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1.  Susceptibility of goats and calves after experimental inoculation or contact exposure to a Canadian strain of Mycoplasma mycoides subsp. mycoides isolated from a goat. 
Transmissibility of Mycoplasma mycoides subsp. mycoides infection from experimentally inoculated goats to other goats and calves was studied. Eight goats and six calves were housed in an 18 m2 room. Six of the goats were inoculated endobronchially with strain D44 isolated from a natural case of polyarthritis in Ontario. These six goats died within a week of Mycoplasma septicemia. The two contact goats or the six calves never showed signs of disease and M. mycoides subsp. mycoides was not recovered from these animals. The contact goats and four calves were killed 25 days after exposure. They were all seronegative, M. mycoides subsp. mycoides was not recovered at necropsy and none had pathomorphological changes attributable to this Mycoplasma. The two remaining calves were inoculated endobronchially with 10(9) CFU of strain D44 and observed for 20 days. They never showed signs of disease and did not have significant lesions at necropsy. Both developed a significant serological response to M. mycoides subsp. mycoides, although this organism was not recovered during the experimental period or at necropsy. This study did not provide evidence for transmission of M. mycoides subsp. mycoides from endobronchially inoculated goats to contact goats or calves and endobronchially inoculated calves did not develop pneumonia. This would suggest that the infection of the goat population in Canada with this pathogen would not be a significant threat to the cattle population.
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PMCID: PMC1235981  PMID: 6365296
2.  Epizootic Pneumonia of Bighorn Sheep following Experimental Exposure to Mycoplasma ovipneumoniae 
PLoS ONE  2014;9(10):e110039.
Background
Bronchopneumonia is a population limiting disease of bighorn sheep (Ovis canadensis). The cause of this disease has been a subject of debate. Leukotoxin expressing Mannheimia haemolytica and Bibersteinia trehalosi produce acute pneumonia after experimental challenge but are infrequently isolated from animals in natural outbreaks. Mycoplasma ovipneumoniae, epidemiologically implicated in naturally occurring outbreaks, has received little experimental evaluation as a primary agent of bighorn sheep pneumonia.
Methodology/Principal Findings
In two experiments, bighorn sheep housed in multiple pens 7.6 to 12 m apart were exposed to M. ovipneumoniae by introduction of a single infected or challenged animal to a single pen. Respiratory disease was monitored by observation of clinical signs and confirmed by necropsy. Bacterial involvement in the pneumonic lungs was evaluated by conventional aerobic bacteriology and by culture-independent methods. In both experiments the challenge strain of M. ovipneumoniae was transmitted to all animals both within and between pens and all infected bighorn sheep developed bronchopneumonia. In six bighorn sheep in which the disease was allowed to run its course, three died with bronchopneumonia 34, 65, and 109 days after M. ovipneumoniae introduction. Diverse bacterial populations, predominantly including multiple obligate anaerobic species, were present in pneumonic lung tissues at necropsy.
Conclusions/Significance
Exposure to a single M. ovipneumoniae infected animal resulted in transmission of infection to all bighorn sheep both within the pen and in adjacent pens, and all infected sheep developed bronchopneumonia. The epidemiologic, pathologic and microbiologic findings in these experimental animals resembled those seen in naturally occurring pneumonia outbreaks in free ranging bighorn sheep.
doi:10.1371/journal.pone.0110039
PMCID: PMC4193846  PMID: 25302992
3.  Mycoplasma ovipneumoniae - A Primary Cause of Severe Pneumonia Epizootics in the Norwegian Muskox (Ovibos moschatus) Population 
PLoS ONE  2014;9(9):e106116.
The Norwegian muskox (Ovibos moschatus) population lives on the high mountain plateau of Dovre and originates from animals introduced from Greenland. In the late summers of 2006 and 2012, severe outbreaks of pneumonia with mortality rates of 25-30% occurred. During the 2012 epidemic high quality samples from culled sick animals were obtained for microbiological and pathological examinations. High throughput sequencing (pyrosequencing) of pneumonic lung tissue revealed high concentrations of Mycoplasma ovipneumoniae in all six animals examined by this method and Pasteurella multocida subsp. multocida in four animals, whereas no virus sequences could be identified. Mycoplasma ovipneumoniae and P. multocida multocida were also isolated by culture. Using real time PCR on lung swabs, M. ovipneumoniae was detected in all of the 19 pneumonic lungs examined. Gross pathological examination revealed heavy consolidations primarily in the cranial parts of the lungs and it also identified one case of otitis media. Histologically, lung lesions were characterized as acute to subacute mixed exudative and moderately proliferative bronchoalveolar pneumonia. Immunohistochemical (IHC) examination revealed high load of M. ovipneumoniae antigens within lung lesions, with particularly intensive staining in the neutrophils. Similar IHC finding were observed in archived lung tissue blocks from animals examined during the 2006 epidemic. An M. ovipneumoniae specific ELISA was applied on bio-banked muskox sera from stray muskoxen killed in the period 2004–2013 and sick muskoxen culled, as well as sera from wild reindeer (Rangifer tarandus tarandus) on Dovre and muskoxen from Greenland. Serology and mycoplasma culturing was also carried out on sheep that had been on pasture in the muskox area during the outbreak in 2012. Our findings indicated separate introductions of M. ovipneumoniae infection in 2006 and 2012 from infected co-grazing sheep. Salt licks shared by the two species were a possible route of transmitting infection.
doi:10.1371/journal.pone.0106116
PMCID: PMC4157772  PMID: 25198695
4.  Safety and Immunogenicity of a Mycoplasma ovipneumoniae Bacterin for Domestic Sheep (Ovis aries) 
PLoS ONE  2014;9(4):e95698.
Background
Mortality from epizootic pneumonia is hindering re-establishment of bighorn sheep populations in western North America. Mycoplasma ovipneumoniae, a primary agent of this disease, is frequently carried asymptomatically by the domestic sheep and goats that constitute the reservoir of this agent for transmission to bighorn sheep. Our long-term objective is to reduce the risk of M. ovipneumoniae infection of bighorn sheep; one approach to this objective is to control the pathogen in its reservoir hosts.
