strain) gave restriction patterns different from each other. Twenty six major DNA restriction bands from a strain cluster of 8 isolates were selected to compare homology with the remaining isolates. A homology in restriction bands ranging from 62 percent to 92 percent was calculated (Tables 2 and 3). The heterogeneity of restriction patterns was much greater in the higher molecular weight ranges (> 12 kb) 155 Table 1. Isolates of M. ovipneumoniae subjected to restriction endonuclease analysis and hybridization.

No Isolate Isolated from Clinical disease

1 2033 Nasal swab 2 S ~ 6 Bronchial wash + 3 126 Bronchial swab + 4 16 Nasal swab + 5 P-21 Bronchial wash + 6 100 Bronchial wash + 7 27 Nasal swab + 8 930 Bronchial wash + 9 P-6/144 Lung swab + 10 349 Bronchial wash + 11 01 Nasal swab _a 12 25 Bronchial wash + 13 100-Nb Nasal swab +

14 06 Nasal swab - 15 288 Bronchial wash + 16 lDE-1 Lung swab + 17 53 Lung swab + 18 927 Nasal swab + 19 B Nasal swab + 20 R-16 Nasal swab +

21 02 Nasal swab - 22 53-N Nasal swab + 23 349-N Nasal swab + 24 930-N Nasal swab + 25 25-N Nasal swab + 26 H-268 Nasal swab + 27 S-19 Bronchial wash + 28 M. ovipneumoniae Laborati 29 38 Bronchial swab +

30 47 Bronchial swab -

31 4 Eness Bronchial swab -

® Presence or absence of clinical disease, M. ovipneumoniae isolated from nasal mucosa. Fig 1. Restriction endonuclease DNA analysis of isolates (1- 16) of M. ovipneumoniae obtained from lambs with a coughing syndrome. M M M M N) Wrfi»{j'io^>JOOVOO|-»tO m o u) m Ln>jvûot0crk0^00i4i^

- &# III W ill III

?Trm

%: ESs:s-:anEarïïE;;]i

.- f-'^y-«^Ay.Tdti' .«.spv .--iCiïtifS

>• -Y ^4ÉdaK9^-t: c. ^ $## Fig 2. Restriction endonuclease DNA analysis of isolates (17- 31) of AT. ovipneumoniae obtained from lambs with a coughing syndrome. H^OOVDVOCNOCnOLT) VD cn CNrHrHrHrHrHrHOOO O KD CNHO(TiCXDt>VDLn'^rO CN H / 160 Table 2. EcoRl restriction fragments of isolates of M. ovipneumoniae using gel electrophoresis.

Isolates of M. ovipneumoniae MM* lb 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

12.7 +° + + + + + + + + + + + + + + +

11 + + - - + + + + + + + + + + +

8.8 - - + + + + + + + - + + + - - - 8.4 + + + + + + + + + + + + + + +

8 .15 + + + - - + + + + + + + + + + +

7.8 + - + + + + + + + + + + + + 4- +

6.8 + + + + + + - + - - + + + - - -

6.4 + + + + + + + + + + - + + + - -

6.0 - + + + + + + + + + + + + + + +

5.55 - - + + + + - + + - + + + + - -

5.2 - + + + + + - + + + + + + + + +

5.0 + + + + + + - + + + - + + - - +

4.7 - + + + + + + + - + + + + + + -

4.5 + - + + + + + + + + - + + + - -

4.0 - + + + + + - + - + - + + + + +

3.6 - + + - - + + + + + - + + + + +

3 .35 + - + - + + + + + + + + + + - +

3.1 - + + + + + + + + + + + + - + +

2.95 - + + + + + + + + + + + + + + +

2.55 + + + + + + + + + - + + + - + + 2 .35 + + + + + + + + + + + + + + + +

1.2 + + + + + + + + + - + + + + + +

Restriction endonuclease DNA analysis gave multiple fragments of each isolate of M. ovipneumoniae. ® Molecular weights of some prominent bands of 8 isolates of one strain used for comparison with other isolates. ^ Isolates of M. ovipneumoniae. ° Presence or absence of corresponding molecular weight bands. 161 Table 3. EcoRl restriction fragments of isolates of M. ovipneumoniae using gel electrophoresis.

Isolates of M. ovipneumoniae MW* 17" 18 19 20 21 22 23 24 25 26 27 28 29 30 31

_C 12.7 + + - + + + + + + - + + +

11 - + + - + - + + + + + + + + +

8.8 + - + + + + - + + + + + + + +

8.4 + + - + - + + + + - - + + + +

8.15 + + + - - + + + + + + + + + +

7.8 + - + + - + + + + + - + + + +

6 . 8 + + + + + + - + + + + + + + - 6.4 + + + + + + + + + + + + + + +

6.0 + + + + + + + + + - + + + + +

5.55 - + + + + - - + + + + + + + + 5.2 + + + + + + + + + + + + + + +

5.0 - - + + + - + + + + + + + - + 4.7 + + + + + + + + + + + + + + +

4.5 + + - - + + + + + + + + + + +

4.0 - + - + + - + + + + - + + - +

3.6 + + - + + + + + + + + - + + + 3 .35 + + + + + + + + + + + + + + + 3.1 + + + + + + + + + + + + + + + 2.95 + + + + + + + + + + + + + + +

2.55 + + + + + + - + + + + + + + + 2.35 + + + + + + + + + + + + + + +

1.2 + + + + + + - + + + + + + + +

Restriction endonuclease DNA analysis gave multiple fragments of each isolates of M. ovipneumoniae. ® Molecular weights of some prominent bands of a group of 8 isolates of one strain used for comparison with other isolates. ^ Isolates of M. ovipneumoniae. ° Presence or absence of corresponding molecular weight band. 162 compared to lower molecular weight fragments (< 6 kb) where there was maximum homology. No two isolates among the remaining 18 showed 100% homology in restriction patterns.

Hybridization with rRNA Probe Hybridization of DNA with ^^P-labeled 16S rRNA provided a procedure for grouping of M. ovipnewnoniae isolates. Restriction endonuclease digestion of DNA by EcoRl provided two or more fragments complementary to 16S rRNA gene (Figs 3 and 4). Based on sizes of the lower molecular weight hybridizing bands 3 groups of M. ovipneumoniae were proposed (Table 4). Fig 3. Autoradiogram of restriction enzyme digests of DNA of M. ovipneumoniae isolates (1-16) showing reaction with a radiolabeled ribosomal RNA probe. 2 3 4 5 6 7 8 9 10 11 121314 15 16 IDE3 Fig 4. Autoradiogram of restriction enzyme digests of DNA of M. ovipneumoniae isolates (17-31) showing reaction with a radiolabeled ribosomal RNA probe. # 171819 2021 22 232425 2627 28 29 âO 31 167 Table 4. Groups of M. ovipneumoniae based on the radiolabeled ribosomal RNA probe for restriction fragments of DNA.

Groups* I so'' 1.5° 2. 5 2.8 4 4.5 5 8 12.7 13.8 16

A 1 + + + 3 + + + 7 + + 13 + + 14 + + + + 18 + + + 26 + + + 28 + + 29 + + + 30 + + B 2 + + + 4 + + + + + 5 + + + 6 + + 8 + + 9 + + + + + 11 + + 12 + + 15 + + 16 + + + + + 17 + + + 19 + + + 20 + + + 21 + + + + 22 + + + 24 + + 25 + + 27 + + 31 + + IDE-3d + + + + + +

C 10 + + + + 23 + + + + ® Proposed groups of M. ovipneumoniae isolates based on the hybridizing bands, ^ Isolates used for hybridization. ° Molecular weights (Df hybridizing bands, Isolate not included in the restriction endonuclease analysis, 168

DISCUSSION

Mycoplasma ovipneumoniae is an important pathogen of ovine pneumonia. It has been associated with a variety of pathological types of pneumonia, but has also been isolated from healthy animals. Previous challenge experimentation with this organism has yielded inconsistent results. This may be due to the presence of multiple strains of this mycoplasma in the sheep population as has been reported by research workers from New Zealand and Scotland (Mew et al., 1985; Thirkell et al., 1990; lonas et al., 1991a,b). The observation of a coughing syndrome associated with M. ovipneumoniae in feeder lambs in Iowa and surrounding states was the foundation for analysis of the causative strains of mycoplasma. Thirty isolates of M. ovipneumoniae, 25 from lambs with a coughing syndrome and 5 from normal animals were subjected to restriction endonuclease DNA analysis or hybridization with a radiolabeled rRNA probe for identification of strains or their variability. Digestion of DNA with EcoRl followed by electrophoretic separation of fragments gave multiple band patterns different from each other (Tables 2 and 3). However, 8 isolates showed 100% homology in band pattern of their DNA, and appeared to belong to one strain cluster. The other isolates though, were different form one another but they had many common bands when 169 compared to the strain cluster of 8 isolates. This homology was more evident particularly in lower molecular weight bands. The heterogeneity was more apparent in bands of molecular weight 12 and above. Similar findings have been reported with isolates of M. ovipneumoniae either from normal sheep (Mew et al., 1985) or from lungs affected with chronic nonprogressive pneumonia (lonas et al., 1991b). This heterogeneity among strains of the same species may be associated with their recovery from diverse habitats as has been reported by Stephens et al. (1983). The 5 isolates from nasal passages gave similar band patterns to the corresponding isolates from lungs of the same animal. This indicated the presence of the same strain cluster of M. ovipneumoniae in both the nostrils and lungs of the same animal. These findings however, differ from previous reports indicating the presence of variable strains in the respiratory tract of the same individual animal (lonas et al., 1985). The presence of multiple strains has been reported with many animal mycoplasmas and bacteria (Marshall et al., 1981; Razin et al., 1983a,b; Khan et al., 1987; Zhao and Yamamoto, 1989) . The isolation of a specific strain cluster of AT. ovipneumoniae from different farms is evidence for the presence of specific strain clusters of this mycoplasma in the sheep population, and heterogeneity of DNA restriction patterns among different isolates may not be only due to 170 spontaneous mutation as suggested earlier (Yogev et al., 1991). There is need to analyze a large number of isolates of M. ovipneumoniae from different areas in order to group them into strain clusters. Restriction endonuclease DNA analysis is a useful procedure to identify or differentiate one strain from another. Furthermore, it would be useful to include more restriction endonucleases run in parallel in order to identify different strains. Also, standardization of the procedure by digitizing densitometry scans of gels would make it possible to compare isolates from different sources. Studies with DNA probes for the detection and identification of different mycoplasmas have been reported (Razin et al., 1983a; Hyman et al., 1987; Geary et al., 1988; McCully and Brock, 1992). The use of the radiolabeled ribosomal RNA probe for restriction-endonuclease-digested DNA in this study has helped to sort isolates of M. ovipneumoniae into fewer groups compared to REA. The numbers and position of DNA band complementary to 16S rRNA differed among the isolates tested, but it was possible to separate them into 3 strain clusters (Table 4). Mollicutes examined to date have 1 to 2 copies of 16S rRNA gene (Amikam et al., 1984; Gobel et al., 1984; Razin, 1985; Sawada et al., 1981; Eligius, 1993). In addition, a highly conserved gene like the gene for 16S rRNA should show few, if any, changes in sequence within a species. The production of multiple hybridizing bands in some isolates 171 in groups A and B especially at higher molecular weight levels appeared to be due to partial digestion of DNA. This was also evident in ethidium bromide stained gels. The grouping based on the rRNA probe however, did not match those found by restriction endonuclease analysis. For example, the isolates Nos. 6 and 13 obtained from one animal belonged to the same restriction endonuclease cluster but fell into different 16S rRNA groups. There is further need to include more isolates of this mycoplasma, have more complete digestion by EcoRl, and use other restriction endonucleases in order to establish firm 16S rRNA groups. Study of other mycoplasmas of ovine origin by this method will help to identify structural similarity in their genetic makeup. 172

