Application of a nested polymerase chain reaction assay to detect Mycoplasma hyopneumoniae from nasal swabs.

The porcine respiratory disease complex (PRDC) is an increasingly important cause of decreased swine productivity and is characterized by slow growth, decreased feed efficiency, anorexia, cough, and dyspnea. Mycoplasma hyopneumoniae is among the most prevalent and important infectious agents associated with PRDC. Understanding of mycoplasmal pneumonia has been hindered by inadequate diagnostic methods. Many of the currently available tests are relatively insensitive or nonspecific when used in a diagnostic laboratory setting or are too costly or difficult for routine diagnostic use. Several polymerase chain reaction (PCR) assays have been described, but they are not sensitive enough to detect the microorganisms in live pigs, from either nasal or tracheal swabs. A nested PCR using 2 species-specific sets of primers from the 16S ribosomal DNA gave positive results with as little as 80 microorganisms and did not cross-react with other mycoplasma species or with other microorganisms commonly found in the respiratory tract of pigs. This assay was better suited for detection of M. hyopneumoniae from nasal swabs than was conventional PCR. Nasal swab samples were taken at different time periods following experimental challenge of 10 susceptible pigs. Only 2 of the 55 swabs examined gave a positive result with conventional PCR, whereas 30 of the 55 swabs gave a positive result using the nested PCR. Twenty of 40 (50%) nasal swabs from pigs experiencing a respiratory disease outbreak where M. hyopneumoniae had been diagnosed also gave a positive result with the nested PCR. To confirm that the amplified product was specific, 4 nested PCR products were purified, sequences were determined and aligned, and they were confirmed to be from M. hyopneumoniae.     

Detection of Mycoplasma hyopneumoniae in lung and nasal swab samples from pigs by nested PCR and culture methods

We examined nasal swab and lung homogenate samples collected from pigs experimentally and naturally infected with Mycoplasma hyopneumoniae for the detection of M. hyopneumoniae by the nested PCR (nPCR) and culture methods. In the 23 experimentally infected pigs, M. hyopneumoniae was commonly detected in nasal swabs by the nPCR and culture methods at 4 weeks after inoculation, and there was a significant correlation (P<0.01) between the titers of viable organisms in nasal swabs and in lung homogenates in the experimentally inoculated pigs. In the naturally infected pigs, on the other hand, discrepancies in detection were found between nasal swab and lung homogenate samples in 17 of 36 cases, although the presence of gross lung lesions correlated relatively well with the detection of organisms from the samples. Our results indicated that the diagnosis of mycoplasmal pneumonia by nPCR in individual pigs with nasal swabs is reliable under these experimental conditions. At present, nPCR with nasal swabs should only be used for monitoring the disease status at the herd level under field conditions.     

Isolation of Mycoplasma hyopneumoniae from different sampling sites in experimentally infected and contact SPF piglets

The purpose of this study was to determine the optimal route of infection and the optimal sampling sites for the recovery of M. hyopneumoniae, the etiological agent of enzootic porcine pneumonia. Virulence of two strains, BQ 14 and 116, isolated in France in 1975 and 2003, respectively, was also compared. Groups of specific pathogen free piglets were experimentally infected by the intratracheal or intranasal route. One non-inoculated pig was placed in each group of infected pigs to study direct transmission. Two groups were kept uninfected. Coughing was recorded daily. Blood samples, nasal, tonsillar and tracheal swabs and tracheobronchiolar washings were collected weekly. Pigs were killed 27-37 days post-infection. Lung lesions were scored and swabs were collected from nasal cavities, tonsils, trachea, lung, liver and spleen. All the samples, collected from live and dead pigs, were cultured for M. hyopneumoniae recovery. Results showed that both experimentally infected pigs and contact pigs developed enzootic pneumonia, whatever the route of infection and the strain tested. Direct contact transmission occurred quickly. No difference between the two routes of infection or between the two strains tested was evidenced, but high individual variations were observed between pigs. Tracheal swabs and tracheobronchiolar washings were the most effective samples to detect M. hyopneumoniae compared to nasal or tonsillar swabs. Our results also suggested that tracheobronchiolar washings could have an influence on the lesion extent observed at necropsy. M. hyopneumoniae could be re-isolated from liver and spleen of experimentally infected pigs and contact pigs.     