Methods
The safety and immunogenicity of M. ovipneumoniae for domestic sheep was evaluated in three experimental immunization protocols: 1) live M. ovipneumoniae (50 ug protein); 2) killed M. ovipneumoniae (50 ug whole cell protein) in oil adjuvant; and 3) killed M. ovipneumoniae (250 ug whole cell protein) in oil adjuvant. Immunogenicity was assessed by two serum antibody measures: competitive enzyme-linked immunosorbent assay (cELISA) (experiments 1–3) and serum growth inhibition (Experiment 3). Passive immunogenicity was also assessed in the third experiment using the same assays applied to blood samples obtained from the lambs of immunized ewes.
Results and Conclusions
Adverse reactions to immunization were generally minor, but local reactions were regularly observed at immunization sites with bacterins in oil adjuvants. No evidence of M. ovipneumoniae specific antibody responses were observed in the first or second experiments and no resistance to colonization was observed in the first experiment. However, the ewes in the third experiment developed strong cELISA serum antibody responses and significant serum M. ovipneumoniae inhibition activity, and these responses were passively transferred to their lambs. The results of these trials indicate that immunization with relatively large antigenic mass combined with an adjuvant is capable of inducing strong active antibody responses in ewes and passively immunizing lambs.
doi:10.1371/journal.pone.0095698
PMCID: PMC3994082  PMID: 24752006
5.  Causes of Pneumonia Epizootics among Bighorn Sheep, Western United States, 2008–2010 
Emerging Infectious Diseases  2012;18(3):406-414.
Mycoplasma ovipneumoniae is a primary pathogen.
Epizootic pneumonia of bighorn sheep is a devastating disease of uncertain etiology. To help clarify the etiology, we used culture and culture-independent methods to compare the prevalence of the bacterial respiratory pathogens Mannheimia haemolytica, Bibersteinia trehalosi, Pasteurella multocida, and Mycoplasma ovipneumoniae in lung tissue from 44 bighorn sheep from herds affected by 8 outbreaks in the western United States. M. ovipneumoniae, the only agent detected at significantly higher prevalence in animals from outbreaks (95%) than in animals from unaffected healthy populations (0%), was the most consistently detected agent and the only agent that exhibited single strain types within each outbreak. The other respiratory pathogens were frequently but inconsistently detected, as were several obligate anaerobic bacterial species, all of which might represent secondary or opportunistic infections that could contribute to disease severity. These data provide evidence that M. ovipneumoniae plays a primary role in the etiology of epizootic pneumonia of bighorn sheep.
doi:10.3201/eid1803.111554
PMCID: PMC3309594  PMID: 22377321
animal diseases; etiology; Mycoplasma ovipneumoniae; pulsed-field gel electrophoresis; ribosomal spacer DNA; bacteria; pneumonia; epizootic; bighorn sheep
6.  Identification by culture, PCR, and immunohistochemistry of mycoplasmas and their molecular typing in sheep and lamb lungs with pneumonia in Eastern Turkey 
This study used cultures, polymerase chain reaction (PCR), and immunoperoxidase to examine samples from 216 lungs from sheep and lambs with macroscopic pneumonia lesions for the presence of Mycoplasma species. DNA was extracted from lung tissue samples and broth cultures with the help of a DNA extraction kit and replicated using genus-specific and species-specific primers for mycoplasma. The lung samples were examined by the immunoperoxidase method using hyperimmune Mycoplasma ovipneumoniae serum. The randomly amplified polymorphic DNA (RAPD) test was used for the molecular typing of M. ovipneumoniae isolates. Mycoplasma was isolated in the cultures of 80 (37.03 %) of a total of 216 lung samples. Genus-specific mycoplasma DNA was identified by PCR in 96 (44.44 %) samples in broth cultures and 36 (16.66 %) directly in the lung tissue. Of these 96 cases in which genus-specific identification was made, 57 (59.37 %) were positive for reaction with species-specific primers for M. ovipneumoniae and 31 (32.29 %) for Mycoplasma arginini. The DNA of neither of the latter two species could be identified in the remaining eight samples (8.33 %) where mycoplasma had been identified. As for the immunoperoxidase method, it identified M. ovipneumoniae in 61 of 216 lung samples (28 %). Positive staining was concentrated in the bronchial epithelium cell cytoplasm and cell surface. RAPD analysis resulted in 15 different profiles. Our results suggest that PCR methods could be successfully used in the diagnosis of mycoplasma infections as an alternative to culture method and identifying this agent at the species level.
doi:10.1007/s11250-013-0394-3
PMCID: PMC3776281  PMID: 23494576
M. ovipneumoniae; PCR; Culture; Pneumonia; Sheep; Lamb
7.  Mycoplasmas isolated from the respiratory tract of horses. 
The Journal of Hygiene  1975;74(3):385-407.
Ten mycoplasmas were isolated from 130 nasopharyngeal swabs from thoroughbred horses with acute respiratory disease and three from 198 apparently normal horses. Two mycoplasmas were isolated from 21 tracheal swabs taken at necropsy. These mycoplasmas, together with six isolated from the equine respiratory tract by other workers, were subjected to biochemical and serological tests. Other properties examined in certain representative strains were appearance under the electron microscope, ability to adsorb or agglutinate the erythrocytes of various animal species and the electrophoretic pattern of the cell proteins. On the basis of these test, mycoplasmas from the equine respiratory tract were divided into seven species. Three species belonged to the genus Acholeplasma, members of which do not require sterol for growth, and were identified as A. laidlawii, A. oculi (formerly A. oculusi) originally isolated from the eyes of goats, and a recently named species A. equifoetale, previously isolated from aborted equine fetuses. Of the four sterol-dependent Mycoplasma species, one was indentified as M. pulmonis, a common rodent pathogen. Another cross-reacted serologically with M. felis and should probably be classified as that species. The other two species probably represent new species peculiar to the horse. One of these, represented by the strains N3 and N11, ferments glucose and is serologically distinct from 19 recognized species of glucose-utilizing mycoplasmas and from two species which do not metabolize either glucose or arginine. The other species, represented by four strains, hydrolyses arginine and, because it is serologically distinct from all the named arginine-hydrolysing Mycoplasma species, the name M. equirhinis sp.nov. is proposed for it. Of the seven species, only M. pulmonis and the glucose-utilizing species represented by N3 and N11 were found exclusively in horses with acute respiratory disease. A. oculi was isolated from an apparently normal horse. The other four species were found in normal horses as well as those with respiratory disease, although three out of the four strains of M. equirhinis were from sick horses.
