Isolation of Mycoplasma and Ureaplasma species from raccoon dogs (Nyctereutes procyonoides viverrinus)

Mycoplasma spp. were isolated from five wild raccoon dogs (Nyctereutes procyonoides viverrinus). On the basis of biochemical properties and serological tests, nine isolates were identified as Mycoplasma edwardii and four were similar to a possibly new Mycoplasma sp. represented by strain LM2 which is negative for both glucose fermentation and arginine hydrolysis. In addition, ureaplasmas were detected from these animals. Ureaplasmas were compared serologically with ureaplasma strains isolated from human, monkey, cattle, goat, sheep, cat, chicken and dog and cross-reacted with one of four serological groups of canine ureaplasmas.     

A Microbiological Study of Pneumonic Calf Lungs

BITSCH, V., N. F. FRIIS and H. V. KROGH: A microbiological study of pneumonic calf lungs. Acta vet. scand. 1976, 17, 32–42.–Fifty pneumonic calf lungs were subjected to microbiologic screening with regard to bacteria, mycoplasmas, and viruses. Of bacteria the species most commonly found were Pasteurella multocida (eight lungs), Pasteurella hemolytica (eight lungs), and Corynebacterium pyogenes (13 lungs). Of special interest was the demonstration of Neisseria spp. in five lungs. Mycoplasma dispar was found in 31 lungs, Mycoplasma bovirhinis in 16 lungs, and Urea-plasma in 26 lungs. Cytopathogenic agents were demonstrated in 14 lungs. Four isolates were found to be bovine respiratory syncytial virus, three were bovine viral diarrhea virus, and two were bovine parainfluenza 3 virus. The remaining five cytopathogenic agents were not identified.     

Genetic Differentiation by Nucleic Acid Homology II. Genotypic Variations Within Two Mycoplasma Species

Somerson, Norman L. (National Institutes of Health, Bethesda, Md.), Paul R. Reich, Barbara E. Walls, Robert M. Chanock, and Sherman M. Weissman. Genetic differentiation by nucleic acid homology. II. Genotypic variations within two Mycoplasma species. J. Bacteriol. 92:311-317. 1966.-A deoxyribonucleic-ribonucleic acid (DNA-RNA) homology technique was used to determine genetic relatedness among the nucleic acids of eight mycoplasmas which were serologically classified as Mycoplasma hominis type 1. The DNA preparations from these organisms were each found to be distinct. No subgrouping of the M. hominis type 1 strains could be demonstrated. In contrast, when the nucleic acids from six serologically related mycoplasmas which were isolated from tissue cultures were studied, the DNA from these species could not be distinguished. The DNA buoyant densities of the tissue culture isolates were similar. These isolates were closely related genetically to a porcine mycoplasma, M. hyorhinis.     

Isolation of a Mycoplasma sp. (Group 7) Previously Associated with Cattle from Guinea Pigs'Genital Tracts

The incidence and persistence of genital mycoplasmas of 25 normal guinea pigs were investigated by culturing swabs collected over a 63-day period. Mycoplasmas were consistently isolated from two of the five males, but only once each from three of the 20 females. The 16 strains isolated were divided into four groups. Representative strains of one group were identified as Mycoplasma sp. (group 7 of Leach 1973), by growth inhibition, growth precipitation and fluorescent antibody tests. The other groups were provisionally classified as Mycoplasma spp. and an Acholeplasma sp.     

Characterization of Canine Mycoplasmas by Polyacrylamide Gel Electrophoresis and Immunodiffusion

Canine mycoplasmas which had been characterized by biological and serological methods were further studied by using polyacrylamide gel electrophoresis (PGE) and double diffusion in agar gel. The three dog mycoplasmas previously characterized, Mycoplasma canis, M. maculosum, and M. spumans showed distinctive patterns by PGE. Five additional representative isolates from dogs had been characterized serologically and biologically into three new groups, A, C, and D. An additional mycoplasma (group B) was indistinguishable from M. canis by growth inhibition and PGE but was more broadly reactive with field isolates serologically. The group A organisms were distinctive in pattern and similar to those studied by Razin and Rottem, tentatively designated M. edwardii. The group C organisms were represented by two isolates which were similar by fluorescent-antibody studies but different by growth inhibition tests. These two isolates were also different from each other by PGE. The group D serotypes were also distinctive by PGE from all other dog mycoplasmas tested. It was found, during these studies, that two different mycoplasmas showed different PGE patterns at different intervals during incubation. Immunodiffusion studies showed a relationship among all the canine mycoplasmas, and bands of nonidentity between the two group C mycoplasmas were demonstrated.     