REFERENCES

Alley, M. R. and Clarke, J. K. 1979. The experimental transmission of ovine chronic non-progressive pneumonia, N. Z. Vet. J. 27:217-220. Alley, M. R. and Clarke, J. K. 1980. The effect of chemotherapeutic agents on the transmission of ovine chronic non-progressive pneumonia. N. Z. Vet. J. 28:77- 80. Amikam, D., Glaser, G., and Razin, S. 1984. Mycoplasmas (Mollicutes) have a low number of rRNA genes. J. Bact. 158:376-378. Andersen, H., Birkelund, D., Christiansen, G., and Freundt, E. A. 1987. Electrophoresis analysis of proteins from MycoplaBma homlnis strains detected by SDS-PAGE: two- dimensional gel electrophoresis and immunoblotting. J. Gen. Microbiol. 133:181-190. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D. (eds). 1992. Short Protocols in Molecular Biology. 2nd Ed. John Wiley and Sons, New York. Chomczynski, P. 1992. One-hour downward alkaline capillary transfer for blotting of DNA and RNA. Anal. Biochem. 14: 134-139. Daniels, M. J. and Meddins, B. M. 1973. Polyacrylamide gel electrophoresis of mycoplasma proteins in sodium dodecyl sulphate. J. Gen. Microbiol. 76:239-242. Eligius, T. 1993. Personal communication. Foggie, A., Jones, G. E., and Buxton, D. 1976. The experimental infection of specific pathogen free lambs with Mycoplasma ovipneumoniae. Res. Vet. Sci. 21:28-35. Geary, S. J., Intres, R., and Gabridge, M. G. 1988. Species- specific biotinylated DNA probe for the detection of Mycoplasma gallisepticum. Mol. Cell Probes, 2:237-244. Gilmour, J. S., Jones, G. E., and Rae, A. G. 1979. Experimental studies of chronic pneumonia of sheep. Comp. Immun. Microbiol. Infect. Dis. 1:285-293. 173 Gobel, U. , Butler, G. H., and Stanbridge, E. J. 1984. Comparative analysis of mycoplasma ribosomal RNA opérons. Isr. J. Med. Sci. 20:762-764. Hyman, H. C., Yogev, D., and Razin, S. 1987. DNA probes for detection and identification of Mycoplaama pneumoniae and Mycoplaama genitalium. J. Clin. Microbiol. 25:726-728. lonas, G., Clarke, J. K., and Marshall, R. B. 1991a. The isolation of multiple strains of Mycoplaama ovipneumoniae from individual pneumonic sheep lungs. Vet. Microbiol. 29:349-360. lonas, G., Mew, A. J., Alley, M. R., Clarke, J. K., Robinson, A. J., and Marshall, R. B. 1985. Colonization of the respiratory tract of lambs by strains of Mycoplaama ovipneumoniae. Vet. Microbiol. 10:533-539. lonas, G., Norman, N. G., Clarke, J. K., and Marshall, R. B. 1991 b. A study of the heterogeneity of isolates of Mycoplaama ovipneumoniae from sheep in New Zealand. Vet. Microbiol. 29:339-347. Jones, G. E., Gilmour, J. S., and Rae, A. 1978. Endobronchial inoculation of sheep with pneumonic lung-tissue suspensions and with bacteria and mycoplasmas isolated from them. J. Comp. Pathol. 88:85-96. Jones, G. E., Gilmour, J. S., and Rae, A. G. 1982a. The effects of different strains of Mycoplaama ovipneumoniae on specific pathogen-free and conventionally-reared lambs. J. Comp. Pathol. 92:267-272. Jones, G. E., Gilmour, J. S., and Rae, A. G. 1982b. The effect of Mycoplaama ovipneumoniae and Paateurella haemolytica on specific pathogen free lambs. J. Comp. Pathol. 92:261-266. Khan, M. I., Lam, K. M., and Yamamoto, R. 1987. Mycoplaama galliaepticum strain variations detected by sodium dodecyl-sulphate polyacrylamide gel electrophoresis. Avian Dis. 31:315-320. Lichtenstein, A. V., Moiseev, V. L., and Zaboikin, M. M. 1990. A procedure for DNA and RNA transfer to membrane filters avoiding weight-induced gel flattening. Anal. Biochem. 15:187-191. 174 Mahairas, G. G. and Minion, F. C. 1989. Random insertion of the gentainicin resistance transposon Tn4001 in Mycoplasma pulmonis. Plasmid, 21:43-47. Maniatis, T., Fritsch, E. F., and Sambrook, J. 1989. Molecular cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold spring Harbor, New York, U.S.A. Marshall, R. B., Wilton, B. E., and Robinson, A. J. 1981. Identification of Leptospira serovars by restriction- endonuclease analysis. Vet. Microbiol. 14:163-166. McCully, M. A. and Brock, K. V. 1992. Development of a DNA hybridization probe for detection of Mycoplasma bovis. J. Vet. Diagn. Invest. 4:464-467. Mew, A. J., lonas, G., Clarke, J. K., Robinson, A. J., and Marshall, R. B. 1985. Comparison of Mycoplasma ovipneumoniae isolates using bacterial restriction endonuclease DNA analysis and SDS-PAGE. Vet. Microbiol. 10:541-548. Razin, S. 1985. Molecular Biology and Genetics of Mycoplasmas (Mollicutes). Microbiol. Rev. 49:419-455. Razin, S., Barile, M. F., Harasawa, R., Amikam, D., and Glaser, G. 1983a. Characterization of the Mycoplasma genome. The Yale J. Biol. Med. 56:357-366. Razin, S., Harasawa, R., and Barile, M. F. 1983b. Cleavage patterns of the mycoplasma chromosome, obtained by using restriction endonucleases, as indicators of genetic relatedness among strains. Int. J. Syst. Bacterid. 33:201-206. Rosendal, S. and Black, F. T. 1972. Direct and indirect immunofluorescence of unfixed and fixed Mycoplasma colonies. Acta. Pathol. Microbiol. Scand. Sect. B. 80:615-622. Sawada, M., Osawa, S., Kobayashi, H., Hori, H., and Muto, A. 1981. The number of ribosomal RNA genes in Mycoplasma capricolum. Mol. Gen. Genet. 196:311-316. Stephens, E. B., Aulakh, G. S., Rose, D. L., and et al., 1983. Intra-species genetic relatedness among strains of Acholeplasma laidlawii and of Acholeplasma axanthum by nucleic acid hybridization. J. Gen. Microbiol. 129:1929- 1934. 175 Thirkell, D., Spooner, R. K., Jones, G. E., and Russell, W. C. 1990. Polypeptide and antigenic variability among strains of Mycoplasma ovipneumoniae demonstrated by SDS-PAGE and immunoblotting. Vet. Microbiol. 21:241-254. Tully, J. G. 1983. Cloning and filtration techniques for mycoplasmas. In: Razin, S. and Tully, J. G. (eds.). Methods in Mycoplasmology. Vol. I. Academic Press, New York. pp. 173-177. Yogev, D., Rosengarten, R., Watson-McKown, R., and Wise, K. S. 1991. Molecular basis of Mycoplaama antigenic variation: a novel set of divergent genes undergo spontaneous mutation of periodic coding regions and 5' regulatory sequences. EMBO. J. 10:4069-4079. Zhao, S. and Yamamoto, R. 1989. Heterogeneity of Mycoplasma iowae determined by restriction enzyme analysis. J. Vet. Diagn. Invest. 1:165-169. 176

ACKNOWLEDGEMENTS

I would like to thank Dr. Chris Minion for permission to use laboratory facilities to conduct restriction endonuclease DNA analysis. 177

PAPER IV. THE EXPERIMENTAL INFECTION OF LAMBS WITH MYCOPLASMA OVIPNEUMONIAE AND A GRAM-NEGATIVE HEMOLYTIC COCCOBACILLUS 178

THE EXPERIMENTAL INFECTION OF LAMBS WITH MYCOPLASMA OVIPNEUMONIAE AND A GRAM-NEGATIVE HEMOLYTIC COCCOBACILLUS

Mumtaz A. Khan D.V.M., M.Sc. (CMS), M.Sc. (TVM) John J. Andrews, D.V.M., Ph.D.* Mammadou Niang, M.Sc. Merlin L. Kaeberle, D.V.M., Ph.D.

* From Veterinary Diagnostic Laboratory, Iowa State University, Ames, lA 50011 179 SUMMARY

Pneumonia is an important disease of animals causing economic losses to the sheep industry. The cause of this problem is multifactorial and comprises both infectious and noninfectious agents. Mycoplasma ovipneumoniae is one the most common organisms isolated from pneumonic lambs but the experimental production of disease with this organism is unsettled. Six mycoplasma free lambs were inoculated intratracheally with M, ovipneumoniae, two of them were super- infected with a Gram-negative hemolytic coccobacillus. Both isolates were recovered from field outbreaks of a coughing syndrome in lambs. The clinical disease in all inoculated lambs was mild compared to natural infection. The macroscopic and microscopic lung lesions were similar to but moderately less severe than those observed with the AT. ovipneumoniae associated coughing syndrome previously described in Paper 1. The histological lesions associated with the Gram-negative coccobacillus were in the form of exudative and proliferative interstitial pneumonia. Mycoplasma ovipneumoniae was reisolated from 4 of 6 animals antemortem and from all animals at postmortem. The Gram-negative coccobacillus was reisolated from both animals at antemortem and only one animal at postmortem. All M. ovipneumoniae inoculated animals were positive for indirect 180 immunohistochemical staining in formalin-fixed lung tissues. Bacterial isolation, pathological changes or immunohistochemical staining were negative in control animals. 181