Effect of Mycoplasma hyopneumoniae colonization at weaning on disease severity in growing pigs

To determine whether Mycoplasma hyopneumoniae colonization at weaning in off-site weaning systems is associated with the severity of respiratory disease due to this agent in growing pigs, we studied 20 groups, each group representing a different week in production, in sow herds at 3 farms of 3000 sows each that had a prevalence of M. hyopneumoniae colonization at weaning higher than 5%. The calculated sample size for assessment at weaning was 39 piglets for each group under study; 39 litters were randomly selected, and 1 piglet was randomly selected from each litter for testing and ear-tagged. In total, 780 piglets were tested. The presence of M. hyopneumoniae in nasal swabs at weaning was established by nested polymerase chain reaction (PCR). All groups were followed until slaughter, at which time blood samples were collected from each ear-tagged pig to test for M. hyopneumoniae antibodies, bronchial swabs were collected for detection of M. hyopneumoniae DNA by nested PCR, and the lung lesion score and percentage of affected lungs in the same animals were calculated. Correlation analyses showed a positive correlation between colonization at weaning and all 4 dependent variables indicating infection at slaughter: average lung lesion score, percentage of affected lungs, presence of M. hyopneumoniae on the bronchial epithelium, and seroconversion. This study provides evidence that severity of the disease can be predicted by the prevalence at weaning in segregated systems. Therefore, strategies focused on reducing colonization at weaning seem to be important elements in the global control of M. hyopneumoniae in segregated production systems.     

Enzootic pneumonia of pigs--a diagnostic dilemma

In an enzootic pneumonia-free Australian pig herd, an outbreak of a severe respiratory disease in the grow-out herd was initially diagnosed as acute tracheitis and pneumonia precipitated by the dusty environment, with a superimposed mixed infection of Pasteurella multocida and Arcanobacterium pyogenes. Culture for Mycoplasma hyopneumoniae, Salmonella sp and fungi was negative. The outbreak persisted. Subsequently, gross lesions consistent with enzootic pneumonia occurred, but histological lesions were equivocal and definitive tests for M hyopneumoniae remained negative. Eighteen months after the initial outbreak, gross and histological lesions were consistent with enzootic pneumonia but serological tests were still negative. Almost 2 years later, one of four nasal swabs was positive by the polymerase chain reaction test for M hyopneumoniae, and then lung samples were sporadically positive. The pneumonic disease became endemic in the herd. Gross lesions consistent with enzootic pneumonia occurred in another herd belonging to the same company nearly 2 years after the initial outbreak. Again, results of laboratory tests were inconsistent. Despite sporadic positive polymerase chain reaction tests for M hyopneumoniae, the respiratory disease resolved within 4 months and there has been no clinical evidence of enzootic pneumonia during the subsequent 4 years. These cases raise important questions about the role of the diagnostic tests and their interpretation, and the ecology of M hyopneumoniae and its role in enzootic pneumonia.     

A nested PCR assay for the detection of Mycoplasma hyopneumoniae in tracheobronchiolar washings from pigs

A nested polymerase chain reaction (PCR) was developed for the detection of Mycoplasma hyopneumoniae, the etiological agent of enzootic pneumonia, in tracheobronchiolar washings from live pigs. Two nested pairs of oligonucleotide primers were designed from the sequence of a specific DNA probe (I 141; accession number U02537). The primer combination was Hp1/Hp3 for the first step PCR while the nested primers (Hp4/Hp6) allowed amplification of a 706 bp fragment. All strains of M. hyopneumoniae tested in this study could be detected by the nested PCR. DNA from other bacterial species isolated from the respiratory tract of pigs or from other mycoplasmal species were not amplified. The detection limit was estimated to be 1 fg, corresponding approximately to one organism, while in the one step PCR previously described 4 x 10(2) organisms were required. The nested PCR was evaluated on 362 tracheobronchiolar lavages collected from pigs at 2, 4 and 6 months of age in eight herds chronically infected with M. hyopneumoniae. The nested PCR was compared to a blocking ELISA performed with sera collected from the same pigs at the same ages, and to an immunofluorescence test at slaughter on 65 lungs from 6-month old pigs. The comparison indicated that the nested PCR was significantly (p<0.05) more sensitive (157 positive results of 362 samples) than ELISA (118 positive results of 362 samples) for detection of M. hyopneumoniae infection. Nested PCR was also significantly more sensitive (54 positive results of 65 samples) than immunofluorescence (29 positive results of 65 samples) for detection of M. hyopneumoniae in pig lungs at slaughter. Moreover, the nested PCR was used to confirm the absence of the mollicute in a pig herd without any history of M. hyopneumoniae infection. Thus, nested PCR appears to be a useful test to assess M. hyopneumoniae infection on pig farms.     