Variations in the surface proteins and restriction enzyme systems of Mycoplasma pulmonis in the respiratory tract of infected rats

Restriction and modification (R-M) systems are generally thought to protect bacteria from invasion by foreign DNA. This paper proposes the existence of an alternative role for the phase-variable R-M systems encoded by the hsd loci of Mycoplasma pulmonis. Populations of M. pulmonis cells that arose during growth in different environments were compared with respect to R-M activity and surface antigen production. When M. pulmonis strain X1048 was propagated in laboratory culture medium, > 95% of colony-forming units (cfu) lacked R-M activity and produced the variable surface protein VsaA. Mycoplasmas isolated from the nose of experimentally infected rats also lacked R-M activity and produced VsaA. In contrast, the cell population of mycoplasmas isolated from the lower respiratory tract of the infected rats was more complex. The most dramatic results were obtained for mycoplasmas isolated from the trachea. At 14 days postinfection, 38% of mycoplasma isolates produced a Vsa protein other than VsaA, and 34% of isolates had active restriction systems. These data suggest that differences in selection pressures in animal tissues affect the surface proteins and the R-M activity of the mycoplasmal cell population. We propose that variations in the production of R-M activity and cell surface proteins are important for the survival of the mycoplasma within the host.     

Mycoplasmas Isolated from the Respiratory Tract of Cattle and Goats in Tanzania

A microbiological study of the mycoplasma flora in the respiratory tracts of cattle and goats in selected regions of Tanzania is described. In the examination of cattle, mycoplasmas were isolated from 60 (17.8%) of the 338 examined lung samples, 8 (47.1%) of the 17 lymph nodes, 4 (13.3%) of the 30 pleural fluid samples and 4 (3.9%) of the 103 nasal swabs examined. All the isolates were identified as Mycoplasma mycoides subsp. mycoides, Small Colony type except for one isolate from pleural fluid which was identified as Mycoplasma arginini. M. mycoides subsp. mycoides, Small Colony type was isolated from samples originating from Dodoma, Iringa, Mbeya, Morogoro and Shinyanga regions where outbreaks of contagious bovine pleuropneumonia had been reported. In the examination of goats, mycoplasmas were isolated from 54 (34.0%) of the 159 examined lung samples, 41 (18.1%) of the 226 nasal swabs and 4 (40.0%) of the 10 pleural fluid samples. The species demonstrated were Mycoplasma capricolum subsp. capripneumoniae, M. mycoides subsp. mycoides, Small Colony type Mycoplasma ovipneumoniae and M. Capricolum subsp. arginini. The isolation of M. capripneumoniae in the Coast and Morogoro regions confirmed the presence of contagious caprine pleuropneumonia in the regions.     

Do mycoplasmas cause human cancer?

A linkage between mycoplasmas and malignancy was mainly proposed in the 1960s when human-associated mycoplasmas were becoming of interest given the novel characterization of the human respiratory pathogen Mycoplasma pneumoniae. Associations with leukemia and other malignancies, however, were largely ascribed to tissue-culture contamination, which is now recognized as a significant potential problem in molecular biology circles. A few epidemiological studies, however, continue to raise concern over such a linkage. As well, in vitro data have demonstrated the potential for some mycoplasmas to induce karyotypic changes and malignant transformation during chronic tissue-culture infestation. As cellular and molecular mechanisms for such transformation become studied, a resurgence of interest in this area is inevitable. A role for mycoplasmas in malignancy of any sort is conjectural, but there remains a need to continue with focussed epidemiological and laboratory investigations.     