INTRODUCTION

Pneumonia is a complex respiratory disease of all animals in all husbandry conditions. It is caused by numerous combinations of infectious agents, compromised host defenses, and environmental factors. Pneumonia causes greater economic losses than any other disease in feedlots, in both cattle and lambs. Numerous infectious agents, usually in combination, discussed in Paper 1, have been isolated from cases of pneumonia and are important components of its multifactorial etiology. Among the number of bacteria, Pasteurella haemolytica and Mycoplasmas are the most common microorganisms isolated from ovine pneumonia. Species of Mycoplasma isolated from ovine pneumonia are generally considered to be mild respiratory pathogens, mainly causing subclinical infections unless coupled with environmental stresses and/or infections by other pathogens (Stalheim, 1983). The damaging effects of Mycoplaamas on the respiratory mucosa and ciliary activity suggest that they may play an important contributory role in the pathogenesis of ovine pneumonia (Wikse, 1990) . Mycoplasma ovipneumoniae is the most important mycoplasma isolated from pneumonic sheep. It was originally isolated in Australia by St. George et al. (1971), and later characterized by Carmichael et al. (1972). Since then it has been isolated 182 from pneumonic as well as normal sheep and goats on all continents of the world (Alley et al., 1975; Stipkovits et al., 1975; Jones et al., 1976, Jones et al., 1978; Jones et al., 1979; Livingston and Gauer, 1979; Bakke, 1982; Davies, 1985). Under natural conditions M. ovipneumoniae associated pneumonia is usually found in combination with viruses or other bacteria. Reports on the existence and association of this mycoplasma with ovine pneumonia in the United States are extremely limited, but there is evidence for its prevalence and association with lamb pneumonia (Kaeberle and Eness, 1988; Eness et al., 1990). Recently we have also consistently isolated M. ovipneumoniae from lambs with a coughing syndrome (Paper 1). Attempts to experimentally induce pneumonia with M. ovipneumoniae in lambs and ewes has yielded inconsistent results (Sullivan et al., 1973; Foggie et al., 1976; Gilmour et al., 1979; Jones et al., 1982a,b). This may be due to variations in the pathogenicity of different strains, nature of experimental animals involved, and the procedure utilized for evaluation of pathogenicity. Analysis of the previous reports indicates that the clinical disease is more readily induced in naturally-reared lambs than in gnotobiotes. In addition, the disease is mild in lambs if infected with M. ovipneumoniae alone as compared to natural outbreaks and/or mixed bacterial infections (Jones et al., 1982). 183 Under laboratory conditions the organisms lose their virulence during multiple passages and adaptation to artificial media (Buddie et al., 1984). In the previous studies it is possible that either strains of M. ovipneumoniae of limited virulence or attenuated organisms were used for experimental production of pneumonia. The purpose of this study was to investigate the pathogenic potential of an isolate of M. ovipneumoniae alone and following super­ infection with field isolates of a Gram-negative hemolytic coccobacillus. The isolate of mycoplasma was identified as M. ovipneumoniae as described in Paper 1 of this manuscript, but the Gram-negative hemolytic coccobacillus could not be classified with available methods. 184

MATERIALS AMD METHODS

Challenge Microorganisms Two types of organisms were used in this challenge experiment : (1) An isolate of M. ovipneuwoniae designated as P-21, was isolated from a naturally infected lamb having typical signs of a coughing syndrome. This isolate of mycoplasma was identified by its growth characteristics as centerless colonies on solid medium and the indirect immunofluorescent technique of Rosendal and Belack, (1972), as described in Paper 1. (2) A pool of six isolates of Gram-negative hemolytic coccobacillus organisms isolated (from lambs # 221, 25, 100, 930, 144, and IDE-3) from a natural outbreak of M. ovipneumoniae associated coughing syndrome was used for super­ infection following M. ovipneumoniae inoculation. These bacteria are abundant in the nasal passages of most lambs with the coughing syndrome and can be routinely isolated from nasal swabs. The pleomorphism of these isolates has hindered identification but many of the isolates possess characteristics described for Neisseria ovis. 185

Cultivation of Microorganisms To avoid possible attenuation by sub-cultivation the M. ovipneumoniae isolate was grown on 3000 ml of modified Friis broth (MPBM described earlier) soon after identification and cloning by the method of Tully (1983). The broth culture was centrifuged to harvest the organisms, and the pelleted organisms washed with broth media without serum and antibiotics. The pellet was diluted to approximately 1 x 10* colony-forming units/ ml with MFBM without serum and antibiotics. The isolates of Gram-negative hemolytic coccobacillus were first grown on 5% blood agar and then on brain heart infusion (BHI, Bacto Brain Heart Infusion, Difco Laboratories, Detroit, MI). After 24 hours of growth the pellet was harvested by centrifugation at 2,000 rpm for 20 minutes followed by twice washing with PBS. A pool of six isolates having a total 5 x 10" colony forming units/ml was made and stored at -70 °C in 1.5 ml aliquots to use for inoculation experiments.

Experimental Animals Eight lambs at the age of 12 weeks were used for experimental production of pneumonia. They were divided randomly into 4 groups comprised of 2 lambs each. All the lambs were Mycoplasma-free as determined by cultural examination (nasal swabs conducted twice prior to challenge). 186 Each group of lambs was maintained in different pens in a building which provided isolation from other animals. The lambs were grouped and identified by tag numbers as group A (tag # 57 and 58), group B (tag # 59 and 60), group C (tag # 61 and 62) and group D (tag # 63 and 64).

Animal Inoculation Animals of groups B, C, and D were inoculated with M. ovipneumoniae and group A lambs were left as uninoculated controls. The area over the trachea in the upper middle third of the neck was clipped and disinfected for inoculation of the challenge dose. A 1.5 inch long 16 gauge needle was inserted into the lumen of the trachea and a 5 ml dose of M. ovipneumoniae organisms was sprayed in. Animals in the control group were injected with the same amount of MFBM without serum and antibiotics at the same sites. Four weeks later, animals of group B were challenged with the Gram-negative hemolytic coccobacillus at an inoculum dose of 1.5 ml by the intratracheal and 1.5 ml by the intranasal route.

Sampling and Clinical Examination All animals were examined daily for the first 2 weeks and then on alternate days for a period of 10 weeks for any clinical evidence of pneumonia. Rectal temperatures and respiration rates were recorded during the same intervals. 187 Nasal swabs were taken at one week intervals for 6 weeks for reisolation of M. ovipneumoniae or other bacteria. The animals of group B were examined for any evidence of postchallenge infection and re-isolation of the Gram-negative coccobacillus until the 10th week of experiment. For reisolation and identification of the Mycoplasma, samples were cultured as described in Paper 1.

Postmortem Findings All lambs in the experiment were euthanized with Beuthanasia-D Special (Pentobarbital 350 mg with Phenytoin 50 mg/ml, Schering-Plough Animal Health Corp., Kenilworth, NJ) at the dose of 10 ml/ animal by intravenous routes after a total period of 6 months post M. ovipneumoniae inoculation. The thorax was opened with aseptic precautions and lung and trachea removed to a sterile tray. Each lung was thoroughly examined for evidences of macroscopic lesions. A block of tissue 2x1 cm was excised from each lobe of each lung together with a section of trachea from all animals for histopathological and immunohistochemical studies. All lung samples were fixed in neutral buffered formalin and processed for each test as described in Paper 1 of this thesis. Tracheal and bronchial swabs were taken for reisolation and identification of microorganisms as described previously. 188

Histopathology Histopathological examination was conducted on all lung samples for evidences of histopathological changes as described before.

Immunohistochemistry The indirect immunohistochemical staining technique of Nakane and Pierce (1967), modified by Doster et al. (1988) as described in Paper 1, was used for the demonstration of M. ovipneumoniae antigens in formalin-fixed tissue sections. 189 RESULTS

Clinical Examination A mild fluctuating daily rectal temperature was observed in all lambs in treated and control groups postinoculation of M. ovipneumoniae, but it remained near the normal limits (39- 40 °C) for this species. Respiration and pulse rates were variable at daily examinations but not significantly different among animals in the various groups. Lung sounds at auscultation were normal in all animals except in lambs # 61 and 62 which were slightly harsh during days 3-5 post M. ovipneumoniae inoculation. No evidence of cough was observed during the course of the trial. A scanty serous to mucopurulent nasal discharge was observed in animals #59, 61 and 62 on days 4-8 postinoculation. Following this all animals remained normal for the remainder of the experimentation period. No observable changes were noted on clinical examination of animals in group B after challenge with the Gram-negative coccobacillus organisms.

Microbiology Results representing isolation of microorganisms during the course of trial period is presented in Table 1. Mycoplaama ovipneumoniae was reisolated discontinuously from challenged animals # 59, 60, 61, and 63 during the course of the 190 Table 1. Clinical and microbiological findings in lambs inoculated with M. ovipneumoniae alone or combined with a Gram-negative hemolytic coccobacillus.

Inoculation Animal Nasal Bacterial isolation Nos. discharge ante and postmortem

_b Control 57 - -

Control 58 - - -

M. ovi® 59 + +

M. ovi 60 - + +

M. ovi 61 + + +

M. ovi 62 + - +

M. ovi 63 - + +

M. ovi 64 - - +

Gram neg° 59 - + +

Gram neg 60 - + -

Isolates of Mycoplasma ovipneumoniae and Gram negative coccobacillus were challenges to a group of lambs. Post challenged reisolation of the organisms and clinical evidence of disease are presented. ® Mycoplasma ovipneumoniae, Presence or absence, ° Gram negative-hemolytic coccobacillus. 191 experiment and later from all inoculated animals from bronchial swabs at postmortem. The Gram-negative coccobacillus was recovered from both lambs at antemortem and from # 59 only during postmortem examinations.

Gross lesions Results representing the development of gross lesions are presented in Table 2. The lungs of infected lambs # 62 and 64 of the control group appeared normal at macroscopic examination. Focal grey areas of consolidation generally confined to the cranial and to some extent middle lobes was observed in lungs of lambs # 61 and 63. Lambs # 59 and 60 developed extensive congestion and consolidation in the anterior lobes and to a lesser extent in the middle lobes of their lungs. Fibrous adhesions of the right cranial lobe to pleural cavity was also observed in the lung of one lamb (# 59). The extent and distribution of lesions however, varied in all infected animals.

Histopathology Histopathological findings and distribution of lesions in lungs of the experimentally infected lambs are presented in Tables 2 and 3. Characteristic mild to moderate histological lesions associated with Mycoplasma infection were observed in lungs of infected lambs similar to Figs 10 and 11 in Paper 1. 192 Table 2. Gross and histopathologic lesions in lungs of experimental lambs.

Lambs Gross findings Histological findings Cg® Cd" Ad° Pled Pvl® Ln^ Mo^ Ns^

57 - - - -

58 ------—

59 + + + + + + + +

60 + + - + + + + +

61 - + - + + + +

62 - - - + + + +

63 - + - + + + +

64 - - - + + + +

Isolates of Mycoplasma ovipneumoniae and a Gram-negative coccobacillus were used to challenge groups of lambs. The table presents pathological findings. ° Congestion, Consolidation, ° Adhesions, ^ Peribronchiolar lymphocytic cuffing, ® Perivascular lymphocytic cuffing, ^ Lymphoid nodule, 3 Macrophage aggregation, ^ Neutrophilic response, ^ presence or absence. 193 Table 3. Histological lesions and IH staining in lungs of lambs infected with M. ovipneumoniae and a Gram negative bacterium.

