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Between 1997 and 2000, a total of 150 healthy cattle and 238 animals with respiratory disease were examined for six Mycoplasma species. Attempts were made to detect Mycoplasma canis, Mycoplasma dispar and Ureaplasma diversum in calves with recurrent disease, and all three of these species were identified in calves with recurrent disease and in healthy lungs. In healthy calves, 84 per cent of bronchoalveolar lavage fluids were mycoplasma free; when cultures were positive, Mycoplasma bovirhinis was the only species isolated. Mycoplasmas were isolated from 78 per cent of animals suffering recurrent respiratory disease and from 65 per cent of acute respiratory cases. Mycoplasma bovis was isolated from bronchoalveolar lavages from 35 per cent of calves suffering recurrent respiratory disease, and from 50 per cent of acute cases, and from 20 per cent of pneumonic cases examined postmortem. M bovis was associated with other Mycoplasma species in 44 per cent of cases. M dispar was also isolated from 45.5 per cent of calves suffering recurrent respiratory disease, often in association with M bovis. M canis was identified for the first time in diseased Belgian cattle. Other mycoplasmas, including Mycoplasma arginini, Mycoplasma alkalescens and U diversum, were isolated less frequently. Associations between mycoplasmas and other pathogens were often observed. Among lungs infected with Pasteurella and/or Mannheimia species, more than 50 per cent were mixed infections with M bovis.

Samples from the mammary tissue of 14 lactating goats (12 naturally infected and two experimentally infected) were examined for the presence of Mycoplasma agalactiae. A monoclonal antibody (5G12) was applied to formalin-fixed, paraffin-wax-embedded sections and labelled by the avidin-biotin peroxidase complex (ABC) method. Histological examination of tissue sections revealed strong immunoreactivity in all animals included in the study. Mycoplasma agalactiae antigen was mainly detected in the cellular debris at the periphery of purulent exudates present within lactiferous sinuses, and lactiferous and interlobular ducts. In addition, M. agalactiae organisms appeared in the cytoplasm of the epithelium of ducts, and in infiltrating macrophages and neutrophils within the ducts, alveoli, interstitial tissue and regional lymph node sinuses. It is concluded that this monoclonal antibody-based immunohistochemical technique is an efficient and specific method for the post-mortem detection of M. agalactiae in cases of clinical mastitis as well as being a useful tool for the study of the route of infection and cellular types involved during mastitis caused by this organism.

Mycoplasma bovis was detected in 134 (18 per cent) of 736 samples of bovine lung tissue collected from fatal pneumonia cases in the Republic of Ireland between April 1995 and December 1998. The cases occurred in 95 herds and recurred in four of them. Other respiratory pathogens were identified in 66 per cent of the M bovis-positive cases, with Pasteurella species, infectious bovine rhinotracheitis and parainfluenza 3 virus being most frequently detected. Mastitis and arthritis were less common clinical signs associated with M bovis infection; 22 cases of M bovis mastitis and five cases of M bovis arthritis were diagnosed in five herds.

Goats aged 3 months were inoculated with a recent isolate of Mycoplasma agalactiae (five animals) or Mycoplasma bovis (five animals) by a combined (intratracheal+intranasal) route. Two control goats were inoculated by the same route with sterile mycoplasma broth. Animals were killed 14 or 21 days after infection. At necropsy, tracheal and lung tissue was taken for pathological and immunohistochemical examination to determine changes in the lymphocyte subpopulations in the bronchus-associated lymphoid tissue (BALT). Consolidation of the lungs was not observed in any animal. M. agalactiae or M. bovis was recovered from the respiratory tract and lung of all but two infected animals. Both Mycoplasma spp. induced a moderate bronchointerstitial pneumonia, characterized by lymphoid hyperplasia of the BALT and infiltration of mononuclear cells into the alveolar walls. The predominant phagocytic cell in the pulmonary parenchyma and the airways was the macrophage. The main cellular type in the BALT was the CD3(+)T lymphocyte, and the ratio of CD4(+): CD8(+)cells was >1. It is likely that cellular immune mechanisms, through the activation of CD4(+)T lymphocytes, plays a prominent role in the acute and subacute phase of these infections., (Copyright 2000 Harcourt Publishers Ltd.)

The use of monoclonal antibodies to detect Mycoplasma californicum was investigated in an antigen capture microtitre format. The finalized test was highly specific and no cross-reactions were detectable with any of the mastitis associated mycoplasma or bacterial antigens tested. Using a concentration step involving centrifugation, the sensitivity of the test could be improved from 10(5)-10(7) to 10(3)-10(5) colony forming units per ml with pure broth cultures, and from 10(7) to at least 10(6) colony forming units per ml in milk samples from two experimentally infected cows. The antigen detected was partially identified by immunoblotting, which demonstrated two polypeptides of 40 and 46 kD.