Isolation and characterization of unusual Mycoplasma spp. from captive Eurasian Griffon (Gyps fulvus) in Sicily

Mycoplasmas have been isolated from birds of prey during clinical examinations, but their significance to the health of raptors is unclear. We report the isolation and characterization of four mycoplasmas found in the upper respiratory tract of four sick Eurasian Griffon (Gyps fulvus) that were housed in a Sicilian rehabilitation center at Ficuzza, near Palermo in Sicily, before reintroduction into the wild. These included Mycoplasma gallinarum, an unidentified mycoplasma highly similar to Mycoplasma glycophilum, and two unidentified mycoplasmas with similarities to Mycoplasma falconis and Mycoplasma gateae.     

Mycoplasma species isolated from harbor porpoises (Phocoena phocoena) and a Sowerby's beaked whale (Mesoplodon bidens) stranded in Scottish waters

Mycoplasma species were recovered from 10 cetacean carcasses that stranded around Scotland. Mycoplasma phocicerebrale was isolated from the lungs of three harbor porpoises (Phocoena phocoena) as well as from the liver of one of these animals. Novel Mycoplasma spp. were isolated from the lungs of five additional harbor porpoises and the kidney of another. In addition an isolate closely related to Mycoplasma species 13CL was obtained from the kidney of a Sowerby's beaked whale (Mesoplodon bidens). The role of these Mycoplasma species in the disease of cetaceans, their host specificity, diversity, and any relation to cetacean strandings are unknown.     

Detection and characterization of Mycoplasma spp. and Salmonella spp. in free-living European tortoises (Testudo hermanni, Testudo graeca, and Testudo marginata)

Free-living and captive chelonians might suffer from upper respiratory tract disease (URTD), a pathology primarily caused by Mycoplasma agassizii. Wild tortoises can also be an important reservoir of Salmonella spp., which are commensal in the host reptile but are potential zoonotic agents. Between July 2009 and June 2010, we screened free-living European tortoises (spur-thighed tortoises Testudo graeca, Hermann's tortoises Testudo hermanni, marginated tortoises Testudo marginata) temporarily housed in a wildlife center in Italy. We molecularly characterized 13 Mycoplasma isolates detected in all Testudo spp. studied, and three PCR-positive animals showed typical URTD clinical signs at the time of sampling. Three Salmonella enterica serotypes (Abony, Potsdam, Granlo), already related to reptile-associated human infections, were also identified. These results highlight the potential role played by wildlife recovery centers in the spread and transmission of pathogens among wild chelonians and to humans.     

Molecular identification of Mycoplasma cynos from laboratory beagle dogs with respiratory disease

In this study, we examined a colony of 20 beagle dogs in a laboratory animal facility. Mycoplasma was detected by consensus PCR assay in 1 dog with respiratory and constitutional symptoms. None of the other dogs were affected. The dog was euthanized and necropsied. In postmortem examinations, gray or plum-colored gross lesions were found on the lung, most commonly in the apical and cardiac lobes. Some lesions showed clear demarcation and consolidation. Microscopic examination showed peribronchiolar lymphoid hyperplasia and interstitial thickening, lesions pathognomonic for mycoplasma pneumonia. To identify canine Mycoplasma species, we used species-specific PCR reactions for M. arginini, M. canis, M. cynos, M. edwardii, M. felis, M. gateae, M. maculosum, M. molare, M. opalescens, M. spumans, Mycoplasma sp. HRC 689, and M. collis. As the result, we identified Mycoplasma cynos by amplification of DNA extracted from lung tissue of the laboratory beagle dog with respiratory disease.     