No® RA'' RM° RC^ LA° LM^ LC^

57 - - - - -

58 + + - + + -

59 ++ H S ++ H S + S ++ H S ++ S + S

60 + + S + H S + H S ++ H S + H S + S

61 ++ H ++ H ++ ++ H ++ +

62 ++ H ++ H + ++ H + +

63 ++ H ++ H + ++ H + H +

64 ++ H + + ++ + _

An isolate of M. ovipneumoniae and a Gram-negative coccobacillus were used to challenge groups of lambs. The table presents the distribution of histological lesions, detection of M. ovipneumoniae by IH technique and type of lesions. ® Lamb numbers, right anterior, ° right middle, * right caudal, ® left anterior, ^ left middle, and ^ left caudal lobes. ^ negative, + peribronchiolar lymphocytic cuffing with mild macrophage infiltration, ++ peribronchiolar, perivascular lymphocytic infiltration, lymphoid nodule and macrophage aggregation, H = Immunohistochemical stain positive, S = exudative lesions. 194 The lungs from animals # 59 and 60 showed exudative and proliferative type lesions along with the peribronchiolar and perivascular cuffing and lymphoid hyperplasia usually observed in AT. ovipneumoniae infected lungs. The histological changes were more pronounced in the anterior lobes with decreasing severity toward caudal lobes in all mycoplasma infected lungs. Moderate peribronchiolar cuffing mainly in the anterior lobes was present in the lung of control animal # 58. Histological lesions were not be observed in control animal # 57.

Iimnunohl s t ochemi stry The data reflecting the results of indirect immunohistochemical staining is presented in Table 3. The presence of M. ovipneumoniae antigen in one or more lobes of all challenged animal was confirmed. However, M. ovipneumoniae antigens in lungs from control animals could not be observed. The M. ovipneumoniae antigen appeared as dark brown bodies associated with cilia of the bronchioles and trachea (Figs 1 and 2) . The dumpiness and loss of cilia was minimal in all infected animals as compared to the previous study reported in Paper 1. Fig 1. Photograph of histological section of lung with negative control indirect immunohistochemical stain in which either normal horse serum or PBS was used instead of specific anti Mycoplasma ovipneumoniae horse serum as primary antibody.

Fig 2. Photograph of histological section of lung with indirect immunohistochemical positive stain. Dark brown staining bodies sticking to ciliated epithelium is indicative of IH positive for M. ovipneumoniae. dumpiness and patchy loss of cilia are also present. 196 197 DISCUSSION

Mycoplasma ovipneumoniae was reisolated from nasal swabs of 4 inoculated animals at various time intervals during the course of the trial. However, it was recovered from bronchial swabs of all inoculated animals at postmortem. The Gram- negative coccobacillus was recovered from one lamb only on ante- and postmortem cultural examinations. Swabs from animals in the control group were negative for M. ovipneumoniae and the inoculated bacteria on both antemortem and postmortem examinations. However, M. arginini was isolated from one lamb during the trial period and at postmortem examination. Clinical disease in inoculated animals was mild and only serous to mucopurulent nasal discharge was observed in 3 of 6 animals without other obvious signs of pneumonia. This situation was not changed even with superinfection with a pool of Gram-negative hemolytic coccobacilli in lambs of group B. The coughing syndrome characteristic of natural outbreaks did not develop in these lambs during the trial period. This may be because these animals were kept in small pens and were not exposed to harsh environment or stressed by handling as observed with outdoor flocks. There may be more than one M. ovipneumoniae strain simultaneously prevalent in natural outbreaks as observed by other research workers (lonas et al., 1991a,b). 198 The gross lesions were limited to focal consolidation mainly in the anterior lobes of lungs of 2 of 4 lambs inoculated with M. ovipneumoniae alone. These mild gross lesions were similar to those in lungs of lambs with the coughing syndrome as described in Paper 1 of this manuscript. The lungs from lambs which were exposed to the Gram-negative bacterium did show congestion, severe consolidation, and adhesions. These latter findings are similar to previous reports indicating the involvement of mixed mycoplasma and other bacteria in the development of exudative pneumonia (Foggie et al., 1976; Alley and Clarke, 1979; Davies et al., 1981) . Microscopic lesions were present in all lung sections of all infected animals. However, the severity of the lesions varied with different lobes and different animals. The lesions were more severe in the anterior lobes as compared to the caudal lobes. These lesions were in the form of peribronchiolar and perivascular cuffing, lymphoid hyperplasia, mild macrophage aggregation, and interstitial thickening. The loss of cilia was less marked compared to the natural disease as reported in Paper 1. While these lesions were mild to moderate in character, they were typical of this mycoplasma infection as discussed in Paper 1 and also reported by other workers (Davies, 1985). Microscopic lesions in lungs of lambs superinfected with 199 a Gram-negative coccobacillus were characteristic of a neutrophilic exudative type pneumonia. They were similar to lesions described by other workers for combined bacterial infections (Jones et al., 1982; Gilmour et al., 1982/ Davies, 1985). However, we did not observe marked necrosis, bacterial colonies, or spindle shaped cells as commonly associated with Paateurella sp. The development of the mild mycoplasmal lesions of peribronchiolar cuffing type in one of the control animals was probably due to M. arginini which was isolated during the later stages of the experiment. This mycoplasma is commonly found in sheep and goats as a saprophyte and may also cause subclinical disease and mild histological changes in the lungs (Cottew, 1983; DaMassa et al., 1992). The experimentation confirmed the applicability of the IH staining technique for detecting Mycoplasma antigens in formalin-fixed lung tissues as described in Paper 1. The presence of M. ovipneumoniae antigens was demonstrated in the ciliated epithelium of bronchioles and trachea in all lungs from infected animals. However, these antigens could not be detected from all lobes of each lung and so it did not synchronize with the histological findings. The dumpiness and loss of cilia as reported with natural disease (Paper 1) was less marked but IH stain consistently demonstrated M. ovipneumoniae in association with intact ciliated cells of the bronchioles. This technique appeared to be specific as it did 200 not give any positive staining in lung sections from control animals or negative control slides. These observations are similar to those reported earlier with other mycoplasmas by workers using the IH technique (Hsu et al., 1981; Imada et al,, 1982; Doster and Lin, 1988). The results of this study indicated that although M. ovipneumoniae may be the main organism isolated from a coughing syndrome of lambs, there may be other factors involved in the production of acute clinical pneumonia as observed in natural outbreaks. Even superinfection with Gram- negative coccobacillus organisms did not enhance the clinical course of the disease. Gross lesions were present in 50% of M. ovipneumoniae-infected lambs although they were mild in nature but were similar to the animals exposed to natural outbreaks of a coughing syndrome. The development of severe consolidation and adhesions in one animal of group B is similar to that seen with other bacterial infections (Davies, 1985) , The histopathological lesions were significant as they were persistently present in all lungs infected with M. ovipneumoniae. The histological changes in lungs due to M. ovipneumoniae infection, i.e., peribronchiolar lymphocytic aggregation and lymphoid hyperplasia and lack of exudative reaction appeared to be a local immune response to superficial infection. These changes may not be part of an invasive or destructive process (Liggitt, 1985; Henson et al., 1984). The 201 histopathological changes observed in lungs of lambs superinfected with the Gram-negative bacteria were similar to those reported earlier in Paper 1 in which there were mixed mycoplasma and a Gram-negative hemolytic coccobacillus infection. With the protocol used we could not produce an acute clinical disease as seen in natural outbreaks of a coughing syndrome. However, the development of pathological changes were pronounced and indicative of an active disease process induced by M. ovipneumoniae infection. The combined histological findings and indirect immunohistochemical staining in formalin-fixed lung tissues appeared to be sensitive and reliable methods for detection of M. ovipneumoniae infection in lambs. There is need to conduct an exhaustive survey to identify the presence of multiple strains of M. ovipneumoniae in individual flocks and animals coacting in the production of a coughing syndrome. 202

