Mycoplasma meleagridis-induced lesions in the tarsometatarsal joints of turkey embryos.

Mycoplasma meleagridis (MM) has the ability to cause bone deformity in turkey poults. However, few pathological lesions have been described and no evidence of MM-induced damage to the bones has been shown. In this study, 17-day-old turkey embryos were inoculated with MM into the allantoic cavity. On the 27th day, eight of the 22 embryos presented with curved toes. Scanning electron microscopy of the tarsometatarsal joints showed fissures in the cartilage. Histological sections of the joints revealed only the infiltration of cells with eosinophilic granules. Immunohistochemical staining (IHS) showed the presence of MM in the aggregates of the bone marrow cells and the cells with eosinophilic granules. Some of these cells were harvested by laser capture microdissection (LCM), lysed, and used as template DNA. With a pair of MM-specific primers in a conventional polymerase chain reaction (PCR), a gene product was amplified, and it comigrated with the MM DNA, which indicates that these captured cells contained MM DNA. Thus, this research shows that inoculation of MM into the turkey embryos produced joint lesions and caused cellular infiltration within the bones.

Interactions between the membranes of turkey cells and Mycoplasma meleagridis.

We used Myoplsma meleagridis (MM) to infect the RP-9 cells and the eggshell membranes and scanning electron microscopy (SEM) and confocal microscopy to study the interactions between the organisms and the cell surfaces. The surface of the RP-9 cells contained numerous projections. After 24 hr of infection with MM, those projections were either lost or aggregated to the side; MM-like particles could be seen on the surface of the cells, and surface fluorescence could be detected by confocal microscopy. On the surface of MM-infected shell membranes were necrotic fibrous tissues and cells detected by SEM and an intense surface fluorescence detected by confocal microscopy. These results indicate that MM infection of the cell surface can result in cellular damage.

Infection of the turkey embryonic trachea with Mycoplasma meleagridis.

Mycoplasma meleagridis was used to infect turkey embryos and tracheal explants. In the embryonic trachea, there was a decrease in the number of cilia and the sloughing of epithelial cells. In the tracheal explants, the deciliation was more severe and erosion of the tracheal surface was also evident; additionally, the cells of the trachea showed prominent perforation. These results indicate that M. meleagridis infection can result in damage to cells and to the surface of the trachea, which may have pathological consequences.

Chemotactic response of lymphocytes in chicken embryos infected with Mycoplasma gallisepticum.

A prominent feature of Mycoplasma gallisepticum (MG) infection is a lymphoproliferative response at the site of infection. In this study, artificial air cells (AACs) were made in eggs containing 16-day old chicken embryos. An MG culture and supernates from MG-infected RP-9 cells, HD-11 cells and monocytes were separately deposited on the membranes of the AAC. After incubation for 5 days, the eggs were opened and the AAC membranes were collected for histopathological examinations. Immunolabelling of MG-infected membranes showed a massive infiltration of lymphocytes possessing CD3 surface markers and the presence of cells that secreted lymphotactin, a chemokine. The supernates from MG-infected cells caused the infiltration of comparatively small numbers of lymphocytes. The culture medium-inoculated AACs had no obvious abnormalities of the membranes. It is suggested, therefore, that MG infection of the embryonic membranes causes the embryos to secrete lymphotactin, which induces the migration and accumulation of lymphocytes to the sites of infection.

Mycoplasma gallisepticum -induced release of macrophage inflammatory protein-1 beta from chicken monocytes-macrophages.

Chicken monocytes and a macrophage-like cell line were used to determine the presence of macrophage inflammatory protein-1 beta (MIP-1 beta). RNA was extracted from these cells and subjected to reverse transcription with an anti-sense primer specific for the whole length of the MIP-1 beta cDNA. After a polymerase chain reaction to amplify the cDNA, a 200 bp gene product was detected, which corresponded to the molecular weight of the MIP-1 beta. The culture supernate of these cells did not have the ability to attract heterophils. However, after infection with Mycoplasma gallisepticum, a potent chemotactic substance in the culture supernate was detected. The chemotactic activity could be neutralized by antibodies prepared against two 10-amino-acid peptides of MIP-1 beta, indicating that the substance was MIP-1 beta.

A pathological and immunohistochemical study of goat kids undergoing septicaemic disease caused by Mycoplasma capricolum subsp. capricolum, Mycoplasma mycoides subsp. capri and Mycoplasma mycoides subsp. mycoides (large colony type).

