Mycoplasma pulmonis of Rodents as a Possible Human Pathogen.

Mycoplasma pulmonis is a naturally occurring respiratory pathogen in rodents. To date, this pathogen was not isolated from humans. This study aimed to evaluate the prevalence and seropositivity to M. pulmonis in humans who have had direct contact with rats. Moreover, the prevalence of M. pulmonis in pet and laboratory rats was assessed. Overall, 131 and 235 oropharyngeal swab samples were collected from human individuals and rats, respectively. In humans, M. pulmonis was detected by PCR in 21 of 86 pet rat keepers (24.42%), 10 of 13 technicians (76.32%), and 8 of 32 (25.0%) veterinarians. In rats, M. pulmonis was identified by PCR in 86 of 122 pet rats (70.49%) and 56 of 113 (49.56%) laboratory rats. Seroprevalence in humans was examined by screening sera from 44 individuals for M. pulmonis-specific IgG using ELISA. In total, 26 out of 44 (59.09%) humans were seropositive to M. pulmonis (4 out of 9 technicians, 8 of 12 veterinarians, and 15 of 23 pet rat keepers).The high antibody titer was found in 4 individuals (2 pet rat keepers and 2 veterinarians), whereas the moderate and low antibody titers were found in 8 and 14 individuals, respectively. The high antibody titer found in humans might indicate an active infection. However, it is unknown whether the presence of M. pulmonis in humans might be associated with disease and whether the foreign Mycoplasma can survive for long in its new environment.

Respiratory Pathology and Pathogens in Wild Urban Rats (Rattus norvegicus and Rattus rattus).

Norway (Rattus norvegicus) and black rats (Rattus rattus) are common peridomestic species, yet little is known about wild rat ecology, including their natural diseases. We describe gross and histological lesions in the respiratory tract of a sample of 711 wild urban rats. A subset was examined for 19 distinct categories of histological lesions in the respiratory tract. Testing for known respiratory pathogens included serology and polymerase chain reaction (PCR) of lung samples. Grossly evident lesions were rare (8/711; 1%). Upper respiratory tract inflammation was present in 93 of 107 (87%) rats and included rhinitis, submucosal and periglandular lymphoplasmacytic tracheitis, and/or tracheal intraluminal necrotic debris and was significantly associated (P < .05) with the presence of cilia-associated respiratory bacillus (CARB), Mycoplasma pulmonis, and increased body mass (odds ratio [OR] = 1.09; 95% confidence interval [CI] = 1.05-1.14 per 10 g). Within the lungs, peribronchiolar and/or perivascular lymphoplasmacytic cuffs were present in 152 of 199 rats (76%) and were also significantly associated (P ≤ .02) with CARB, M. pulmonis, and increased body mass (OR = 1.20; 95% CI = 1.14-1.27 per 10 g). Rats were frequently coinfected with M. pulmonis and CARB, and lesions associated with these pathogens were histologically indistinguishable. Pneumocystis sp was detected in 48 of 102 (47%) rats using PCR but was not significantly associated with lesions. This description of pathology in the respiratory system of wild rats demonstrates that respiratory disease is common. Although the impact of these lesions on individual and population health remains to be investigated, respiratory disease may be an important contributor to wild rat morbidity and mortality.

Rapid identification of Mycoplasma pulmonis isolated from laboratory mice and rats using matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Mycoplasma species identification is based on biochemical, immunological, and molecular methods that require several days for accurate identification. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a novel method for identification of bacteria and has recently been introduced into the clinical microbiology laboratory as a rapid and accurate technique. This method allows a characteristic mass spectral fingerprint to be obtained from whole inactivated mycoplasmal cells. In this study, we evaluated the performance of the MALDI-TOF MS for the identification of Mycoplasma by comparison with standard sequence analysis of 16S rRNA. We developed the first database of MALDI-TOF MS profiles of Mycoplasma species, containing Mycoplasma pulmonis, M. arthritidis, and M. neurolyticum, which are the most common pathogens in mice and/or rats, and species-specific spectra were recorded. Using the database, 6 clinical isolates were identified. Six tracheal swabs from 4 mice and 2 rats were cultured on PPLO agar for 4 to 7 days, and the colonies were directly applied to analyze the protein profiles. Five strains were identified as M. pulmonis, and 1 strain from a mouse was identified as M. neurolyticum (spectral scores were >2.00); the results were consistent with the results of the 16S rRNA gene sequence analysis (homologies>97.0%). These data indicate that MALDI-TOF MS can be used as a clearly rapid, accurate, and cost-effective method for the identification of M. pulmonis isolates, and this system may represent a serious alternative for clinical laboratories to identify Mycoplasma species.

A serological survey to evaluate contemporary prevalence of viral agents and Mycoplasma pulmonis in laboratory mice and rats in western Europe.