Application and field validation of a PCR assay for the detection of Mycoplasma hyopneumoniae from swine lung tissue samples.

A PCR assay was validated for the detection of Mycoplasma hyopneumoniae in porcine lung tissue. The detection limit of the assay was 0.18 colony-forming units/g of lung sample spiked with M. hyopneumoniae. In field validation, 426 pigs from 220 cases were examined for M. hyopneumoniae infection by M. hyopneumoniae PCR and a fluorescent antibody (FA) test. In total, 103 pig lungs (24.2%) were positive in the PCR test, and 69 pig lungs (16.2%) were positive in the FA test, among which, 62 pigs were positive for both PCR and FA test. Most of the PCR-positive but FA test-negative cases had lesions compatible with M. hyopneumoniae infection. With Bayesian modeling, the diagnostic sensitivity and specificity of the PCR were determined to be 97.3% and 93.0%, respectively.     

An assessment of the duration of Mycoplasma hyopneumoniae infection in an experimentally infected population of pigs

Mycoplasma hyopneumoniae (M. hyopneumoniae) is the primary pathogen of enzootic pneumonia (EP), a highly prevalent respiratory disease that affects pigs worldwide. Previous studies have demonstrated that M. hyopneumoniae infection can be longer than 185 days; however, the total duration of infection has not been determined yet. Therefore, the objective of this study was to determine the duration of M. hyopneumoniae infection in asymptomatic carriers. To achieve our goal, 60 pigs were inoculated with M. hyopneumoniae strain 232 and the persistence of M. hyopneumoniae in the respiratory tract was assessed by detection of the bacterial DNA in bronchial swabs and the ability of the infected pigs to transmit the pathogen to sentinels. Infection of the inoculated animals was demonstrated by the detection of M. hyopneumoniae DNA in nasal swabs, seroconversion to the bacteria and the onset of dry coughing. Experimentally infected pigs shed M. hyopneumoniae prior to and after the cough was observed. M. hyopneumoniae DNA was detected in 100% of experimentally infected pigs at 94 days post infection (dpi), 61% at 214dpi and 0% at 254dpi. Experimentally infected pigs transmitted the bacteria to sentinels at 80 and 200dpi. Results of this study have demonstrated that M. hyopneumoniae infected pigs can be incubatory as well as convalescent carriers of the pathogen and that convalescent carriers can remain infectious for up to 200 days. Total clearance of M. hyopneumoniae in the group was evidenced, demonstrating that duration of M. hyopneumoniae infection lasts less than 254 days.     

Screening of Hungarian cattle herds for Mycoplasma mycoides subspecies mycoides small colony infection with negative results

At abattoirs and farms, 1248 sera were collected from animals representing 121 farms, and examined by complement fixation test using Mycoplasma mycoides subspecies mycoides small colony type (MmmSC) antigen. All sera were negative except seven from four farms, giving ++ reactions in the serum dilution of 1:10. On retesting, these sera and additional 30 sera collected repeatedly in both farms gave negative results. In isolation attempts, 953 lung samples collected from slaughtered cattle at the same abattoirs, and 326 nasal swabs collected from 11 herds proved to be negative for the presence of MmmSC, but M. bovis was isolated frequently. In the small farms 23.95% of the animals had pleurisy and/or pneumonia while in the large herds 34.69% had lesions. DNA extracted from 50 nasal swabs and 430 lung samples was examined by polymerase chain reaction (PCR) using M. mycoides cluster-specific primers. DNA from further 325 lung samples was tested by the more specific M. mycoides subspecies mycoides small colony/large colony/capri specific primers and 196 samples by nested PCR specific for MmmSC. All gave negative results. The detection level of cluster-specific primers and the more specific primers was 33.4 pg of DNA, whereas that of nested PCR was 0.33 pg.     