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PMCID: PMC2130589  PMID: 807616
8.  Association of Mycoplasma ovipneumoniae Infection with Population-Limiting Respiratory Disease in Free-Ranging Rocky Mountain Bighorn Sheep (Ovis canadensis canadensis)▿  
Journal of Clinical Microbiology  2007;46(2):423-430.
Bronchopneumonia is a population-limiting disease in bighorn sheep in much of western North America. Previous investigators have isolated diverse bacteria from the lungs of affected sheep, but no single bacterial species is consistently present, even within single epizootics. We obtained high-quality diagnostic specimens from nine pneumonic bighorn sheep in three populations and analyzed the bacterial populations present in bronchoalveolar lavage specimens of seven by using a culture-independent method (16S rRNA gene amplification and clone library analyses). Mycoplasma ovipneumoniae was detected as a predominant member of the pneumonic lung flora in lambs with early lesions of bronchopneumonia. Specific PCR tests then revealed the consistent presence of M. ovipneumoniae in the lungs of pneumonic bighorn sheep in this study, and M. ovipneumoniae was isolated from lung specimens of five of the animals. Retrospective application of M. ovipneumoniae PCR to DNA extracted from archived formalin-fixed, paraffin-embedded lung tissues of historical adult bighorn sheep necropsy specimens supported the association of this agent with bronchopneumonia (16/34 pneumonic versus 0/17 nonpneumonic sheep were PCR positive [P < 0.001]). Similarly, a very strong association was observed between the presence of one or more M. ovipneumoniae antibody-positive animals and the occurrence of current or recent historical bronchopneumonia problems (seropositive animals detected in 9/9 versus 0/9 pneumonic and nonpneumonic populations, respectively [P < 0.001]). M. ovipneumoniae is strongly associated with bronchopneumonia in free-ranging bighorn sheep and is a candidate primary etiologic agent for this disease.
doi:10.1128/JCM.01931-07
PMCID: PMC2238132  PMID: 18057131
9.  Respiratory disease in a colony of rats 
The Journal of Hygiene  1972;70(3):387-407.
Mycoplasma pulmonis was isolated from the pneumonic lung of a rat. Two groups of mycoplasma-free rats were inoculated, one with a culture of the M. pulmonis strain which had been cloned four times (group A) and the other with a lung homogenate of the rat from which the strain had been isolated (group B). A third group (C) consisted of uninoculated control animals. Each group was kept in strict isolation and allowed to breed so that the progeny was naturally exposed to any pathogens present in the inoculated animals. After different periods of exposure, rats were autopsied, respiratory tracts and inner ears were cultured for mycoplasmas and bacteria, and sera were tested for complement-fixing antibodies to murine mycoplasmas.
In group-A rats, M. pulmonis was consistently isolated from the inner ears or lungs from 50 to 715 days after exposure. Complement-fixing antibody to M. pulmonis was detected 20 days after inoculation, but in the naturally exposed progeny antibody took longer than 50 days to develop. Antibodies to the other known mycoplasmas of murine origin, M. arthritidis and M. neurolyticum, were never found. Purulent otitis interna was consistently found from day 55 onwards, while lung lesions were first observed at 85 days and persisted to 715 days. Pulmonary lesions developed more slowly in inoculated parents than in exposed progeny. Similar results were found in group-B rats, which were examined up to 441 days after inoculation. Uninoculated group-C rats were examined up to 768 days of age, but M. pulmonis was not recovered; of the 54 animals whose serum was tested all were negative to the three species of mycoplasmas, except one which had a titre of 16 with M. pulmonis. Pneumonia, bronchiectasis or lymphoreticular hyperplasia were not seen in any of these control rats. Bacterial respiratory pathogens were not isolated from rats in any of the groups, nor was antibody to Sendai virus detected.
The results suggest that M. pulmonis alone can cause pneumonia and bronchiectasis in rats since mechanical carry-over of another pathogen with the initial cloned inoculum is very unlikely and there was no evidence for the participation of any other rat pathogen. The respiratory disease induced by the cloned culture was comparable with that induced by the lung homogenate, and with the well-known syndrome of chronic respiratory disease and bronchiectasis in the rat.
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PMCID: PMC2130201  PMID: 4506989
10.  Experimental peste des petits ruminants (goat plague) in goats and sheep. 
In order to study the pathomorphology and immunohistochemistry of peste des petits ruminants, four goats and two sheep were inoculated intranasally with the Malig-Yemen strain of peste des petits ruminants virus. The animals developed fever, nasal discharge, oral erosions, cough and diarrhea. One goat and one sheep died and one moribund goat was killed. Three animals survived the infection. At necropsy, erosive stomatitis, pneumonia and gastroenteritis were found. Histopathologically the pneumonocytes and epithelial cells of the ileum had eosinophilic cytoplasmic and nuclear inclusions. By an indirect immunoperoxidase method, the nuclei and cytoplasm of the ileal epithelial cells of one goat contained positively (brown) stained antigen, which corresponded to viral nucleocapsids by electron microscopy. Virus appeared to be released through the microvilli of the epithelial cells. We also confirmed the formation of giant cells due to peste des petits ruminants virus.
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PMCID: PMC1255399  PMID: 3280108
11.  Characterization of the Strain-Specific and Common Surface Antigens of Mycoplasma arginini 
Infection and Immunity  1980;29(2):442-451.