To evaluate current prevalence rates of 24 viruses and of the bacterium Mycoplasma pulmonis, the authors retrospectively surveyed serological data obtained from laboratory mice and rats housed in more than 100 western European institutions. Serum samples were submitted to the authors' institution for testing between January 2007 and June 2008. The prevalence of an infection was defined as the percentage of tested samples that yielded positive results for a specific agent. In mice, the most commonly detected infectious agents were murine norovirus (prevalence of 31.8%), mouse hepatitis virus (5.5%), mouse rotavirus (1.7%) and parvoviruses (1.0%). In rats, parvoviruses (12.1%) and M. pulmonis (3.6%) were the most prevalent infectious agents. Most rodent parvovirus infections could be attributed to mouse parvovirus in mice and to rat minute virus or to Kilham rat virus in rats. These data suggest the importance of up-to-date animal health monitoring programs and should stimulate the scientific community to further improve the microbiological quality of laboratory rodents.

Detection of Mycoplasma pulmonis in laboratory rats and technicians.

Five species of mycoplasma are associated with several rat diseases. Mycoplasma pulmonis is the most important and most studied, possibly causing disease in rats and undermining the validity of laboratory experiments. M. pulmonis was isolated in 144/240 laboratory rats and identified by PCR in 155/240. This species was also detected in 12 human individuals (technicians of a laboratory animal house hold) in contact with these rats. The results were confirmed by sequencing of DNA products. Mycoplasma species are host specific; however, M. pulmonis was identified in humans, suggesting a case of unspecific colonization. Statistical analysis shows a greater risk for M. pulmonis colonizing individuals who are exposed to infected rats in animal facilities than individuals who do not. The detection of M. pulmonis in humans indicates a new status for this mollicute mycoplasmas in animal-holding facilities.

Prevalence of naturally occurring viral infections, Mycoplasma pulmonis and Clostridium piliforme in laboratory rodents in Western Europe screened from 2000 to 2003.

In this report prevalence rates of rodent viruses in laboratory animals are presented based on routine serological screening of mouse and rat colonies from European institutes. The prevalences found during the period 2000-2003 are compared with those reported for 1981-1984 and 1990-1993. It is shown that some infections were eliminated from laboratory animal colonies (e.g. K-virus and polyomavirus) by taking preventative measures whereas other infections such as mouse hepatitis virus and parvoviruses remained at a high rate. Further decreases in prevalence rates in the last 10 years were found for infections such as pneumonia virus of mice, reovirus type 3, Sendai virus, sialodacryoadenitis/rat coronavirus and Mycoplasma pulmonis. The introduction of new detection methods showed that mouse parvovirus and rat parvovirus, both members of the Parvoviridae family, remain a major threat to laboratory mice and rats. Guinea pig cytomegalovirus and para-influenza virus appeared to be the most prevalent agents among laboratory guinea pigs. The importance of a standardized, up-to-date screening programme is discussed.

Detection of Mycoplasma pulmonis by fluorogenic nuclease polymerase chain reaction analysis.

Mycoplasma pulmonis induces persistent infections in laboratory mice and rats and can contaminate biological materials. We developed a fluorogenic nuclease polymerase chain reaction (fnPCR) assay to detect M. pulmonis specifically. Primer and probe sequences for the assay were targeted to 16S rRNA sequences specific to M. pulmonis. The assay consistently detected the equivalent of fewer than 10 copies of template DNA. When evaluated against a panel of 24 species of bacteria, the M. pulmonis assay detected only M. pulmonis isolates. Evaluation of 10-fold serial dilutions of cultured M. pulmonis showed that the M. pulmonis fnPCR assay and culture on Dutch agar had comparable sensitivity in detecting viable M. pulmonis organisms, whereas the mouse antibody production test displayed positive serologic results at dilutions higher than those in which viable organisms could be detected. Finally, the M. pulmonis fnPCR assay was able to detect M. pulmonis DNA in nasopharyngeal wash fluid and trachea, lung, and uterus tissue collected from mice naturally infected with M. pulmonis but did not detect the organism in similar samples collected from uninfected, negative control mice. The M. pulmonis fnPCR assay provides a high-throughput, PCR-based method to detect M. pulmonis in infected rodents and contaminated biological materials.

Differential identification of mycoplasma pulmonis and M. arthritidis using PCR-based RFLP.

Mycoplasma pulmonis and Mycoplasma arthritidis were differentially identified using PCR-restriction fragment length polymorphism (RFLP). A genus-specific sequence of mycoplasma was amplified by PCR and the PCR products were digested with the restriction enzyme SmaI. Each PCR product from the four isolates of M. pulmonis was digested with SmaI into two fragments; however, there was no digestion in the PCR product from M. arthritidis. This method might be useful to differentiate infection of M. pulmonis from that of M. arthritidis.

Polymerase chain reaction with a primer pair in the 16S-23S rRNA spacer region for detection of Mycoplasma pulmonis in clinical isolates.

To develop a diagnostic tool to identify Mycoplasma pulmonis (M. pulmonis) in clinical isolates, we developed a polymerase chain reaction (PCR) assay using primers specific for the 16S-23S rRNA intergenic spacer region (SR) of M. pulmonis. One pair of PCR primers reacted specifically with two reference strains of M. pulmonis tested and seven samples isolated from naturally infected rats. The primer pair did not produce PCR products of the correct size from any other rodent or human mycoplasmas or cellular DNA from rodent lungs. Specificity of the PCR assay was confirmed by Southern blotting with probe specific for the SR of M. pulmonis. The PCR assay for detection of M. pulmonis established in this study is suitable for diagnosis of M. pulmonis infection in clinical cases.