Dynamics and persistence of Mycoplasma hyopneumoniae infection in pigs

The purpose of this study was to describe the dynamics (shedding and transmission) of Mycoplasma hyopneumoniae infection within a population of swine and to determine the duration of the infection (persistence) through the identification of the agent in bronchial samples. Sixty-three 2-month-old pigs were used in this study. The pigs (n = 28) were experimentally infected by the intratracheal route with M. hyopneumoniae and considered as seeder pigs. The remaining pigs (n = 32) were not inoculated and randomly allocated to 2 different groups: direct contact exposure pigs (n = 12) and indirect contact exposure pigs (n = 20). Blood samples and nasal swabs were collected throughout the study on days 0, 28, 35, 42, 49, 63, 91, and 119 postinfection. To assess the duration of M. hyopneumoniae infection, 9 seeder and 6 contact exposure pigs were slaughtered at days 155 (group 1), 170 (group 2), and 185 (group 3) postinfection. Direct contact pigs showed evidence of infection on day 28 by polymerase chain reaction (PCR) and on day 35 by serology. The indirect contact exposure pigs presented a very delayed and slow seroconversion pattern; they did not present evidence of transmission until 42 d after the infection of seeder pigs. Identification of M. hyopneumoniae in bronchial swabs was confirmed by nested-PCR from days 155 to 185 postinfection. At the last slaughter date, 77.7% and 100% of the seeders and contact exposure pigs, respectively, tested positive. The results of this study reconfirmed direct infection of M. hyopneumoniae and suggest that indirect transmission can occur in a population. Finally, duration of the infection in this study was longer than previously described.     

Exploratory field study on Mycoplasma hyopneumoniae infection in suckling pigs

The present study focused on Mycoplasma hyopneumoniae (M. hyopneumoniae) detection by nPCR in nasal swabs of 507 suckling pigs. These animals came from 69 sows (from 1 to 8 parity number) of a farrow-to-finish herd with Enzootic Pneumonia (EP) problems at finishing stages. At 1 and 3 weeks of age (still in the farrowing units), nasal swabs and blood samples were taken from all piglets. Moreover, from these 507 animals, 37 randomly selected pigs were necropsied at 3 weeks of age. From those necropsied pigs, M. hyopneumoniae presence was tested in bronchial and tonsillar swabs. At 1 week post-farrowing, blood samples from sows were collected and used to detect M. hyopneumoniae antibodies. From the 69 analysed sows, 19 (27.5%) were seropositive. Global percentage of pigs with M. hyopneumoniae detection in nasal swabs at 1 and 3 weeks of age was 1.5% (8 out of 507) and 3.8% (19 out of 507), respectively. From these nPCR positive pigs, 89% (24 out of 27) were seronegative and 11% were seropositive. From necropsied animals, the pathogen DNA was detected in two pigs at bronchus level and in another pig at tonsil. In this study, sow parity was not statistically related with sow seropositivity and piglet colonization. These results confirm that M. hyopneumoniae infection may be detected not only in nasal cavities of naturally infected suckling piglets but also at their low respiratory tract airways. Our results suggest that M. hyopneumoniae detection in lower and upper respiratory tract could be an indicator that respiratory problems associated to EP may start relatively early in the production system. In consequence, sow-to-piglet and/or piglet-to piglet transmission in farrowing barns should not be underestimated.     

Chronological study of Mycoplasma hyopneumoniae infection, seroconversion and associated lung lesions in vaccinated and non-vaccinated pigs

A field trial was conducted to study Mycoplasma hyopneumoniae (Mh) infection dynamics by nested polymerase chain reaction (nPCR) and serology in pigs of a farm affected by enzootic pneumonia (EP). Moreover, correlation of Mh detection at different respiratory tract sites with presence of EP gross and microscopic lung lesions was assessed. These parameters were studied and compared between vaccinated (two doses at 1 and 3 weeks of age versus one dose at 6 weeks of age) and non-vaccinated pigs. Animals were monitored from birth to slaughter by nPCR from nasal swabs and by serology. From 3 to 22 weeks of age, an average of three pigs per treatment and per batch were necropsied (n = 302). The remaining pigs were sent to the slaughter (n = 103). Nasal, bronchial and tonsillar swabs were taken from the necropsied/slaughtered pigs; gross and microscopic EP-suggestive lung lesions were also assessed. Single and double vaccination resulted in earlier seroconversion and higher percentage of Mh seropositive pigs compared to control group. At slaughter, double vaccinated pigs showed lower percentage of EP-compatible gross lung lesions and lower Mh prevalence at upper respiratory tract sites (nasal cavity and tonsil) than control pigs.     