A combination of quantitative immunoelectrophoresis and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was used to determine location and molecular weights of surface membrane antigens of four strains of Mycoplasma arginini. Two major surface antigens were identified for M. arginini by absorption of antiserum with whole cells: one surface antigen was strain specific, electrophoretically fast, and prominently located on the surface, whereas the other surface antigen was common to the four strains and of intermediate electrophoretic mobility. Three of the four strains of M. arginini (G-230, 23243, and 27389) possessed immunologically strain-specific antigens which did not cross-react, whereas the leonis strain lacked an immunologically detectable unique surface antigen. A monospecific antiserum prepared against immune precipitates of the strain-specific antigen of strain G-230 detected three polypeptides of 74,000, 44,000, and 17,000 daltons in SDS-polyacrylamide gels of membrane preparations. All four strains shared the common surface antigen which appeared considerably more hydrophobic than the strain-specific surface antigen because it could only be demonstrated by charge-shift immunoelectrophoretic conditions (addition of deoxycholate to the nonionic detergent). Monospecific antiserum to the common antigen of strain G-230 reacted with all four M. arginini strains, but did not react with two other arginine-utilizing species, and recognized three polypeptides of 40,000, 29,000, and 20,000 daltons in membranes of strain G-230. Whereas the common surface antigen is a likely target for conventional serological reactions used for identification of the species M. arginini, strain-specific antigen cannot fulfill this role but must participate in other surface reactions.
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PMCID: PMC551138  PMID: 7216419
12.  Identification of a mycoplasmal protein which binds immunoglobulins nonimmunologically. 
Infection and Immunity  1991;59(6):2147-2151.
Immunoblotted protein samples from several strains of Mycoplasma hominis and from one strain of Mycoplasma arginini each contain a polypeptide of a molecular mass of 95,000 to 105,000 Da which binds immunoglobulin nonimmunologically. Immunoblots from these organisms were probed with alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin, conjugated goat immunoglobulin G (IgG) Fab fragments, and conjugated goat IgG Fc fragments. The polypeptide bound the goat anti-rabbit molecules and the Fab fragments but not the Fc fragments. These reactions could be blocked with nonimmune unconjugated goat IgG and unconjugated human IgM. Controls probed with alkaline phosphatase alone did not stain. Binding of the conjugated preparations to whole mycoplasmal cells was dependent on concentrations of both conjugate and cells for the goat anti-rabbit preparation and for Fab. The mycoplasmal polypeptide may be a light-chain-specific reactant.
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PMCID: PMC257979  PMID: 2037376
13.  Natural peste des petits ruminants virus infection in Black Bengal goats: virological, pathological and immunohistochemical investigation 
BMC Veterinary Research  2014;10(1):263.
Background
Peste des Petits Ruminants (PPR), also known as Goat Plague, occurs in goats, sheep and related species. It is caused by a morbillivirus in the family Paramyxoviridae. In Bangladesh PPR is endemic and it causes serious economic losses. Pathology of PPR has been reported in different goat and sheep breeds from natural and experimental infections. Field results are better indicators of pathogenicity of the circulating virus. The severity of the disease varies with species, breed and immune status of the host. Pathological investigations of natural outbreaks of PPR in Balck Bengal goats are very limited. The current investigation was aimed at describing pathology and antigen localization in natural PPR infections in Black Bengal goats.
Results
A total of 28 outbreaks were investigated clinically and virologically. Average flock morbidity and mortality were 75% and 59%, respectively, with case fatality rate of 74%. Necropsy was conducted on 21 goats from 15 outbreaks. The major gross lesions were congestion of gastrointestinal tract, pneumonia, engorged spleen, and oedematous lymphnodes. Histopathological examination revealed severe enteritis with denudation of intestinal epithelium, severe broncho-interstitial pneumonia with macrophages within lung alveoli and extensive haemorrhages with depletion of lymphoid cells and infiltration of macrophages in the sinuses of spleen. In lymph nodes, the cortical nodules were replaced by wide sinusoids with severe depletion of lymphocytes, infiltration of mononuclear cells and some giant cells in sub-capsular areas and medullary sinuses. PPR virus antigen was found in pneumocytes and alveolar macrophages in lungs. Viral RNA could be detected by RT-PCR in 69 out of 84 nasal swab, 59 out of 84 blood and 21 out of 21 lymph node samples. Sequence analyses revealed closeness of Bangladeshi strains with other recent Asian isolates.
Conclusion
Natural outbreaks of PPR in Black Bengal goats in Bangladesh resulted in 75% and 59% flock morbidity and mortality, respectively, with a case fatality rate of 74%. The striking histo-morphologic diagnosis of PPR was acute pneumonia and severe gastro-enteritis. A detailed experimental pathological study on Black Bengal goats infected with recent isolates is required.
doi:10.1186/s12917-014-0263-y
PMCID: PMC4233235  PMID: 25394771
PPR; Natural outbreak; Pathology; Antigen detection; Nucleic acid detection
14.  Role of enteric pathogens in the aetiology of neonatal diarrhoea in lambs and goat kids in Spain. 
Epidemiology and Infection  1996;117(1):203-211.
Faeces samples from diarrhoeic and non-diarrhoeic lambs and goat kids aged 1-45 days were examined for enteric pathogens. Cryptosporidium parvum was detected in both diarrhoeic lambs (45%) and goat kids (42%) but not in non-diarrhoeic animals. F5+ (K99+) and/or F41+ Escherichia coli strains were isolated from 26% and 22% of the diarrhoeic lambs and goat kids, respectively, although these strains, which did not produce enterotoxins ST I or LT I, were found with similar frequencies in non-diarrhoeic animals. A F5-F41-ST I+ E. coli strain was isolated from a diarrhoeic lamb (0.6%). Verotoxigenic E. coli was isolated from both diarrhoeic and non-diarrhoeic lambs (4.1% and 8.2%, respectively) and there was no association between infection and diarrhoea. The prevalence of group A rotavirus infection in diarrhoeic lambs was very low (2.1%). Groups A and B rotaviruses were detected in three (8.1%) and five (13.5%) diarrhoeic goat kids from two single outbreaks. Group C rotaviruses were detected in four non-diarrhoeic goat kids. An association of diarrhoea and infection was demonstrated only for group B rotavirus. Clostridium perfringens was isolated from 10.8% of the diarrhoeic goat kids but not from non-diarrhoeic goat kids or lambs. Salmonella arizonae was isolated from a diarrhoeic goat kid (2.7%) and the clinical characteristics of the outbreaks where these two latter enteropathogens were found different from the rest. Picobirnaviruses were detected in a diarrhoeic lamb. No coronaviruses were detected using a bovine coronavirus ELISA. No evidence was found of synergistic effect between the agents studied. Enteric pathogens were not found in four (8.7%) and three (20%) outbreaks of diarrhoea in lambs and goat kids, respectively.