REFERENCES

Alley, M. R. and Clarke, J. K. 1979. The experimental transmission of ovine chronic non-progressive pneumonia. N. Z. Vet. J. 27:217220. Alley, M. R., Quinlan, J. R., and Clarke, J. K. 1975. The prevalence of Mycoplaama ovipneumoniae and Mycoplasma arginini in the respiratory tract of sheep. N. Z. Vet. J. 23:137-141. Bakke, T. 1982. The occurrence of mycoplasmas and bacteria in lungs of sheep in southern Norway. Acta. Vet. Scand. 23:235-247. Buddie, B. M., Herceg, M., and Davies, D. H. 1984. Experimental infection of sheep with Mycoplasma ovipneumoniae and Pasteurella haemolytica. Vet. Microbiol. 9:543-558. Carmichael, L. E., St. George, T. D., Sullivan, N. D., and Horsfall, N. 1972. Isolation, propagation and characterization studies of an ovine mycoplasma responsible for proliferative interstitial pneumonia. Cornell Vet. 62:654-679. Cottew, G. S. 1979. Caprine-ovine mycoplasmas. In: Tully, J.G., and Whitcomb, R.F. (eds.). The Mycoplasmas. Vol. II. Academic Press, New York. pp. 103-132. Cottew, G. S. 1983. Recovery and identification of caprine and ovine mycoplasmas. In: Tully, J. G. and Razin, S. (eds.). Methods in Mycoplasmology. Vol. II. Academic Press, New York. pp. 91-104. Davies, D. H. 1985. Aetiology of pneumonia of young sheep. Prog. Vet. Microbiol. Immun. 1:229-248. Davies, D. H., Jones, B. A. H., and Thurley, D. C. 1981. infection of specific pathogen free lambs with para­ influenza virus type 3, Pasteurella haemolytica and Mycoplaama ovipneumoniae. Vet. Microbiol. 6:295-308. Doster, R. and Lin, B. C. 1988. Identification of Mycoplasma hyopneumoniae from formalin-fixed lung, using an indirect immunoperoxidase method. Am. J. Vet. Res. 49:1719-1721. 203 DaMassa, A. J., Wakenell, P. S., and Brooks, D. L. 1992. Mycoplasma of goats and sheep. J. Vet. Diagn. Invest. 4:101-113. Eness, G. P., Kaeberle, M. L., and Hummel, D. 1990. Association of Mycoplaama ovipneumoniae and respiratory disease in feeder lambs. Beef-Sheep Research Report. Iowa State University, Ames, lA. pp. 44-45. Foggie, A., Jones, G. E., and Buxton, D. 1976. The experimental infection of specific pathogen free lambs with Mycoplaama ovipneumoniae. Res. Vet. Sci. 21:28-35. Foggie, A., Jones, G. E., and Buxton, D. 1976. The experimental infection of specific pathogen free lambs with Mycoplasma ovipneumoniae. Res. Vet. Sci. 21:28-35. Gilmour, J. S., Jones, G. E., and Rae, A. 1979. Experimental studies of chronic pneumonia of sheep. Comp. Immunol. Microbiol. Infect. Dis. 1:285-293. Henson, M. P., Larsen, G. L., Henson, J. E., Newman, S. L., Musson, R. A., and Leslie, C. C. 1984. Resolution of pulmonary inflammation. Federation Proc. 43:2799-2806. Hsu, S. M., Raine, L., and Fanger, H. 1981. The use of avidin- biotin peroxidase complex in immunoperoxidase techniques. Am. J. Clin. Pathol. 75:816-861. Imada, Y., Nonomura, I., and Furta, K. 1982. Indirect immunoperoxidase technique for the assay of antibodies against Mycoplasma gallisepticum and M. synoviae in chicken serum. Natl. Inst. Anim. Health Q. 22:16-22. lonas, G., Clarke, J. K., and Marshall, R. B. 1991a. The isolation of multiple strains of Mycoplasma ovipneumoniae from individual pneumonic sheep lungs. Vet. Microbiol. 29:349-360. lonas, G., Norman, N. G., Clarke, J. K., and Marshall, R. B. 1991b. A study of the heterogeneity of isolates of Mycoplaama ovipneumoniae from sheep in New Zealand. Vet. Microbiol. 29:339-347. Jones, G. E., Buxton, D., and Barker, D. B. 1979. Respiratory infection in housed sheep, with particular reference to mycoplasmas. Vet. Microbiol. 4:47-59. 204 Jones, G. E., Foggie, A., Mould, D. L., and Livitts, S. 1976. The comparison and characterization of glycolytic mycoplasmas isolated from the respiratory tract of sheep. J. Med. Microbiol. 9:39-52. Jones, G. E., Gilmour, J. S., and Rae, A. 1978. Endobronchial inoculation of sheep with pneumonic lung-tissue suspensions and with the bacteria and the mycoplaamas isolated from them. J. Comp. Pathol. 88:85-96. Jones, G. E., Gilmour, J. S., and Rae, A., 1982a. The effects of different strains of Mycoplasma ovipneumoniae on specific pathogen free and conventional reared lambs. J. Comp. Pathol. 92:267-272. Jones, G. E., Gilmour, J. S., and Rae, A., 1982b. The effect of Mycoplasma ovipneumoniae and Pasteurella haemolytica on specific pathogen free lambs. J. Comp. Pathol. 92:261- 266. Kaeberle, M. L. and Eness, P. 1988. Serological analysis of pneumonia in feeder lambs. Beef-Sheep Research Report. Iowa State University, Ames, lA. pp. 54-55. Liggitt, H. D. 1985. Defense mechanisms in the bovine lung. Symp. Bovine Reap. Dis. 1:347-360. Livingston, C. W. Jr. and Gauer, B. B. 1979. Isolation of Mycoplasma ovipneumoniae from Spanish an Angora goats. Am. J. Vet. Res. 40:407-408. Nakane, P. K. and Pierce, G. B. 1967. Enzyme labelled antibodies: Preparation and application for the localization of antigens. J. Histoch. Cytochem. 14:929- 931. Rosendal, S. and Black, F. T. 1972. Direct and indirect immunofluorescence of unfixed and fixed Mycoplasma colonies. Acta. Pathol. Microbiol. Scand. Sect. B. Microbiol. Immunol. 80:615-622. Stalheim, O. H. 1983. Mycoplasmal respiratory diseases of : a review and update. J. Am. Vet. Med. Assoc. 182:403-406. St. George, T. D., Sullivan, N. D., Love, J. A., and Horsfall, N. 1971. Experimental transmission of pneumonia in sheep with a mycoplasma isolated from pneumonic sheep lung. Aust. Vet. J. 47:282-283. 205 Stipkovits, L., Belak, S., Palfi, V., and Tury, F. 1975. Isolation of Mycoplasma ovipneumoniae from sheep with pneumonia. Acta. Vet. Acad. Sci. Hung. 25:267-273. Sullivan, N. D., St.George, T. D., and Horsfall, N. 1973. A proliferative interstitial pneumonia of sheep associated with mycoplasma infection. 2. The experimental exposure of young lambs to infection. Aust. Vet. J. 49:63-68. Tully, J. G. 1983. Cloning and filtration techniques for mycoplasmas. In: Razin, S., and Tully, J.G. (eds.). Methods in Mycoplasmology. Vol. I. Academic Press, New York. pp. 173-177. Wikse, S. E. 1990. The bronchopneumonias. Respiratory disease complex in cattle, sheep, and goats. In: Smith, B.P. (ed.). Large Animal Internal Medicine. The C.V. Mosby Co., Philadelphia, pp. 582-594. 206

ACKNOWLEDGEMENTS

I would like to thank Mammadou Niang for his friendly and constant cooperation throughout the period of this research project. 207

GENERAL SUMMARY

Ovine pneumonia is an important disease problem causing major losses to the sheep industry of the world. The etiology of this disease is multifactorial and comprises both infectious and noninfectious agents. Pasteurella sp. and M. ovipneumoniae are among the commonly isolated organisms from both pneumonic and normal lambs. The study was conducted in an attempt to correlate M. ovipneumoniae with outbreaks of a "coughing syndrome" in lambs. Primary emphasis was directed to isolation and characterization of causative organisms, their in vitro and in vivo roles in the development of the disease, and validity of strain variability of M. ovipneumoniae. The disease is characterized by paroxysmal cough, mucoid nasal discharge, intolerance to exercise, and occasional development of rectal prolapses in some animals. A similar disease in lambs has not previously been reported in the literature. This suggested that the causative agent may be a different strain of M. ovipneumoniae from those reported previously. Mycoplasma ovipneumoniae was isolated from 70% of the total lambs investigated during the study. Persistent isolation of this mycoplasma in diseased lambs was one of the evidences of its involvement in a coughing syndrome. Gross pathology in the form of focal consolidation especially in cranial lobes of lungs, was present in 43% of lambs naturally 208 infected with M. ovipneumoniae. However, in 75% of the cases having mixed M. ovipneumoniae and other bacterial infections, these lesions were in the form of severe consolidation and fibrinous adhesions. Histological lesions were present in all lung sections obtained from M. ovipneumoniae infected animals. These were in the form of peribronchial and perivascular lymphocytic cuffing, lymphoid hyperplasia, interstitial thickening, and patchy loss of ciliated epithelium. The primary infiltrating cells were lymphocytes, plasma cells, and macrophages. The presence of these histological lesions in lambs with the coughing syndrome were classical and facilitated the diagnosis of M. ovipneumoniae infection in lambs. The use of indirect immunohistochemical staining in formalin-fixed lung tissues verified 84% of M. ovipneumoniae infected lungs. This technique proved to be a sensitive and specific method for in situ detection of M. ovipneumoniae. Antigens of M. ovipneumoniae (Heat killed antigen, SDS treated antigen, and M. ovipneumoniae membrane fragments) have limited mitogenic capability to transform ovine lymphocytes to blast cells. This mitogenicity appeared to be nonspecific. This reactivity may contribute to the lymphocytic response in the form of typical histological lesions observed in tissue sections of infected lungs. Challenge of a group of mycoplasma-free lambs with an 209 isolate of M. ovipneumoniae produced mild clinical disease. However, typical gross lesions were present in 50% of the challenged lambs. All infected lambs developed the classical histological lesions of M. ovipneumoniae infection and the IH technique confirmed the presence of this mycoplasma in tissue sections of all lungs from challenged animals. Restriction endonuclease DNA analysis indicated extensive heterogeneity among isolates of M. ovipneumoniae. Eight of 30 isolates possessed homologous patterns indicative of a single strain cluster. However, major to minor differences were present among the remaining isolates. Analysis of these isolates indicated 62 percent to 92 percent homology in band patterns when compared to the 8 isolates of one strain cluster. Hybridization of restriction DNA digests of 30 isolates with a radiolabeled rRNA probe detected multiple bands of different molecular weights complementary to 16S rRNA gene, and this bracketed the isolates into 3 groups. The study did not define the exact mechanisms involved in acute disease development. Variability in strains of M. ovipneumoniae is obviously one contributing factor in disease production. A competent hybridization probe for identification of various strains of this mycoplasma would help to reveal the epidemiological pattern of the disease. The importance of this disease entity warrants further investigation of the pathogenesis and approaches to control the infection in lambs. 210