Detection of Mycoplasma mycoides subspecies mycoides in tissues from an outbreak of contagious bovine pleuropneumonia by culture, immunohistochemistry and polymerase chain reaction

Postmortem observations of 37 cattle from an outbreak of contagious bovine pleuropneumonia (CBPP) in north Italy in 1993 were made at the abattoir, where samples of lung and tracheobronchial lymph node tissues were taken for culture and identification of Mycoplasma mycoides subspecies mycoides (MmmSC), immunohistochemistry with the peroxidase anti-peroxidase (PAP) system, and molecular detection by the polymerase chain reaction (PCR) amplification of specific DNA from MmmSC. Nasal swabs were also taken for testing by PCR Lung pathology typical of CBPP was observed in 38 per cent of the animals, and MmmSC was isolated from 19 per cent DNA of MmmSC was detected by PCR in 64 per cent of lung samples and 35 per cent of the nasal swabs. Staining of lung tissue and lymph node tissue by PAP was positive in 27 per cent and 30 per cent of cases, respectively, and was a useful back-up test. These results suggest that PCR amplification from lung tissue may be used as a rapid and accurate confirmatory test for cases with pathology resembling CBPP.     

Evaluation and comparison of various PCR methods for detection of Mycoplasma gallisepticum infection in chickens

Four genetic Mycoplasma gallisepticum (MG) polymerase chain reactions (PCRs) (16s rRNA PCR, three newly developed PCR methods that target surface protein genes [mgc2, LP (nested) and gapA (nested)]) were compared for analytical specificity and sensitivity and for diagnostic sensitivity (Se) and specificity of detection from tracheal swabs. The licensed MG DNA Test Kit Flock Chek test (IDEXX, Laboratories, Inc., Westbrook, ME) was as well evaluated for the diagnostic specificity and sensitivity of detection from tracheal swabs. Analytical specificity was evaluated for the four generic PCR methods using a panel of DNA samples from microorganisms that may be isolated from the trachea of commercial poultry and other fowl. PCR methods mgc2, nLP, and ngapA only amplified DNA from MG, whereas 16S rRNA PCR amplified DNA from MG and Mycoplasma imitans. The analytical sensitivity of the four generic PCR methods expressed in color-changing units (CCU)/amplification reaction was estimated for each PCR method and ranged from 4 to 400 CCU/reaction; the sensitivities of single PCR methods 16S rRNA and mgc2 were estimated at 40 CCU/reaction, the nLP at 400 CCU/reaction, and the ngapA at 4 CCU/reaction. The diagnostic sensitivity and specificity of MG detection from tracheal swab pools, as compared to isolation from choanal cleft swabs, was evaluated for the five PCR methods using three groups of birds exposed to vaccine strains ts-11 and 6/85 and to challenge strain R. All PCR methods were able to detect the vaccine strains and the challenge strain R directly from tracheal swabs, indicating that PCR primers from the different methods amplified divergent MG strains. Isolation and PCR results correlated satisfactorily among the three experimentally infected groups, with agreement values (k) ranging from 0.52 to 1.00. The ngapA, IDEXX, and mgc2 PCRs showed the best sensitivity (Se) ratios for detection of M. gallisepticum strains as compared to isolation. Compared to the ngapA and IDEXX PCR methods, the mgc2 PCR has a faster turnaround time, since this test consists of a single amplification reaction and the amplification product is detected by gel electrophoresis. Therefore, among the PCR methods evaluated in this study, the mgc2 PCR is the method of choice to further validate in the field.     