PMCID: PMC2271684  PMID: 8760970
15.  Secreted Sialidase Activity of Canine Mycoplasmas 
Veterinary microbiology  2009;137(3-4):380-383.
Through a survey of the phylogenetic distribution of sialidase among mycoplasmas, we detected activity secreted by the type strains of three of eleven species frequently or first isolated from dogs. The specific activity of washed cells of the type strains of Mycoplasma canis, Mycoplasma cynos, and Mycoplasma molare ranged from 5.2 ± 0.8 × 10-6 to 1.1 ± 0.3 × 10-5 enzymatic units per colony-forming unit (U/CFU). Cells of M. molare strain H542T had twice the specific activity (P < 0.05) of M. canis strain PG14T or M. cynos strain H831T. Significant differences in sialidase activity existed among nine clinical isolates of M. canis, ranging from not detectable to 2.1 ± 0.1 × 10-5 U/CFU. The type strains of other species previously isolated from dogs (Mycoplasma arginini, Mycoplasma bovigenitalium, Mycoplasma edwardii, Mycoplasma felis, Mycoplasma gatae, Mycoplasma maculosum, Mycoplasma opalescens, and Mycoplasma spumans) did not exhibit either secreted or cell-associated sialidase activity. Neither specific nor degenerate PCR primers complementary to the three known mycoplasmal sialidase alleles were able to amplify orthologs in M. canis, M. cynos, or M. molare, further evidence that the secreted sialidase of those species is distinct from the strictly cell-associated sialidases of Mycoplasma alligatoris, Mycoplasma synoviae, and Mycoplasma gallisepticum. This is the first report of a well-known bacterial virulence factor whose expression varies among strains of certain Mycoplasma species that infect dogs.
doi:10.1016/j.vetmic.2009.01.009
PMCID: PMC2684937  PMID: 19201110
16.  Evidence for Transmission of Bluetongue Virus Serotype 26 through Direct Contact 
PLoS ONE  2014;9(5):e96049.
The aim of this study was to assess the mechanisms of transmission of bluetongue virus serotype 26 (BTV-26) in goats. A previous study, which investigated the pathogenicity and infection kinetics of BTV-26 in goats, unexpectedly revealed that one control goat may have been infected through a direct contact transmission route. To investigate the transmission mechanisms of BTV-26 in more detail an experimental infection study was carried out in which three goats were infected with BTV-26, three goats were kept uninfected, but were housed in direct contact with the infected goats, and an additional four goats were kept in indirect contact separated from infected goats by metal gates. This barrier allowed the goats to have occasional face-to-face contact in the same airspace, but feeding, watering, sampling and environmental cleaning was carried out separately. The three experimentally infected goats did not show clinical signs of BTV, however high levels of viral RNA were detected and virus was isolated from their blood. At 21 dpi viral RNA was detected in, and virus was isolated from the blood of the three direct contact goats, which also seroconverted. The four indirect barrier contact goats remained uninfected throughout the duration of the experiment. In order to assess replication in a laboratory model species of Culicoides biting midge, more than 300 Culicoides sonorensis were fed a BTV-26 spiked blood meal and incubated for 7 days. The dissemination of BTV-26 in individual C. sonorensis was inferred from the quantity of virus RNA and indicated that none of the insects processed at day 7 possessed transmissible infections. This study shows that BTV-26 is easily transmitted through direct contact transmission between goats, and the strain does not seem to replicate in C. sonorensis midges using standard incubation conditions.
doi:10.1371/journal.pone.0096049
PMCID: PMC4010411  PMID: 24797910
17.  The effect of Clostridium perfringens type C strain CN3685 and its isogenic beta toxin null mutant in goats 
Veterinary Microbiology  2012;157(3-4):412-419.
Clostridium perfringens type C is an important cause of enteritis and/or enterocolitis in several animal species, including pigs, sheep, goats, horses and humans. The disease is a classic enterotoxemia and the enteric lesions and associated systemic effects are thought to be caused primarily by beta toxin (CPB), one of two typing toxins produced by C. perfringens type C. This has been demonstrated recently by fulfilling molecular Koch’s postulates in rabbits and mice. We present here an experimental study to fulfill these postulates in goats, a natural host of C. perfringens type C disease. Nine healthy male or female Anglo Nubian goat kids were inoculated with the virulent C. perfringens type C wild-type strain CN3685, an isogenic CPB null mutant or a strain where the cpb null mutation had been reversed. Three goats inoculated with the wild-type strain presented abdominal pain, hemorrhagic diarrhea, necrotizing enterocolitis, pulmonary edema, hydropericardium and death within 24 h of inoculation. Two goats inoculated with the CPB null mutant and two goats inoculated with sterile culture media (negative controls) remained clinically healthy during 24 h after inoculation and no gross or histological abnormalities were observed in the tissues of any of them. Reversal of the null mutation to partially restore CPB production also increased virulence; 2 goats inoculated with this reversed mutant presented clinical and pathological changes similar to those observed in goats inoculated with the wild-type strain, except that spontaneous death was not observed. These results indicate that CPB is required for C. perfringens type C to induce disease in goats, supporting a key role for this toxin in natural C. perfringens type C disease pathogenesis.
doi:10.1016/j.vetmic.2012.01.005
PMCID: PMC3348370  PMID: 22296994
Beta toxin; Clostridium perfringens type C; enterotoxemia; goats
18.  Q Fever in Pregnant Goats: Pathogenesis and Excretion of Coxiella burnetii 
PLoS ONE  2012;7(11):e48949.