REFERENCES

Adair, B. M., FcFerran, J. B., and McKillop, E. R. 1982. A sixth species of ovine adenovirus isolated from lambs in New Zealand. Archs. Virol. 74:269-275. Alley, M. R. and Clarke, J. K. 1977. The influence of microorganisms on the severity of lesions in chronic ovine pneumonia. N.Z. Vet. J. 27:217-220. Alley, M. R. and Clarke, J. K. 1979. The experimental transmission of ovine chronic non-progressive pneumonia. N. Z. Vet. J. 27:217-220. Alley, M. R., Quinlan, J. R., Clarke J. K. 1975. A survey of the prevalence of Mycoplasma ovlpneumoniae and M. arginini in the respiratory tract of sheep in New Zealand. N. Z. Vet. J. 23:137-141. Almagor, M., Yatziv, S., and Kahane, I. 1984. Inhibition of host cell catalase by Mycoplasma pneumoniae: a possible mechanism for cell injury. Infect. Immun. 41:251-256. Amar, A., Rottem, S., and Razin, S. 1978. Disposition of membrane proteins as affected by changes in the electrochemical gradient across mycoplasma membranes. Biochem. Biophys, Res. Commun. 84:306-312. Amikam, D., Razin, S., and Glaser, G. 1982. Ribosomal RNA genes in Mycoplasma. Nucleic Acids Res. 10:4215-4222. Annon. 1979. Subcommittee on the taxonomy of Mollicutes. Proposal of minimal standards for descriptions of new species of the class Mollicutes. Int. J. Syst. Bacteriol. 29:172-180. Araake, M. 1982. Comparison of ciliostasis by mycoplasmas in mouse and chicken tracheal organ cultures. Microbiol. Immunol. 26:1-14. Bains, R. M. and Miller, K. D. 1988. Peroxidase labelling in immunocytochemistry: a critical comparison of five systems. Med. Lab. Sci. 45:240-244. Bakke, T. 1982. The occurrence of mycoplasma and bacteria in lungs from sheep in Southern Norway. Acta. Vet. Scand. 23:235-247. 211 Bakke, T. and Nostvold, S. 1982. An investigation of ovine pneumonia in four herds from central Norway. I. Prevalence of pneumonia and microbiological findings. Acta. Vet. Scand. 23:248-258. Ball, H. J., and Todd, D. 1978. Comparison of antigens of pneumonia-associated mycoplasma species by gel diffusion. Infect. Immu. 21:954-958. Banai, M., Razin, S., Bredt, W., and Kahane, I. 1980. Isolation of binding sites to glycophorin from Mycoplasma pneumoniae membranes. Infect. Immun. 30:628-634. Banai, M., Kahane, I., Razin, S., and Bredt, W. 1978. Adherence of Mycoplasma gallisepticum to human erythrocytes. Infect. Immun. 21:365-372. Banerjee, M., Singh, N., and Gupta, P. P. 1979. Isolation of mycoplasmas and acholeplasmas from pneumonic lesions in sheep and goats in India. Zentbl. Vet. Med. 268:689-695. Barbour, A. G. 1990. Antigenic variation of a relapsing fever Borrelia species. Annu. Rev. Microbiol. 44:155-171. Barile, M. F. and Leventhal, B. G. 1968. Possible mechanism for Mycoplasma inhibition of lymphocyte transformation induced by phytohaemagglutinin. Nature, 219:750-752. Baseman, J. B., Cole, R. M., Krause, D. C., and Leith, D, K. 1982. Molecular basis for cytadsorption of Mycoplasma pneumoniae. J. Bact. 151:1514-1522. Belak, S. and Palfi, V. 1974. An adenovirus isolated from sheep and its relationship to type 2 bovine adenovirus. Arch. Ges. Virusforsch. 46:336-369. Bennett, R. H. and Jasper, D. E. 1977. Immunosuppression of humoral and cell-mediated responses in calves associated with inoculation of Mycoplasma bovis. Am. J. Vet. Res. 38:1731-1738. Biberfeld, G. and Nilson, E. 1978. Mitogenicity of Mycoplasma fermentans for human lymphocytes. Infect. Immun. 21:48- 54. Biberfeld, G., Arneborn, P., Forsgren, M., Van Stedingk, L. v., and Blomquist, S. 1983. Non specific polyclonal antibody response induced by Mycoplasma pneumoniae. Yale J. Biol. Med. 56:639-642. 212 Biberfeld, F. and Gronowicz, E. 1978. Mycoplasma pneumoniae in polyclonal B-cell activation. Nature, 261:238-239. Bosworth, T. J. and Lovell, R. 1944. The occurrence of haemolytic cocco-bacilli in the nose of normal sheep and cattle. J. Comp. Path. 54:168-171. Bradbury, J. M. and Ideris, A. 1982. Abnormalities in turkey poults following infection with Mycoplasma iowae. Vet. Rec. 110:559-560. Bredt, W., Wellek, B., Brunner, H., and Loos, M., 1977. Interaction between Mycoplasma pneumoniae and first component of complement. Infect. Immun. 15:7-12. Brogden, A., Rose, D., Cutlip, R. C., Lehmkuhl, H. D., and Tully, J.G. 1988. Isolation and identification of mycoplasmas from the nasal cavity of sheep. Am. J. Vet. Res. 49:1669-1672. Brunner, H., Feldner, J., and Bredt, W. 1985. Effect of monoclonal antibodies to the attachment tip on experimental Mycoplasma pneumoniae infection of hamsters: A preliminary report. Israel J. Med. Sci. 17:599-603 Buddie, B. M., Herceg, M., and Davies, D. H. 1984. Experimental infection of sheep with Mycoplasma ovipneumoniae and Pasteurella haemolytica. Vet. Microbiol. 9:543-558. Buttery, S. H., Lloyd, L. C., and Titchen, D. A. 1976. Acute respiratory, circulatory, and pathological changes in the calf after intravenous injections of galactan from Mycoplasma mycoides ssp. mycoides. J. Med. Microbiol. 9:379-391. Campbell, R. S. P., Rae. A., Sharp, J. M., and Wise, K. S. 1979. Infection of tracheal organ cultures with ovine adenovirus type 2. J. Comp. Pathol. 89:107-114. Carmichael, L. E., St.George, T. D., Sullivan, N. D., and Horsefall, N. 1972. Isolation, propagation and characterization studies of an ovine mycoplasma responsible for proliferative interstitial pneumonia. Cornell Vet. 62:654 Cassell, G. H., Lindsey, J. R., Quercash, R. G., and Baker, H. J. 1973. Murine mycoplasma respiratory disease. Am. N. Y. Acad. Sci. 225:395-412. 213 Cassell, G. H., Clyde, W. A. Jr., and Davis, J. K. 1985. Mycoplasmal respiratory infections. In: Razin, S. and Barile, M. P. (eds.). The Mycoplasma. Vol. IV. Academic Press, New York. pp. 65-106. Chan, H. W., and Ross, R. F. 1984. Restriction endonuclease analysis of two porcine mycoplasma deoxyribonucleic acids: sequence-specific methylation in Mycoplasma hyopneumoniae genome. Int. J. Syst. Bacterid. 34:16-20. Christiansen, G. and Erno, H. 1990. Ribosomal RNA gene used in diagnosis of caprine mycoplasmas. lOM Lett. 1:176-177. Christiansen, C. 1987. The mycoplasma genome; part I. Microbiol. Sci. 4:168-172. Clarke, J. K., Brown, V. G., and Alley, M. R. 1974. Isolation and identification of mycoplasmas from the respiratory tract of sheep in New Zealand. N. Z. Vet. J. 22:117-121. Clyde, W. A. Jr. 1963. Hemolysis in identifying Eaton's pleuropneumonia like organisms. Science, 13 9:55 Clyde, W. A. Jr. and Thomas, L. 1973. Tropism of Mycoplasma gallisepticum for arterial walls. Proc. Natl. Acad. Sci. 70:1545-1549. Cohen, G. and Somerson, N. L. 1969. Glucose dependent secretion and destruction of hydrogen peroxide by Mycoplasma pneumoniae. J. Bacterid. 98:547-551. Cole, B. C., Aldridge, K. E., and Ward, J. R. 1977. Mycoplasma dependent activation of normal lymphocytes: Mitogenic potential of mycoplasmas for mouse lymphocytes. Infect. Immun. 18:393-399. Cole, B. C., Naot, Y., Stanbridge, E. J., and Wise, K. S. 1985. Interaction of mycoplasmas and their products with lymphoid cells in vitro. In: Razin, S. and Barile, M. F. (eds.). The Mycoplasma. Vol. IV. Academic Press, New York. pp. 203-257. Cole, B. C. and Ward, J. R., 1973. Interaction of Mycoplasma arthritidis and other mycoplasmas with murine peritoneal macrophages. Infect. Immun. 7:691-699. 214 Cole, B. C., Washburn, L. R., Sullivan, G. J., and Ward, J. R. 1982. Specificity of a mycoplasma mitogen for lymphocytes from human and various animal hosts. Infect. Immun. 36:662-666. Collier, A. M. 1979. Mycoplasma in organ cultures. In: Tully, J. G. and Whitcomb, R. F. (eds.).The Mycoplasma. Vol. II. Cambridge University Press, Cambridge, pp. 479-493. Collier, A. M., and Clyde, W. A. 1971. Relationship between MycoplaBma pneumoniae and human respiratory epithelium. Infect. Immun. 3:694-701. Colvin, R. B., Bhan, A. K., and McCluskey, R. T. 1988. Diagnostic Immunopathology. Raven Press, New York, NY., U.S.A. Coons, A. H., Creech, H. J., and Jones, R. N. 1941. Immunologic properties of an antibody containing a fluorescent group. Proc. Soc. Exp. Biol. Med. 47:200-202. Copperman, R. and Morton, H. E. 1966. Reversible inhibition of mitosis in lymphocyte cultures by non-viable mycoplasma. Proc. Soc. Exp. Biol. Med. 123:790-795. Cottew, G. S. 1971. Characterization of mycoplasmas isolated from sheep with pneumonia. Aust. Vet. J. 47:591-596. Cottew, G. S. and Yeats, F. R. 1981. Occurrence of mycoplasmas in clinically normal goats. Aust. Vet. J. 57:52-53. DaMassa, A. J., Wakenell, P. S., and Brooks, D. L. 1992. Mycoplasmas of goats and sheep. J..Vet. Diagn. Invest. 4:101-113. Davies, D. H. 1985. Aetiology of pneumonias of young sheep. Prog. Vet. Microbiol. Immun. 1:229-248. Davies, D. H. 1980. Isolation of parainfluenza type 3 virus from pneumonic lambs. N. Z. Vet. J. 28:148. Davies, D. H., Jones, B. A. H., and Thurley, D. C. 1981. Infection of specific pathogen free lambs with parainfluenza virus type 3, Pasteurella haemolytica and Mycoplasma ovipneumoniae. Vet. Microbiol. 6:295-308. Denny, F. W., Taylor-Robinson, D., and Allison, A. C. 1972. The role of thymus dependent immunity in Mycoplasma pulmonis infection of mice. J. Med. Microbiol. 5:327-336. 215 Dhingra, P. N., Agarwal, L. P., Mahajan, V. M., and Adalakha, S. C. 1980. Isolation of Chlamydia from pneumonic lungs of buffaloes, cattle and sheep. Zentbl. Vet. Med. 27:680- 682 . Dybvig, K., Swinton, D., Maniloff, J., and Hattman, J. 1982. Cytosine methylation of the sequence GATC in a mycoplasma. J. Bacterid. 