Real-time PCR investigation of potential vectors, reservoirs, and shedding patterns of feline hemotropic mycoplasmas

Three hemotropic mycoplasmas have been identified in pet cats: Mycoplasma haemofelis, "Candidatus Mycoplasma haemominutum," and "Candidatus Mycoplasma turicensis." The way in which these agents are transmitted is largely unknown. Thus, this study aimed to investigate fleas, ticks, and rodents as well as saliva and feces from infected cats for the presence of hemotropic mycoplasmas, to gain insight into potential transmission routes for these agents. DNA was extracted from arthropods and from rodent blood or tissue samples from Switzerland and from salivary and fecal swabs from two experimentally infected and six naturally infected cats. All samples were analyzed with real-time PCR, and some positive samples were confirmed by sequencing. Feline hemotropic mycoplasmas were detected in cat fleas and in a few Ixodes sp. and Rhipicephalus sp. ticks collected from animals but not in ticks collected from vegetation or from rodent samples, although the latter were frequently Mycoplasma coccoides PCR positive. When shedding patterns of feline hemotropic mycoplasmas were investigated, "Ca. Mycoplasma turicensis" DNA was detected in saliva and feces at the early but not at the late phase of infection. M. haemofelis and "Ca. Mycoplasma haemominutum" DNA was not amplified from saliva and feces of naturally infected cats, despite high hemotropic mycoplasma blood loads. Our results suggest that besides an ostensibly indirect transmission by fleas, direct transmission through saliva and feces at the early phase of infection could play a role in the epizootiology of feline hemotropic mycoplasmas. Neither the investigated tick nor the rodent population seems to represent a major reservoir for feline hemotropic mycoplasmas in Switzerland.     

Fulminant Mycoplasma pneumoniae pneumonia.

The frequency of fulminant pneumonia due to Mycoplasma pneumoniae is relatively rare despite the high prevalence of Mycoplasma species infection in the general population. We recently encountered such a case and have reviewed the English-language literature on cases of M pneumoniae pneumonia that have resulted in respiratory failure or death. Due to host factors or on epidemiologic grounds, fulminant cases seem to be more common in young healthy adults, in males, and possibly in smokers among the 46 patients we found. An enhanced host cellular immune response may be responsible for the development of severe cases. A spectrum of small airways disease is characteristic, including cellular bronchiolitis and bronchiolitis obliterans with and without organizing pneumonia. Based largely on anecdotal experience, corticosteroid use may be salutary in patients with respiratory failure. For reasons that are not well known, the incidence of pulmonary thromboembolism is increased in fatal cases.     

Clinical and microbiologic characterization of hemorrhagic pneumonia due to extraintestinal pathogenic Escherichia coli in four young dogs

Over a 21-month period, three Beagle dogs and one mixed-breed dog at our facility developed fatal pneumonia. The four dogs, all purpose bred, came from three vendors and had received the standard canine vaccines prior to shipment. In each instance, the affected dog had been shipped to our facility within the past 10 days. Three cases presented as a peracute clinical syndrome, and all had gross and microscopic findings consistent with hemorrhagic pneumonia. Escherichia coli was isolated from the lungs of all four dogs. Results of testing of lung tissue for canine parainfluenza virus and canine adenovirus were negative. Escherichia coli was also isolated from blood of three of the four dogs. Serotyping of the E. coli isolates indicated that two were serotype 06 and two were 04. Isolates from all four dogs were positive for the virulence factors alpha hemolysin and cytotoxic necrotizing factor 1 and for the adhesin factor class-III papG allele. These traits place the isolates in the class of extraintestinal pathogenic E. coli, which is being increasingly implicated as a cause of extraintestinal infections in animals and humans and may represent a zoonotic risk to humans working with research dogs.     

Detection of Mycoplasma ovipneumoniae and M. arginini in bighorn sheep using enrichment culture coupled with genus- and species-specific polymerase chain reaction

Mycoplasma species are of interest as possible primary pathogens in the pneumonia complex of bighorn sheep (Ovis canadensis). Previous investigations have not commonly detected low frequencies of Mycoplasma spp. from free-ranging bighorn sheep, possibly due to the fastidious and slow growth of these organisms. We developed a culture protocol that employed an average initial 3-day enrichment culture in liquid Hayflick broth in a CO(2)-enhanced atmosphere. The broth was plated to solid Hayflick medium and the cultures observed for growth for up to 30 days. Polymerase chain reaction (PCR) was performed on DNA isolated from the enrichment broth and on isolates obtained from culture using Mycoplasma genus-specific PCR assays and species-specific PCR assays for M. arginini and M. ovipneumoniae. Some cultures that grew on Hayflick plates were picked as single colonies but were mixed because two organisms may grow together and appear as a single colony. Culture and PCR tests produced similar results for M. arginini, but for M. ovipneumoniae, culture alone was less accurate than PCR. Use of genus-specific primers also may allow detection of other species in samples negative for M. arginini and M. ovipneumoniae. Two methods of transport from field to laboratory (Port-a-Cul? tubes, cryoprotectant in liquid N(2) and Fisher Transport System) gave similar results under our study conditions.     