Coxiella burnetii is an intracellular bacterial pathogen that causes Q fever. Infected pregnant goats are a major source of human infection. However, the tissue dissemination and excretion pathway of the pathogen in goats are still poorly understood. To better understand Q fever pathogenesis, we inoculated groups of pregnant goats via the intranasal route with a recent Dutch outbreak C. burnetii isolate. Tissue dissemination and excretion of the pathogen were followed for up to 95 days after parturition. Goats were successfully infected via the intranasal route. PCR and immunohistochemistry showed strong tropism of C. burnetii towards the placenta at two to four weeks after inoculation. Bacterial replication seemed to occur predominantly in the trophoblasts of the placenta and not in other organs of goats and kids. The amount of C. burnetii DNA in the organs of goats and kids increased towards parturition. After parturition it decreased to undetectable levels: after 81 days post-parturition in goats and after 28 days post-parturition in kids. Infected goats gave birth to live or dead kids. High numbers of C. burnetii were excreted during abortion, but also during parturition of liveborn kids. C. burnetii was not detected in faeces or vaginal mucus before parturition. Our results are the first to demonstrate that pregnant goats can be infected via the intranasal route. C. burnetii has a strong tropism for the trophoblasts of the placenta and is not excreted before parturition; pathogen excretion occurs during birth of dead as well as healthy animals. Besides abortions, normal deliveries in C. burnetii-infected goats should be considered as a major zoonotic risk for Q fever in humans.
doi:10.1371/journal.pone.0048949
PMCID: PMC3494687  PMID: 23152826
19.  Animal model of Mycoplasma fermentans respiratory infection 
BMC Research Notes  2013;6:9.
Background
Mycoplasma fermentans has been associated with respiratory, genitourinary tract infections and rheumatoid diseases but its role as pathogen is controversial. The purpose of this study was to probe that Mycoplasma fermentans is able to produce respiratory tract infection and migrate to several organs on an experimental infection model in hamsters. One hundred and twenty six hamsters were divided in six groups (A-F) of 21 hamsters each. Animals of groups A, B, C were intratracheally injected with one of the mycoplasma strains: Mycoplasma fermentans P 140 (wild strain), Mycoplasma fermentans PG 18 (type strain) or Mycoplasma pneumoniae Eaton strain. Groups D, E, F were the negative, media, and sham controls. Fragments of trachea, lungs, kidney, heart, brain and spleen were cultured and used for the histopathological study. U frequency test was used to compare recovery of mycoplasmas from organs.
Results
Mycoplasmas were detected by culture and PCR. The three mycoplasma strains induced an interstitial pneumonia; they also migrated to several organs and persisted there for at least 50 days. Mycoplasma fermentans P 140 induced a more severe damage in lungs than Mycoplasma fermentans PG 18. Mycoplasma pneumoniae produced severe damage in lungs and renal damage.
Conclusions
Mycoplasma fermentans induced a respiratory tract infection and persisted in different organs for several weeks in hamsters. This finding may help to explain the ability of Mycoplasma fermentans to induce pneumonia and chronic infectious diseases in humans.
doi:10.1186/1756-0500-6-9
PMCID: PMC3544566  PMID: 23298636
Animal model; Mycoplasma; Mycoplasma fermentans; Respiratory infection
20.  Carriage of Mycoplasma pneumoniae in the Upper Respiratory Tract of Symptomatic and Asymptomatic Children: An Observational Study 
PLoS Medicine  2013;10(5):e1001444.
In order to determine the possible asymptomatic carriage of Mycoplasma pneumoniae in the upper respiratory tracts of children, Emiel Spuesens and colleagues investigate the prevalence of M. pneumoniae in symptomatic and asymptomatic children at a hospital in The Netherlands.
Please see later in the article for the Editors' Summary
Background
Mycoplasma pneumoniae is thought to be a common cause of respiratory tract infections (RTIs) in children. The diagnosis of M. pneumoniae RTIs currently relies on serological methods and/or the detection of bacterial DNA in the upper respiratory tract (URT). It is conceivable, however, that these diagnostic methods also yield positive results if M. pneumoniae is carried asymptomatically in the URT. Positive results from these tests may therefore not always be indicative of a symptomatic infection. The existence of asymptomatic carriage of M. pneumoniae has not been established. We hypothesized that asymptomatic carriage in children exists and investigated whether colonization and symptomatic infection could be differentiated by current diagnostic methods.
Methods and Findings
This study was conducted at the Erasmus MC–Sophia Children's Hospital and the after-hours General Practitioners Cooperative in Rotterdam, The Netherlands. Asymptomatic children (n = 405) and children with RTI symptoms (n = 321) aged 3 mo to 16 y were enrolled in a cross-sectional study from July 1, 2008, to November 30, 2011. Clinical data, pharyngeal and nasopharyngeal specimens, and serum samples were collected. The primary objective was to differentiate between colonization and symptomatic infection with M. pneumoniae by current diagnostic methods, especially real-time PCR. M. pneumoniae DNA was detected in 21.2% (95% CI 17.2%–25.2%) of the asymptomatic children and in 16.2% (95% CI 12.2%–20.2%) of the symptomatic children (p = 0.11). Neither serology nor quantitative PCR nor culture differentiated asymptomatic carriage from infection. A total of 202 children were tested for the presence of other bacterial and viral pathogens. Two or more pathogens were found in 56% (63/112) of the asymptomatic children and in 55.5% (50/90) of the symptomatic children. Finally, longitudinal sampling showed persistence of M. pneumoniae in the URT for up to 4 mo. Fifteen of the 21 asymptomatic children with M. pneumoniae and 19 of the 22 symptomatic children with M. pneumoniae in this longitudinal follow-up tested negative after 1 mo.
Conclusions
Although our study has limitations, such as a single study site and limited sample size, our data indicate that the presence of M. pneumoniae in the URT is common in asymptomatic children. The current diagnostic tests for M. pneumoniae are unable to differentiate between asymptomatic carriage and symptomatic infection.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Pneumonia (a form of acute respiratory infection) is the single largest cause of death in children worldwide, killing an estimated 1.2 million children aged five and under every year, particularly in South Asia and sub-Saharan Africa. In these settings, bacterial infections with Streptococcus pneumoniae and Haemophilus influenzae are the most common causes of bacterial pneumonia. However, in high-income settings, bacterial infection with Mycoplasma pneumoniae is a major cause of upper and lower respiratory tract infections in children: over one-third of childhood cases of community-acquired pneumonia that require admission to a hospital are caused by M. pneumoniae. Currently, diagnosis of M. pneumoniae infections relies on the detection of antibodies against M. pneumoniae in the blood or detection of bacterial DNA in samples from the upper respiratory tract through polymerase chain reaction (PCR) tests.