151:1420-1424. Elazhary, M. A. S. Y., Silim, A., and Dea, S. 1984. Prevalence of antibodies to Bovine respiratory syncytial virus. Bovine viral diarrhea. Bovine herpes virus-1 and Bovine parainfluenza-3 virus in sheep and goats in Quebec. Am. J. Vet. Res. 45:1660-1662. Eness, P., Kaeberle, M. L., and Hummel, D. 1990. Association of Mycoplasma ovipneumoniae and respiratory disease in feeder lambs. Beef-Sheep Research Report. Iowa State University, Ames, Iowa. pp. 44-45. Erno, H. and Jurmanova, K. 1973. Bovine mycoplasma: Serological studies by double immuno-diffusion, growth precipitation and growth inhibition. Acta. Vet. Scand. 14:524-527. Fernald, G. W. 1979. Humoral and cellular immune responses to mycoplasmas. In: Tully, J. G. and Whitcomb, R. F. (eds.). The Mycoplasmas. Vol. II. Academic Press, New York. pp. 399-423. Foggie, A. 1977. Chlamydial infections in mammals. Vet. Rec. 100:315-317. Foggie, A., Jones, G. E., and Buxton, D. 1976. The experimental infection of specific pathogen free lambs with Mycoplasma ovipneumoniae. Res. Vet. Sci. 21:28-35. Foggie, A. and Angus, K. W. 1972. Observation on the distribution of Mycoplasma arginini as a respiratory tract infection in sheep and its pathogenicity for specific pathogen free lambs. Vet. Rec. 90:312-313. Friis, N. F., and Jensen, P. T. 1984. Serological comparison of type strains of porcine, bovine, and ovine mycoplasmas with atypical colony morphology. Acta. Vet. Scand. 25:29- 35. Gabridge, M. G. 1975. Oxygen consumption by trachea organ cultures infected with Mycoplasma pneumoniae. Infect. Immun. 12: 544-549. 216 Gabridge, M. G. 1982. A review of the morphological and biochemical features of the attachment process in infections with Mycoplasma pneumoniae. Review Infect. Dis. 4(S) ;179-184. Gabridge, M. G. and Schneider, P. R. 1975. Cytotoxic effect of Mycoplaama fermentans on mouse thymocytes. Infect. Immun. 11:460-465. Geary, S. J., Tourtellotte, M. E., and Cameron, J. A. 1981. Inflammatory toxin from Mycoplasma bovia: Isolation and characterization. Science, 212:1032-1033. Gilmour, N. J. L. 1978. Pasteurellosis in sheep. Vet. Rec. 102:100-102. Gilmour, N. J. 1980. Experimental pulmonary infection of sheep caused by Pasteurella haemolytica biotype T. Vet. Rec. 106:507-508. Gilmour, J. S., Jones, G. E., and Rae, A. G. 1979. Experimental studies of chronic pneumonia of sheep. Comp. Immun. Microbiol. Infect. Dis. 1:285-293. Gilmour, N. J. L. 1980. Pasteurella haemolytica infection in sheep. Vet. Quart. 2:191-198. Gilmour, J. S., Jones, G. E., Keir, W. A., et al. 1982. Long term pathological and microbiological progress in sheep of experimental disease resembling atypical pneumonia. J. Comp. Pathol. 92:229-238. Ginsburg, H. and Nicolet, J. 1973. Extensive transformation of lymphocytes by a mycoplasma organism. Nature (London), New Biol. 246:143-146. Goltz, J. P., Rosendal, S., McCraw, B. M., and Ruhnke, H. L, 1986. Experimental studies on the pathogenicity of Mycoplasma ovipneumoniae and M. arginini for the respiratory tract of goats. Am. J. Vet. Res. 50:59-67. Gourlay, R. N. and Howard, C. J. 1982. Respiratory mycoplasmosis. Adv. Vet. Sci. Comp. Med. 26:289-332. Haines, D. M. and Chelack, B. J. 1991. Technical consideration for developing enzyme immunohistochemical staining procedure for formalin fixed paraffin embedded tissues for diagnostic pathology. J. Vet. Diagn. Invest. 3:101- 112. 217 Hamdy, A. H. and Pounden, W. D. 1959. Experimental production of pneumonia in lambs. Am. J. Vet. Res. 20:78-83. Hartwig, N. R. and Eness, P. G. 1989. Causes of death loss in Iowa sheep. Proc. Iowa Vet. Med. Ass. 107th Annual Meeting, Des Moines, lA. pp. 141-143. Hill, A. C. 1978. Demonstration of Mycoplasmas in tissue by the immuno-peroxidase technique. J. Infect. Dis. 137:152- 154. Hollingdale, M. R. and Lemcke, R. M. 1970. Antigenic differences within the species of Mycoplasma hominis. J. Hyg. 68:469-477. Hore, D. E., Stevenson, R. G., Gilmour, N. J. L., Vantsis, J. T., and Thompson, D. A. 1968. Isolation of parainfluenza virus from the lungs and nasal passages of sheep showing respiratory disease. J. Comp. Pathol. 78:259-265. Howard, C. J. and Taylor, G. 1985. Humoral and cell mediated immunity. In: Razin, S. and Barile, M. F. (eds.). The Mycoplasma. Vol. IV. Academic Press, New York. pp. 259- 292. Hsu, S., Raine, L., and Fanger, H. 1981. A comparative study of the peroxidase antiperoxidase method and a Avidin- biotin complex method for studying polypeptide hormones with radioimmunoassay antibodies. Am. J. Clin. Path. 75:734-738. Hu, P. C., Collier, A. M., and Baseman, J. B. 1975. Alterations in the metabolism of hamster tracheas in organ culture after infection by virulent Mycoplaama pneumoniae. Infect. Immun. 11:704-711. Hu, P. C., Collier, A. M., and Baseman, J. B. 1977. Surface parasitism by Mycoplasma pneumoniae of respiratory epithelium. J. Exp. Med. 45:1328-1343. Huang, S., Minassain, H., and More, J. D. 1976. Application of immunofluorescent staining in paraffin section improved by trypsin digestion. Lab. Invest. 35:383-391. lonas, G., Norman, N. G., Clarke, J. K., and Marshall, R. B. 1991b. A study of the heterogeneity of isolates of Mycoplasma ovipneumoniae from sheep in New Zealand. Vet. Microbiol. 29:339-347. 218 lonas, G., Clarke, J. K., and Marshall, R. B. 1991. The isolation of multiple strains of Mycoplasma ovipneumoniae from individual pneumonic sheep lungs. Vet. Microbiol. 29:349-360. lonas, G., Mew, A. J., Alley, M. R., Robinson, A. J., and Marshall, R. B. 1985. Colonization of the respiratory tract of lambs by strains of Mycoplasma ovipneumoniae. Vet. Microbiol. 10:533-539. Jannette. J. C. 1989. Immunohistology in Diagnostic Pathology. CRC Press, Boca Raton, FL. Johnson, J. L. 1984. Nucleic acid in bacterial classification. In: Krieg, N. R. and Holt, J. G. (eds.). Bergey's Manual of Systemic Bacteriology, Vol. I. Williams and Wilkins, Baltimore, U.S.A. pp. 8-17. Jones, G. E. 1983. Mycoplasmas of sheep and goats: A synopsis. Vet. Rec. 113:619-620. Jones, G. E., Buxton, D., and Marker, D. B. 1979. Respiratory infections in housed sheep, with particular reference to mycoplasmas. Vet. Microbiol. 4:47-59. Jones, G. E., Foggie, A., Mould, D. L., and Livitt, S. 1976. The comparison and characterization of glycolytic mycoplasmas isolated from the respiratory tract of sheep. J. Med. Microbiol. 9:39-52. Jones, G. E., and Gilmour, J. S. 1983. Atypical pneumonia. In: Martin, W.B. (éd.). Diseases of Sheep. Blachwell Scientific Publications, Oxford, pp. 17-23. Jones, G. E., Gilmour, J. S., and Rae, A. G. 1982. The effect of Mycoplasma ovipneumoniae and Paateurella haemolytica on specific pathogen free lambs. J. Comp. Path. 92:261-266. Jones, G. E., Gilmour, J. S., and Rae, A. G. 1982. The effects of different strains of Mycoplasma ovipneumoniae on specific pathogen-free and conventionally-reared lambs. J. Comp. Pathol. 92:267-272. Jones, G. E., Keir, W. A., and Gilmour, J. S. 1985. The pathogenicity of Mycoplasma ovipneumoniae and M. arginini in ovine and caprine tracheal organ cultures. J. Comp. Path. 95:477-487. 219 Kaeberle, M. L. and Eness, P. 1988. Serological analysis of pneumonia in feeder lambs. Beef-Sheep Research Report. Iowa State University, Ames, lA. pp. 54-55. Kahane, I. 1984. In vitro studies on the mechanism of adherence and pathogenicity of mycoplasmas. Israel J. Med. Sci. 9:210-216. Kahane, I., Banai, M., Razin, S., and Feldner, Jr. 1982. Attachment of mycoplasmas to host cell membranes. Review Infect. Dis. 4(s): 185-191. Kenny, G. E. 1975. The antigens of the Mycoplasmatales and Chlamydia. In: Sela, M. (ed.). The Antigens. Vol. III. Academic Press, New York. pp. 449-478. Khan, M. I., Lam, K. M., and Yamamoto, R. 1987. Mycoplasma galliaepticum strain variations detected by sodium dodecyl-sulfate polyacrylamide gel electrophoresis. Avian Dis. 31:315-320. Kluve, B., Merrick, W. C., Stanbridge, E. J., and Gershman, H. 1981. Mycoplasmas induce collagenase in BALB/c 3T3 cells. Nature, 292(5826) ; 855-857. Lehmkuhl, H. D. and Cutlip, R. C. 1982. Characterization of Parainfluenza type 3 virus isolated from the lung of a lamb with pneumonia. Am. J. Vet. Res. 43:626-628. Liggitt, H. D. 1985. Defense mechanisms in the bovine lung. Vet. Clin. N. Am. 1:347-361. Livingston, C. W., and Gauer, B. B. 1979. Isolation of Mycoplasma ovipneumoniae from Spanish and Angora goats. Am. J. Vet. Res. 40:407-408. Lloyd, L. C., Buttery, S. H., and Hudson, J. R. 1971. The effect of the galactan and other antigens of Mycoplasma mycoides var mycoldes on experimental infection with that organism in cattle. J. Med. Microbiol. 4:425-439. Lukacs, K., Roth, J. A., and Kaeberle, M. L. 1985. Activation of neutrophils by antigen-induced lymphokine, with emphasis on antibody-independent cytotoxicity. J. Leuk. Biol. 38:557-572. Malone, F. E., McCullough, S. T., McLoughlin, M. P., Ball, H. J., O'Hagan, J., and Neil, S. D. 1988. Infectious agent in respiratory disease of housed, fattening lambs in Northern Ireland. Vet. Rec. 27:203-207. 220 Messier, S., Ross, R. F., and Paul, P. S. 1990. Humoral and cellular immune responses of pigs inoculated with Mycoplaama hyopneumoniae. Am. J. Vet. Res. 51:52-58. Mew, A. J., lonas, G., Clarke, J. K., Robinson, A. J., and Marshall, R. B. 1985. Comparison of Mycoplasma ovipneumoniae isolates using bacterial restriction endonuclease DNA analysis and SDS-PAGE. Vet. Microbiol. 10:541-548. Meyer, T. F., and Van Putten, J. P. M. 1989. Genetic mechanisms and biological implications of phase variation in pathogenic neisseriae. J. Clin. Microbiol. Rev. 2(S) :139-145. Mohan, K., Obwolo, M. J., and Hill, F. W. 1992. Mycoplasma ovipneumoniae infection in Zimbabwean goats and sheep. J. Comp. Pathol. 107:73-79. Mori, Y., Hamaoka, T., Sato, S., and Takeuchi, S. 1988. Immunoblotting analysis of antibody response in swine, experimentally inoculated with Mycoplasma hyopneumoniae. Vet. Immunol. Pathol. 19:239-250. Nakane, P. K. and Pierce, G. B. Jr. 1967. Enzyme labelled antibodies: preparation and application for the localization of antigens. J. Histochem. Cytochem. 