Lesions associated with a novel Mycoplasma sp. in California sea lions (Zalophus californianus) undergoing rehabilitation

From July 1999 to November 2001, Mycoplasma sp. was cultured from lesions in 16 California sea lions (Zalophus californianus) undergoing rehabilitation. The Mycoplasma sp. was the likely cause of death of four animals in which it was associated with either pneumonia or polyarthritis. The most common lesion associated with this bacterium was subdermal abscessation, found in 12 animals. Other lesions included intramuscular abscesses, septic arthritis, and lymphadenopathy. Infection was associated with a leukocytosis and left shift in 12 animals. Animals with abscesses improved clinically after surgical lancing, irrigation, and systemic antibiotic therapy. The mycoplasma isolates had a consistent 16S rRNA sequence dissimilar from other Mycoplasma spp. and represent a novel species, Mycoplasma zalophi proposed sp. nov.     

Potential pathogens carried by Spanish ibex (Capra pyrenaica hispanica) in southern Spain

The Spanish ibex (Capra pyrenaica hispanica) population of southern Spain was surveyed for potential pathogens associated with the conjunctiva, external ear canal, as well as reproductive and upper respiratory tracts. We sampled 321 ibex (131 adult males, 100 adult females, and 90 yearlings); these included 271 apparently healthy animals and 50 that were naturally infected with Sarcoptes scabiei. A total of 688 bacterial isolates were identified (377 gram-negatives, 225 gram-positives, and 86 Mycoplasma spp.); sex, age, location, infection with S. scabiei, and disposition of the animal (free-ranging versus captive) were evaluated as risk factors for infection. Infections with Mycoplasma agalactiae and Mycoplasma arginini were associated with age, having a higher frequency of isolation in young animals. With Escherichia coli, Mannheimia haemolytica, Pasteurella multocida biotype A, and Staphylococcus aureus, significantly higher isolation rates were associated with adults. The isolation frequency for E. coli was higher in females, whereas Moraxella bovis isolations were mostly associated with males. The presence of mange increased the risk of infection with both Streptococcus equi subsp. zooepidemicus and M. haemolytica. The geographic origin of sampled animals was related to the isolation of Branhamella ovis, M. agalactiae, and all Pasteurella sp. Isolations of M. haemolytica, P. multocida biotype A, E. coli, and B. ovis were more prevalent in samples from free-ranging rather than captive animals. Of the gram-positive bacteria, S. aureus represented the predominant species isolated from nasal, vaginal, and ocular samples. Mycoplasma agalactiae and M. arginini were the predominant Mycoplasma spp., and both were associated most often with the external ear canal. The most frequently isolated gram-negative bacteria included E. coli, M. haemolytica, P. multocida biotype A, and B. ovis. Isolation rates of gram-negative species varied by source. In nasal samples, M. haemolytica and P. multocida biotype A were isolated most frequently, whereas in ocular and vaginal samples, B. ovis and E. coli, respectively, were most frequently isolated.     