This paper studies the pathological and immunohistochemical findings in 12 kids experimentally infected with Mycoplasma capricolum subsp. capricolum (Mcc), M. mycoides subsp. capri (Mmc) and M. mycoides subsp. mycoides (large colony type), (MmmLC). For the demonstration of Mcc, Mmc and MmmLC antigens an immunoperoxidase technique based on the labelled streptavidin biotin method was used in the 12 kids inoculated with mycoplasmas and the three control kids inoculated with mycoplasma medium. All 12 kids, inoculated by different routes, developed subcutaneous swelling at the point of inoculation and terminated in fatal septicaemia from 1 to 5 days post inoculation. The histopathological findings consisted of cellulitis at the point of inoculation, acute diffuse interstitial pneumonia, arthritis and multifocal necrotic purulent splenitis in some kids. The Mcc, Mmc and MmmLC antigens were detected immunohistochemically in all kids with specific punctiform labelling inside the cytoplasm of the leucocytes or extracellularly at the inoculation point, respiratory airways, spleen, liver, joints, tonsils and lymph nodes. The results obtained in this study showed that the inoculation of these mycoplasmas by parenteral routes caused mycoplasmaemia. Moreover, the immunohistochemical results appear fully to confirm that the mycoplasmas were the cause of the death of the kids because of a septicaemic state.

Polymerase chain reaction and restriction endonuclease digestion for selected members of the "Mycoplasma mycoides cluster" and Mycoplasma putrefaciens.

A specific diagnostic method using the polymerase chain reaction, together with restriction endonuclease digestion patterns, was developed for members of the "Mycoplasma mycoides cluster" that normally occur in the United States (i.e., Mycoplasma mycoides subsp. mycoides Large Colony and Mycoplasma capricolum subsp. capricolum in addition to "cluster" mycoplasma, bovine serogroup 7, and Mycoplasma putrefaciens. The digestion of "cluster" polymerase chain reaction DNA (1,225 bp) amplification products with restriction enzymes AseI and SspI gave mycoplasma species-specific patterns for all strains of M. mycoides subsp. mycoides Large Colony, M. capricolum subsp. capricolum, and bovine group 7 tested. Moreover, we found a nonspecific amplification product for M. putrefaciens that occurred with the oligonucleotide primers used for the "M. mycoides cluster" reaction. However, the restriction endonuclease digestion patterns observed with the restriction enzymes AluI, AseI, and SspI for M. putrefaciens were different than the digestion patterns obtained for the other "cluster" mycoplasmas. This report confirms the usefulness of polymerase chain reaction DNA amplification allied with restriction enzyme digestion profile analysis for the rapid and specific identification of mycoplasmas belonging to the "M. mycoides cluster" and M. putrefaciens.

Immunohistochemical and ultrastructural characteristics of Mycoplasma capricolum subsp. capricolum in caprine abortion: a case report.

Described in this study are the immunohistochemical and ultrastructural findings in a case of caprine abortion due to the experimental infection of the dam with strain GM13 of Mycoplasma capricolum subsp. capricolum. Mycoplasma antigens were seen mainly in choriallantoic trophoblasts and in the lumen of blood vessels in the allantoic membrane. Examination with an electron microscope showed that the chorioallantoic trophoblasts were filled with typical mycoplasma organisms. No other bacteria were observed in any of the samples. Our results confirm by immunohistochemical and electron microscopic techniques that Mycoplasma capricolum subsp. capricolum can cause caprine abortion and that the process can occur without premonitory signs.

Experimental reproduction of Mycoplasma gallisepticum disease in chukar partridges (Alectoris graeca).

An outbreak of conjunctivitis and severe respiratory disease occurred in an integrated chukar partridge (Alectoris graeca) operation that involved about 8000 birds. The main clinical features were conjunctivitis and sinusitis and frequent mouth breathing, but almost no gasping or coughing. In 1000 breeders, egg production declined from 73% to 20%. Morbidity reached 100%, and losses from mortality and culling approached 60%. At necropsy, a conjunctivitis (often bilateral) and extensive caseated sinusitis were common. There was an occasional slight mucoid tracheitis, but no significant air sac lesions were noted. Mycoplasma gallisepticum, designated strain GM1125, was isolated and identified. Exposure of susceptible chukars to GM1125 reproduced the field disease. GM1125 was reisolated from the conjunctiva of all exposed birds 12 days postinfection, but infrequently from there or the respiratory system 36 days postexposure, even though clinical disease was still present. The experimental disease was confined to the conjunctiva and the upper respiratory tract. An occasional mucoid tracheitis was noted, but generally, the lungs and air sacs were not involved. Infection was followed by an appreciable serological response to M. gallisepticum.

DNA diversity among isolates of Campylobacter jejuni detected by PCR-based RAPD fingerprinting.