14:929- 931. Naot, Y. 1982. In Vitro studies on the mitogenic activity of mycoplasmal species toward lymphocytes. Rev. Infect. Dis. 4(S) :205-209. Naot, Y. Ginsburg, H. 1978. Activation of B lymphocytes by mycoplasma antigens. Immunology, 34:715-720. Naot, Y., Merchav, S., Ben-David, E., and Ginsburg, H. 1979a. Mitogenic activity of Mycoplasma pulmonis I. Stimulation of rat B and T lymphocytes. Immunology, 36:399-406. Naot, Y., Merchav, S., and Ginsburg, H. 1979b. Mycoplasma neurolyticum. A potent mitogen for rat B lymphocytes. Eur. J. Immunol. 9:185-189. Naot, Y., Simon-Tov, R., and Ginsburg, H. 1979c. Mitogenic activity of Mycoplasma pulmonis. II. Studies on the biochemical nature of the mitogenic factor. Eur. J. Immunol. 9:149-159. 221 Okoh, A. E. J. and Ocholi, R. A. 1986. Disease associated with Mycoplasma mycoides subspecies mycoides in sheep in Nigeria. Vet. Rec. 118:212. Panangala, V. S., Moray, M. A., Gresham, M. M., and Toivio- Kinnucan, M. 1992. Antigenic variation of Mycoplaama gallisepticum, as detected by use of monoclonal antibodies. Am. J. Vet. Res. 53:1139-1144. Pfeffer, A., Thurley, D. C., Boyes, B. W. 1983. The prevalence and microbiology of pneumonia in a flock of lambs. N. Z. Vet. J. 31:196-202. Pfeffer, A. 1981. The pathology of small lesions of atelectasis and consolidation in the anterior lobes of the lungs of young sheep. J. Comp. Pathol. 91:165-174. Razin, S. 1969. Structure and function in mycoplasma. Annu. Rev. Microbiol. 23:317-356. Razin, S. (1978). The Mycoplasmas. Microbiol. Rev. 42:414-470 Razin, S. 1989. Molecular approach to mycoplasma phylogeny. In: Whitcomb, R. F. and Tully, J. G. (eds.). The Mycoplasma. Vol. VI. Academic Press, Inc., San Diego, CA. U.S.A. pp. 33-69. Razin, S. 1992. Mycoplasma Taxonomy and Ecology. In : Maniloff, J., McElhaney, R. N., Finch, L. R., and Baseman, J. B. (eds.). Mycoplasma, Molecular Biology and Pathogenesis. Am. Soc. Microbiol. Washington, D.C. pp. 3-22. Razin, S., Harawawa, R. and Barile, M. F. 1983a. Cleavage patterns of the mycoplasma chromosome, obtained by using restriction endonucleases, as indicators of genetic relatedness among strains. Int. J. Syst. Bacteriol. 33:201-206. Razin, S. and Rottem, S. 1967. Identification of mycoplasma and other microorganisms by polyacrylamide gel electrophoresis of cell proteins. J. Bacteriol. 94:1807- 1810. Razin, S. and Rottem, S. 1976. Techniques for the manipulation of mycoplasma membranes. In: Haddy, A.H. (ed). Biochemical Analysis of Membranes. Chapman and Hall, London, pp. 3-26. Razin, S. and Tully, J. G., 1983b. Methods in Mycoplasmology. Vol. I. Academic Press, New York. 222 Razin, S. and Yogev, D. 1986. Genetic relatedness among Ureaplasma urealyticum serotypes (serovars). Pediatr. Infect. Dis. 5(S): 300-304 . Rickert, R. R. and Maliniak, R. M. 1989. Intralaboratory quality assurance of immunohistochemical procedures. Arch. Pathol. Lab. Med. 113:673-679. Roberts, D., Windsor, R. S., Masiga, W. N., and Kariavu, C. G. 1973. Cell mediated immune response in cattle to Mycoplaama mycoides var mycoidea. Infect. Immun. 8:349- 354. Rosenbusch, R. F. 1992. Membrane bound superoxide dismutases in colonizing mycoplasmas. 9th International Congress of the International Organization for Mycoplasmology. Ames, Iowa. Abst. No. W12/3. p. 72. Rosendal, S. 1984. Pathogenetic mechanisms of Mycoplasma mycoides subsp mycoides septicemia in goats. Isr. J. Med. Sci. 20:970-971. Rosendal, S. 1984a. Effect of the caprine variant of Mycoplasma mycoides subspecies mycoides on endothelium, monocytes, and complement of guinea pig, calf, sheep, and goat serum. Am. J. Vet. Res. 45:2396-2402. Rosendal, S. 1986. Mycoplasma. In: Carton, L.G. and Thoen, C.O. (eds.). Pathogenesis of Bacterial Infection in Animals. Iowa State University Press, Ames, lA. pp. 205- 215. Rosengarten, R. and Wise, K. S. 1990. Phenotypic switching in mycoplasmas : Phase variation of diverse surface lipoproteins. Science, 247:315-318. Sabato, A. R., Cooper, D. M., and Thong, Y. H. 1981. Transitory depression of immune function following Mycoplasma pneumoniae infection in children. Pediatr. Res. 15:813-816. Schneider, E. L. and Stanbridge, E. J. 1975. Comparison of methods for the detection of mycoplasma contamination of cell cultures: A review. In Vitro. 11:20-34. Seid, R. C. Jr., Smith, P. F., Guevara, G., Hochstein, H. D., and Barile, M. F. 1980. Endotoxin like activities of mycoplasmal lipopolysaccharides (lipoglycans). Infect. Immun. 29:990-994. 223 St.George, T. D. and Carmichael, L. E. 1975. Isolation of Mycoplasma ovipneumoniae from sheep with chronic pneumonia. Vet. Rec. 97:205-206. St. George, T. D. and Horsfall, N. 1973. A proliferative interstitial pneumonia of sheep associated with mycoplasma infection. 1. Natural history of the disease in a flock. Aust. Vet. J. 49:57-62. Stamp, J. T. and Nisbet, D. I. 1963. Pneumonia of sheep. J. Comp. Path. 73:319-328. Stanbridge, E. 1971. Mycoplasmas and cell cultures. Bacterid. Rev. 35:206-227. Stanbridge, E. J. 1982. Mycoplasma lymphocyte interaction and their possible role in immunopathologic manifestations of mycoplasmal disease. Rev. Infect. Dis. 4(S) : 219-226. Stanbridge, E. J. and Weiss, R. L. 1978. Mycoplasma capping on lymphocytes. Nature, 276(5688) :583-587. Stevenson, R. G. 1969. Respiratory diseases of sheep. Vet. Bull. 39:747-759. Stipkovits, L., Belak, S., Palfi, V., and Tury, E. 1975. Isolation of Mycoplasma ovipneumoniae from sheep with pneumonia. Acta. Vet. Acad. Scand. Hung. 25:267-273. Sullivan, N. D., St. George, T. D., and Horsefall, N. 1973. A proliferative interstitial pneumonia of sheep associated with mycoplasma infection. I. Natural history of the disease in a flock. Aust. Vet. J. 49:57-62. Tajima, M., Nunoya, T., and Yagihashi, T. 1979. An ultrastructural study on the interaction of Mycoplasma gallisepticum with the chicken tracheal epithelium. Am. J. Vet. Res. 40:1009-1014. Taylor, G., Taylor-Robinson, D., and Fernald, G. W. 1974. Reduction in the severity of Mycoplasma pneumoniae induced pneumonia in hamsters by immunosuppressive treatment with anti-thymocyte sera. J. Med. Microbiol. 7:343-348. Taylor-Robinson, D., Furr, P. M., Davies, H. A., Manchee, R. J., Mouches, C., and Bove, J, M. 1981. Mycoplasma adherence with particular reference to the pathogenicity to Mycoplasma pulmonis. Israel J. Med. Sci. 17:599-603. 224 Thirkell, D., Spooner, R. K., Jones, G. E., and Russel, W. C. 1990. The humoral immune response of lambs experimentally infected with Mycoplasma ovipneumoniae. Vet. Microbiol. 24:143-153. Thirkell, D., Spooner, R. K., Jones, G. E., Russell, W. C., and Voice, M. W. 1991. Cross reacting antigens between Mycoplasma ovipneumoniae and other species of mycoplasma of animal origin, shown by ELISA and immunoblotting with reference antisera. Vet. Microbiol. 26:249-261. Thirkell, D., Spooner, R. K., Jones, G. E., and Russell, W. C. 1990. Polypeptide and antigenic variability among strains of Mycoplasma ovipneumoniae demonstrated by SDS- PAGE and immunoblotting. Vet. Microbiol. 21:241-254. Thomas, L., Aler, P., Bitensky, M. W., Davidson, M., and Gesner, B. 1966. Studies of PPLO infection. II. The neurotoxin of Mycoplasma neurolyticum. J. Exp. Med. 124:1067-1082. Thomas, L. H., Howard, C. J., Parsons, K. R., and anger, H. S. 1987. Growth of Mycoplasma bovis in organ cultures of bovine fetal trachea and comparison with Mycoplasma dispar. Vet. Microbiol. 13:189-200. Thomasen, A. C. and Heron, I. 1979. Effect of mycoplasmas on phagocytosis and immunocompetence in rats. Acta. Pathol. Microbiol. Scand. 87(S):67-71. Tully, J. G. 1964. Production and biological characterization of an extracellular neurotoxin from Mycoplasma neurolyticum. J. Bacteriol. 88:381-388. Uppal, P. K. and Chu, A. P. 1977. Attachment of Mycoplasma gallisepticum to tracheal epithelium of fowls. Res. Vet. Sci. 22:259-260. Villemot, J. M., Provost, A., and Queval, R. 1962. Endotoxin from Mycoplasma mycoides. Nature, 193:906-907. Watson, H. L., Dybvig, K., Blalock, D. K., and Cassell, G. H. 1989. Subunit structure of the variable V-I antigen of Mycoplasma pulmonis. Infect. Immun. 57:1684-1690. Watson, H. L., McDaniels, L. S., Blalock, D. K., Fallon, M. T., and Cassell, G. H. 1988. Heterogeneity among strains of a high rate of variation within strains of a major surface antigen of Mycoplasma pulmonis. Infect. Immun. 56:1358-1363. 225 Whittlestone, P. 1976. Effect of climatic conditions on enzootic pneumonia of pigs. Int. J. Biometeorol. 20:42- 48. Yogev, D., Levisohn, S., Kleven, S. H., Halachmi, D. and Razin, S. 1988. Ribosomal RNA gene probes detected intraspecies heterogeneity in Mycoplasma gallisepticum and M. synoviae. Avian Dis. 32:220-231. Zhao, S., and Yamamoto, R. 1989. Heterogeneity of Mycoplasma iowae determined by restriction enzyme analysis. J. Vet. Diagn. Invest. 1:165-169 226

ACKNOWLEDGEMENTS

I wish to express my gratitude to my major professor Dr. Merlin L. Kaeberle for his patience, guidance, support, and contributions during the period of my studies and the execution of this research project. I would also like to thank Drs. Ricardo F. Rosenbusch, Moustafa A. Gabal, Wallace M. Wass, and John J. Andrews for their technical guidance, encouragement, moral support, and for serving on my committee. My deepest appreciations goes to my wife and daughters for their constant love and sacrifices during my absence distant from home. My gratitude is also extended to my other family members and friends for their energizing support when needed. My special remembrance and prayers goes to my loving mother who wanted to see me return but Heavenly God called her, in my absence, to His heaven.