Why Was This Study Done?
Other bacteria, such as Streptococcus pneumoniae, are commonly present in children without causing infection, a situation known as asymptomatic carriage. However, to date, it is unknown whether M. pneumoniae is also commonly carried in the upper respiratory tract of children without causing symptoms or leading to infection. The possibility of asymptomatic carriage of M. pneumoniae could have major implications for the interpretation of the results of diagnostic tests and also for clinical management. So in this study conducted in The Netherlands, the researchers investigated whether asymptomatic carriage of M. pneumoniae exists and also whether symptomatic infection could be differentiated from asymptomatic carriage by current diagnostic methods.
What Did the Researchers Do and Find?
Between 2008 and 2011, the researchers recruited children aged between three months and 16 years attending a hospital in Rotterdam for an elective surgical procedure (asymptomatic group) or admitted with a respiratory tract infection (symptomatic group). All children had blood tests and respiratory samples (nasopharyngeal swab) taken on admission and were tested for other pathogens. The researchers invited children who tested positive for M. pneumoniae by PCR to attend for further follow-up and tested them monthly for the presence of M. pneumoniae DNA in the upper respiratory tract until the test was negative on two occasions. Using these methods, the researchers recruited 726 children over the study period—405 in the asymptomatic group and 321 in the symptomatic group. The researchers found that the prevalence of M. pneumoniae did not differ between the asymptomatic group and the symptomatic group, with prevalences of 21.2% and 16.2%, respectively (the prevalence of M. pneumoniae also did not differ significantly between those with lower versus upper respiratory infection). There were also no differences in prevalence in the asymptomatic and symptomatic groups when diagnosed using blood tests. The researchers found a high rate of multiple, coexisting bacterial and viral pathogens in both asymptomatic and symptomatic children: two or more pathogens were found in 56% (63/112) of the asymptomatic children and in 55.5% (50/90) of the symptomatic children. Furthermore, season and the year of enrollment affected the prevalence of M. pneumoniae in the asymptomatic group, ranging from 3% during the spring of 2009 to 58% during the summer of 2010. Finally, of the 21 children from the asymptomatic group who participated in the follow-up study, 15 (71%) tested negative within one month, and in the symptomatic group, 19 of 22 children (86%) tested negative after the first visit.
What Do These Findings Mean?
These findings show that M. pneumoniae is carried at high rates in the upper respiratory tracts of healthy children, and that this asymptomatic carriage cannot be differentiated from symptomatic respiratory tract infection by diagnostic tests (serology or PCR). As the prevalence of M. pneumoniae varied between year and season, carriage of M. pneumoniae may follow a cyclic epidemic pattern. This study is from a single study site in one city in The Netherlands, with a relatively small number of children, and so these findings may not be generalizable to other populations. However, as this study suggests that current diagnostic tests do not discriminate between carriage and infection, clinicians may need to reconsider the clinical significance of a positive test result. Future studies are needed to address this diagnostic challenge and also to investigate possible factors that may affect the progression of asymptomatic carriage of M. pneumoniae to symptomatic infection.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001444.
MicrobeWiki has more information on M. pneumoniae
Lab Tests Online explains current tests for M. pneumoniae
doi:10.1371/journal.pmed.1001444
PMCID: PMC3653782  PMID: 23690754
21.  Mycoplasma species recovered from the reproductive tracts of western Canadian cows. 
Samples of cervico-vaginal mucus from 633 animals from 110 herds were cultured and yielded the following mycoplasmas: T-strain--88: Mycoplasma bovigenitalium--79, Mycoplasma spp. (Leach Group 7)--7, Acholeplasma laidlawii--4, Mycoplasma bovirhinis--2 and one not typable. Uterine exudates and endometrial scrapings from 80 infertile cows in two herds were examined. Four animals were positive, M. bovigenitalium was isolated three times, A. laidlawii and Mycoplasma arginini once each. Sixty-five normal uterine contents from pregnant cows were examined, one yielded M. bovigenigalium and the same organism was recovered from the fetal kidney. T-strain mycoplasma, M. bovigenitalium and other Mycoplasma spp. appear to be a part of the normal flora of the cervico-vaginal region of clinically normal one and two year old bred heifers in Alberta and Saskatchewan. Although M. arginini was not recovered from the cervico-vaginal region, a single recovery was made from the uterus of an infertile cow.
PMCID: PMC1277433  PMID: 1125831
22.  Simultaneous Detection and Identification of Common Cell Culture Contaminant and Pathogenic Mollicutes Strains by Reverse Line Blot Hybridization 
We have developed a reverse line blot (RLB) hybridization assay to detect and identify the commonest mollicutes causing cell line contamination (Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycoplasma orale, and Acholeplasma laidlawii) and human infection (Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma parvum, and Ureaplasma urealyticum). We developed a nested PCR assay with “universal” primers targeting the mollicute 16S-23S rRNA intergenic spacer region. Amplified biotin-labeled PCR products were hybridized to membrane-bound species-specific oligonucleotide probes. The assay correctly identified reference strains of 10 mollicute species. Cell cultures submitted for detection of mollicute contamination, clinical specimens, and clinical isolates were initially tested by PCR assay targeting a presumed mollicute-specific sequence of the 16S rRNA gene. Any that were positive were assessed by the RLB assay, with species-specific PCR assay as the reference method. Initially, 100 clinical and 88 of 92 cell culture specimens gave concordant results, including 18 in which two or more mollicute species were detected by both methods. PCR and sequencing of the 16S-23S rRNA intergenic spacer region and subsequent retesting by species-specific PCR assay of the four cell culture specimens for which results were initially discrepant confirmed the original RLB results. Sequencing of amplicons from 12 cell culture specimens that were positive in the 16S rRNA PCR assay but negative by both the RLB and species-specific PCR assays failed to identify any mollicute species. The RLB hybridization assay is sensitive and specific and able to rapidly detect and identify mollicute species from clinical and cell line specimens.
doi:10.1128/AEM.70.3.1483-1486.2004
PMCID: PMC368316  PMID: 15006769
23.  Experimental Contagious Caprine Pleuropneumonia: A Long Term Study on the Course of Infection and Pathology in a Flock of Goats Infected with Mycoplasma capricolum subsp. capripneumoniae 
Acta Veterinaria Scandinavica  2004;45(3):167-179.