A PCR-based randomly amplified polymorphic DNA method was used to amplify Campylobacter jejuni DNA using a single oligonucleotide primer derived from either a homologous source or from Mycoplasma gallisepticum. The method was able to detect the heterogeneity of amplified DNA from human, chicken and turkey sources and can be used as a tool to study the epidemiology of Campylobacter jejuni infection.

In ovo pathogenicity of Mycoplasma iners strain Oz.

Embryonating chicken eggs were inoculated with the type strain (PG30) of Mycoplasma iners and with an additional strain of this species designated Oz. Marked gross and histopathologic lesions were observed in the embryos inoculated with strain Oz but not in those infected with strain PG30. Gross lesions were manifested by an enlargement of one or more joints that often contained a caseous exudate, both in the joint space and periarticularly. In the order of frequency, the most common lesions were found in the following joints: tibiotarsal, distal humoral, proximal humoral, femoral, mandibular, tibiofemoral, and phalangial. Similar caseous foci were also seen in the liver and, rarely, in the heart. Histologically, the most prominent lesion found in the joints and viscera was a multifocal caseous necrosis accompanied by the formation of granulomas. Many joints had progression of the inflammatory cells outward from the joint spaces along the periosteal surfaces, and the articular cartilages contained erosions and focal necrosis.

Mycoplasma infection in a commercial goat dairy caused by Mycoplasma agalactiae and Mycoplasma mycoides subsp. mycoides (caprine biotype).

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

Mycoplasma auris sp. nov., Mycoplasma cottewii sp. nov., and Mycoplasma yeatsii sp. nov., new sterol-requiring mollicutes from the external ear canals of goats.

Three mycoplasma strains, designated GIHT (T = type strain), UIAT, and VIST, were isolated from the external ear canals of goats and were shown to be serologically distinct from each other and from previously described Acholeplasma, Entomoplasma, Mesoplasma, and Mycoplasma species. Using light and transmission electron microscopy, we showed that the cells of these organisms were small, pleomorphic, coccoid, nonmotile, and nonhelical and that each cell was surrounded by a single cytoplasmic membrane. There was no evidence of a cell wall, and the organisms grew freely in media containing penicillin at concentrations of 1,000 U/ml or more and thallous acetate (final concentration, 1:4,000) and produced the "fried-egg" morphology typical of most mollicutes. Growth occurred both aerobically and anaerobically (as determined by the GasPak method). The ability to catabolize glucose and mannose and the ability to hydrolyze arginine varied among the three strains. All three strains required sterol for growth, and none of the strains hydrolyzed urea. The guanine-plus-cytosine contents of the DNAs of strains UIAT, VIST, and GIHT were determined to be 26.9, 27.0, and 26.6 mol%, respectively. Our data indicate that the three strains represent new Mycoplasma species, for which we propose the names Mycoplasma auris, Mycoplasma cottewii, and Mycoplasma yeatsii. The type strain of M. auris is UIA (= ATCC 51348 = NCTC 11731), the type strain of M. cottewii is VIS (= ATCC 51347 = NCTC 11732), and the type strain of M. yeatsii is GIH (= ATCC 51346 = NCTC 11730).

Pathogenicity of Campylobacter jejuni for turkeys and chickens.

A Campylobacter jejuni isolate obtained from a turkey liver, designated C101, and a C. jejuni isolate obtained from the feces of a chicken, designated C111, were used to inoculate their respective hosts. Isolate C101 depressed weight gain by 20% when inoculated into newly hatched poults or 4-day-old poults. It also caused death, hepatic necrosis, and generalized hemorrhages in turkey embryos. The chicken-derived isolate, C111, did not reduce weight gain in newly hatched chicks, but it did induce mortality in chicken embryos. The supernatant of the cultures of both C. jejuni isolates also caused mortality in embryos.

Characteristics of an unusual mycoplasma isolated from a case of caprine mastitis and arthritis with possible systemic manifestations.

A mycoplasma designated strain GM790A was isolated from milk and internal organs of 2 lactating goats showing mastitis and arthritis. The isolate was not related serologically to any of the currently known ovine-caprine mycoplasmas, except an isolate designated Mycoplasma sp. G, first recorded from the external ear canal of clinically normal goats in Australia. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and restriction enzyme DNA studies of strain GM790A and Mycoplasma sp. G revealed similar but not identical patterns. The inoculation of strain GM790A into the teat canal of 2 lactating goats resulted in an abrupt diminution of lactation leading to mastitis and agalactia in about 3 days. A maximum of 1 x 10(7) colony-forming units (CFU) of the mycoplasma were shed per ml of mammary secretion. Milk production partially resumed at a low level 3 weeks postinoculation, the longest period tested, but the milk still contained 1 x 10(2) CFU of the agent. The results of this study indicate that strain GM790A possesses pathogenic potential for the goat and most probably represents a new species of the genus Mycoplasma.