Twelve strains of M. bovigenitalium and two of three strains of M. canadense caused an infection resulting in a pathogenic effect when experimentally inoculated into the ovine mammary gland. Differences in the pathogenesis were quantified by the duration of continuous mycoplasma excretion and the duration of high milk cell levels, but variation in the susceptability of the experimental animals prevented the establishment of firm conclusions on the relative virulence of the strains. Seven M. bovigenitalium and two M. canadense strains were eventually eliminated naturally from the infected glands, but four M. bovigenitalium strain infections ultimately became sub-clinical with intermittent mycoplasma excretion and low milk cell levels.

In a survey of 332 fresh and 137 processed bovine semen samples and 25 preputial washes, mycoplasmas and, or, ureaplasmas were isolated from 46 per cent, 31 per cent and 80 per cent, respectively. Intermittent isolation from different semen collections from the same bull indicated that at least three collections per bull were necessary to determine whether infection was present. When stored processed samples were examined Mycoplasma canadense and M bovigenitalium were isolated from straws taken as long ago as 1975. Addition of lincomycin and spectinomycin to the semen extender eliminated the isolation of mycoplasmas and reduced the rate of isolation of ureaplasmas.

A mycoplasma-like organism was isolated from five cows with chronic incurable mastitis from one dairy herd. This organism was shown to be Mycoplasma californicum and was used to infect three cows by intramammary inoculation. The challenge organism was reisolated from all the infected quarters and the ensuing inflammatory response produced a chronic mastitis with a permanent drop in milk yield. Antibiotic treatment in one animal was unsuccessful.

The aminopeptidase activity of arginine-utilizing mycoplasmas was investigated with 20 aminoacyl beta-naphthylamide substrates. High levels of arginyl-beta-naphthylamide hydrolysis were demonstrated in 6 of 11 species when extracts of concentrated washed organisms were used. Relatively low arginine aminopeptidase activity was demonstrated with similar extracts from 22 species not utilizing arginine. The high level of arginine aminopeptidase activity could also be demonstrated with unwashed, unconcentrated samples of the same 6 species and also with Mycoplasma arthritidis. The procedure for preparing the extract of M. arthritidis appeared to remove the arginine aminopeptidase activity which was demonstrated to be present in the untreated culture. Fluorogenic and chromogenic tests were developed whereby this distinctive arginine aminopeptidase activity could be demonstrated within 4 h with the use of small volumes of broth culture (10 microliter) or single colonies, thus providing a rapid test for early characterization of some Mycoplasma species.

Comparison of fluorocarbon-extracted antigens of six mycoplasma species by double immunodiffusion and counterimmunodiffusion techniques revealed a close reciprocal relationship among Mycoplasma dispar, M. ovipneumoniae, and M. hyopneumoniae. A lesser degree of cross-reaction was also demonstrated between these three species and M. hyorhinis and M. bovoculi. The interrelationships were more clearly demonstrated by double immunodiffusion than by counterimmunodiffusion.

Aminopeptidase activity was demonstrated in extracts of Acholeplasma laidlawii, Mycoplasma bovirhinis, M. bovis and M. dispar. The enzyme specificity, which differed amongst the mycoplasma species examined, was characterized using 19 aminoacyl-beta-naphthylamides as substrates. The differences were detected after 4 h incubation with the substrates and potentially could be employed to aid in the characterization of certain mycoplasma species.

In an examination of arginine-utilizing Mycoplasma species, simple fluorogenic and chromogenic tests were used to demonstrate high levels of arginine aminopeptidase activity in unwashed, unconcentrated broth and agar cultures in all strains of 5 species and low levels of activity in all strains of 14 species. Four species contained strains which exhibited either high or low levels of activity.

Samples from the mammary tissue of 14 lactating goats (12 naturally infected and two experimentally infected) were examined for the presence of Mycoplasma agalactiae . A monoclonal antibody (5G12) was applied to formalin-fixed, paraffin-wax-embedded sections and labelled by the avidin–biotin peroxidase complex (ABC) method. Histological examination of tissue sections revealed strong immunoreactivity in all animals included in the study. Mycoplasma agalactiae antigen was mainly detected in the cellular debris at the periphery of purulent exudates present within lactiferous sinuses, and lactiferous and interlobular ducts. In addition, M. agalactiae organisms appeared in the cytoplasm of the epithelium of ducts, and in infiltrating macrophages and neutrophils within the ducts, alveoli, interstitial tissue and regional lymph node sinuses. It is concluded that this monoclonal antibody-based immunohistochemical technique is an efficient and specific method for the post-mortem detection of M. agalactiae in cases of clinical mastitis as well as being a useful tool for the study of the route of infection and cellular types involved during mastitis caused by this organism. [ABSTRACT FROM AUTHOR]