Contagious caprine pleuropneumonia (CCPP) is a major threat to goat farming in parts of Africa and Asia. It classically causes acute high morbidity and mortality early in infection, but little is known of its long term epizootiology and course. In this study, 10 goats were inoculated with Mycoplasma capricolum subsp. capripneumoniae (M. capripneumoniae) and then mixed with 15 goats for contact transmission. The disease course was monitored in each goat for 56–105 days, whereafter the goats were killed and necropsied. Varying features signifying infection occurred in altogether 17 goats (7 inoculated, 10 in-contact). Clinical signs were severe in 8 goats but no fatalities occurred. Only 6 goats had serum antibody titres against M. capripneumoniae in ELISA. Fourteen goats (5 inoculated, 9 in-contact) had chronic pleuropulmonary lesions compatible with CCPP at necropsy and 7 of those showed M. capripneumoniae antigen in the lung by immunohistochemistry. Neither cultivation nor PCR tests were positive for the agent in any goat. The results indicate that the clinical course of CCPP in a flock may be comparatively mild, M. capripneumoniae-associated lung lesions may be present at a late stage of infection, and chronic infection may occur without a significant serological response.
doi:10.1186/1751-0147-45-167
PMCID: PMC1820987  PMID: 15663077
goat; Mycoplasma; contagious pleuropneumonia; ELISA; immunohistochemistry; serology; pathology.
24.  Epidemic caprine keratoconjunctivitis: recovery of Mycoplasma conjunctivae and its possible role in pathogenesis. 
Infection and Immunity  1977;18(3):806-815.
Clinical, microbiological, serological, histological, and therapeutic aspects of two separate outbreaks of caprine keratoconjunctivitis are described. The disease was characterized by a high rate of contagion, rapid onset, intense lacrimation, conjunctival hyperemia, and corneal opacity with neovascularization. In addition, many of the animals developed respiratory illness during the second epidemic. The only organism consistentlyisolated was Mycoplasma conjunctivae. A total of 23 strains were isolated from 18 inflamed conjunctivae, one normal conjunctiva, and the nasal secretions of four goats with concomitant respiratory illness. The convalescent sera of goats in the first outbreak had neutralizing antibody titers to M. conjunctivae that ranged from 1:32 to 1:256. In the milder second outbreak the antibody titers ranged from 1:4 to 1:32 in animals with only ocular disease and from 1:4 to 1:64 in animals with only respiratory disease. Whereas little change was noted in antibody titers of goats with only localized eye disease, 43% of the goats with respiratory disease showed significant fourfold rises. The histological picture was consistent with acute corneal infection. Animals requiring antibiotic treatment appeared to respond favorably to a combination of oxytetracycline and polymyxin B, but not to penicillin. These findings suggest that M. conjunctivae is one cause of epidemic caprine keratoconjunctivitis.
Images
PMCID: PMC421305  PMID: 591066
25.  Innate Immune Response to Rift Valley Fever Virus in Goats 
Rift Valley fever (RVF), a re-emerging mosquito-borne disease of ruminants and man, was endemic in Africa but spread to Saudi Arabia and Yemen, meaning it could spread even further. Little is known about innate and cell-mediated immunity to RVF virus (RVFV) in ruminants, which is knowledge required for adequate vaccine trials. We therefore studied these aspects in experimentally infected goats. We also compared RVFV grown in an insect cell-line and that grown in a mammalian cell-line for differences in the course of infection. Goats developed viremia one day post infection (DPI), which lasted three to four days and some goats had transient fever coinciding with peak viremia. Up to 4% of peripheral blood mononuclear cells (PBMCs) were positive for RVFV. Monocytes and dendritic cells in PBMCs declined possibly from being directly infected with virus as suggested by in vitro exposure. Infected goats produced serum IFN-γ, IL-12 and other proinflammatory cytokines but not IFN-α. Despite the lack of IFN-α, innate immunity via the IL-12 to IFN-γ circuit possibly contributed to early protection against RVFV since neutralising antibodies were detected after viremia had cleared. The course of infection with insect cell-derived RVFV (IN-RVFV) appeared to be different from mammalian cell-derived RVFV (MAM-RVFV), with the former attaining peak viremia faster, inducing fever and profoundly affecting specific immune cell subpopulations. This indicated possible differences in infections of ruminants acquired from mosquito bites relative to those due to contact with infectious material from other animals. These differences need to be considered when testing RVF vaccines in laboratory settings.
Author Summary
Rift Valley fever (RVF) is a mosquito-transmitted disease of ruminants and man, which occurs in Africa, Saudi Arabia and Yemen but could spread to other areas. There isn't much information on some aspects of the immune response to this disease and how it affects cells of the immune system in the natural animal hosts. To fill in some of this knowledge gap, we studied RVF in goats experimentally infected with the RVF virus. We also compared RVF virus grown in an insect cell-line and that grown in a mammalian cell-line for differences in the course of infection. Virus was present in the blood of the goats one day after infection. Some goats had fever coinciding with the time when the virus level in the blood was highest. Some cells in the blood dropped in number possibly as a direct effect of virus. Infected goats secreted cytokines (interferon gamma and interleukin-12), which possibly contributed to protection against RVF. Virus from an insect cell-line appeared to have more obvious effects in infected goats suggesting that differences may exist in infections of ruminants acquired from mosquito bites compared to those due to contact with infectious material from other animals.
doi:10.1371/journal.pntd.0001623
PMCID: PMC3335883  PMID: 22545170

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