One Test Microbial Diagnostic Microarray for Identification of Mycoplasma mycoides subsp. mycoides and Other Mycoplasma Species

The present study describes the use of microarray technology for rapid identification and differentiation of Mycoplasma mycoides subsp. mycoides from other mycoplasmas that may be pathogenic to ruminants, including those of the Mycoplasma mycoides cluster, genetically and antigenically strictly correlated with Mycoplasma mycoides subsp. mycoides. A microarray containing genetic sequences of 55 different bacterial species from Acholeplasma, Mycoplasma, Spiroplasma and Ureaplasma genera was constructed. Sequences to genes of interest were collected in FASTA format from NCBI. The collected sequences were processed with OligoPicker software. Oligonucleotides were then checked for their selectivity with BLAST searches in GenBank. The microarray was tested with ATCC/NCTC strains of Mycoplasma spp. of veterinary importance in ruminants including Mycoplasma belonging to the mycoides cluster as well as Mycoplasma mycoides subsp. mycoides and Mycoplasma mycoides subsp. capri field strains. The results showed that but one ATCC/NCTC reference strains hybridized with their species-specific sequences showed a profile/signature different and distinct from each other. The heat-map of the hybridization results for the nine genes interrogated for Mycoplasma mycoides subsp. mycoides demonstrated that the reference strain Mycoplasma mycoides subsp mycoides PG1 was positive for all of the gene sequences spotted on the microarray. CBPP field, vaccine and reference strains were all typed to be M. mycoides subsp. mycoides, and seven of the nine strains gave positive hybridization results for all of the nine genes. Two Italian strains were negative for some of the genes. Comparison with non-Mycoplasma mycoides subsp. mycoides reference strains showed some positive signals or considerable homology to Mycoplasma mycoides subsp. mycoides genes. As expected, some correlations were observed between the strictly genetically and antigenically correlated Mycoplasma mycoides subsp. mycoides and Mycoplasma mycoides subsp. capri strains. Specifically, we observed that some Italian Mycoplasma mycoides subsp. mycoides strains were positive for two out of the three Mycoplasma mycoides subsp. capri genes, differently from what has been observed for other European or African Mycoplasma mycoides subsp. mycoides strains. This study highlighted the use of microarray technology as a simple and effective method for a single-step identification and differentiation of Mycoplasma mycoides subsp. mycoides from other mycoplasmas that may be pathogenic to ruminants, including those of the Mycoplasma mycoides cluster, genetically and antigenically strictly correlated with Mycoplasma mycoides subsp. mycoides. The opportunity to discriminate several mycoplasmas in a single analysis enhances diagnostic rapidity and may represent a useful tool to screen occasionally mycoplasmas affecting animal farming in territories where diagnostic laboratory support is limited. The heat-map of the hybridization results of the comparative genomic hybridizations DNA-designed chip clearly indicates that the microarray performs well for the identification of the tested Mycoplasma mycoides subsp. mycoides reference and field strains, discriminating them from other mycoplasmas.     

An epizootic of Mycoplasma ovipneumoniae infection in captive Dall's sheep (Ovis dalli dalli)

In late spring of 1986, 10 of 23 Dall's sheep (Ovis dalli dalli) at the Metropolitan Toronto Zoo were moved to a new exhibit, where all developed severe respiratory signs refractory to anthelmintic and antibiotic therapy. In July, two animals died with chronic active bronch-pneumonia, and a third was euthanized because of pneumonia several months later. Bacteria were not isolated from the lungs of the first, steptococci and Pasteurella hemolytica were isolated from the other two, respectively; Mycoplasma ovipneumoniae was isolated from both. Pulmonary lesions in all three sheep were consistent with Mycoplasma sp. infection. Nasal swabs of the remaining animals yielded no consistent bacterial isolates; however, four of eight sheep were positive for M. ovipneumoniae. Viral cultures yielded an as yet unidentified herpesvirus. Sheep in the original and new herds had no serologic titers to parainfluenza-3, equine viral rhinopneumonitis, or infectious bovine rhinotracheitis, and had variable titers against bovine respiratory syncytial virus. No titers against M. ovipneumoniae were present in 13 sheep still in the original exhibit, but titers varied from 1:32 to 1:256 in eight pneumonic sheep. Sera taken from three sheep before or early in the outbreak were all negative for antibody to M. ovipneumoniae. Two of the affected Dall's sheep had been in contact with domestic sheep in the winter of 1985-1986, and M. ovipneumoniae was subsequently cultured from the domestic flock. Exposure to a new pathogen, and environmental and social stress in a new exhibit may have resulted in this severe disease in Dall's sheep.