PCR detection of Porcine circovirus type 2 DNA in whole blood,serum, oropharyngeal swab,nasal swab,and feces from experimentally infected pigs and field cases

Porcine circovirus type 2 (PCV2) shedding patterns were investigated by polymerase chain reaction (PCR) for the detection of PCV2 DNA, and the diagnostic suitability of a sample for the PCR was examined by using different types of samples. In the experimental infection, sixteen pigs were inoculated intranasally with PCV2. The samples, including oropharyngeal and nasal swabs, feces, whole blood and serum became positive for PCV2 DNA by PCR immediately after the inoculation, and almost all samples remained positive during the observation period, post-inoculation-day 70. Field samples were collected from 313 pigs in five different age groups. The overall percentages of positive samples in the whole blood, nasal swabs, and feces detected by PCR were 30.4%, 19.2%, and 20.4%, respectively. The frequency of positive samples increased after the nursery stages and reached a peak in the 3 to 4-month-old pigs. These results indicate that PCV2 infection may occur after weaning, that PCV2 DNA may be present in whole blood for a long period after infection, and that whole blood and serum are the most suitable sample types for the PCR analysis of PCV2.     

Evaluation of a nested PCR test and bacterial culture of swabs from the nasal passages and from abscesses in relation to diagnosis of Streptococcus equi infection (strangles)

REASONS FOR PERFORMING STUDY: Streptococcus equi is the cause of strangles in horses. To improve diagnostic sensitivity, development and evaluation of DNA-based methods are necessary. OBJECTIVES: To evaluate diagnostic methods and observe the pattern of bacterial shedding during natural outbreaks. METHODS: Two herds with natural outbreaks of strangles were visited over a period of 15 weeks and 323 samples originating from 35 horses investigated. The diagnostic use of a nested PCR test was evaluated using a collection of 165 isolates of Lancefield group C streptococci (species specificity) and swabs from nasal passages or from abscesses from horses infected with S. equi (diagnostic sensitivity). RESULTS: All 45 S. equi isolates tested positive in the nested PCR, whereas no amplicon was formed when testing the other 120 Lancefield group C isolates. A total of 43 samples were collected from 11 horses showing clinical signs of strangles during the study period. The diagnostic sensitivity for PCR test was 45% and 80% for samples from the nasal passages and abscesses, respectively; the corresponding diagnostic sensitivity for cultivation was 18% and 20%. The diagnostic sensitivity was significantly higher for PCR than for bacterial cultivation. Furthermore, the shedding of S. equi in 2 infected horse populations was evaluated. An intermittent shedding period of S. equi of up to 15 weeks was recorded in this part of the study. It was also shown that shedding of S. equi occurred both from horses with and without clinical signs. CONCLUSIONS AND POTENTIAL RELEVANCE: The nested PCR test represents a species-specific and -sensitive method for diagnosis of S. equi from clinical samples. It may, however, be desirable in future to develop detection methods with high diagnostic sensitivity and specificity without the potential problems inherent in nested PCR.     

Field validation of a real-time PCR test for the detection of Mycoplasma hyopneumoniae in nasal swabs of live pigs

Enzootic pneumonia (EP) of pigs, caused by Mycoplasma hyopneumoniae has been a notifiable disease in Switzerland since May 2003. The diagnosis of EP has been based on multiple methods, including clinical, bacteriological and epidemiological findings as well as pathological examination of lungs (mosaic diagnosis). With the recent development of a real-time PCR (rtPCR) assay with 2 target sequences a new detection method for M. hyopneumoniae became available. This assay was tested for its applicability to nasal swab material from live animals. Pigs from 74 herds (average 10 pigs per herd) were tested. Using the mosaic diagnosis, 22 herds were classified as EP positive and 52 as EP negative. From the 730 collected swab samples we were able to demonstrate that the rtPCR test was 100% specific. In cases of cough the sensitivity on herd level of the rtPCR is 100%. On single animal level and in herds without cough the sensitivity was lower. In such cases, only a positive result would be proof for an infection with M. hyopneumoniae. Our study shows that the rtPCR on nasal swabs from live pigs allows a fast and accurate diagnosis in cases of suspected EP.     

Use of touch-down polymerase chain reaction to enhance the sensitivity of Mycobacterium bovis detection.

The confirmatory diagnosis of Mycobacterium bovis (M. bovis) in animal samples is carried out by culture in Stonebrink media. However, culture is very slow because of the extremely long duplication time of the bacillus and difficult because of the scarcity of bacilli in diagnostic samples. This study describes the development of a single-tube touch-down polymerase chain reaction (PCR) protocol for the detection of M. bovis using primers that target the IS6110 element. Spiked water and milk as well as routine diagnostic samples (milk and nasal swabs) from M. bovis-positive cattle were tested. This protocol allows the rapid and sensitive detection of M. bovis in bovine samples by enhancing the sensitivity of standard PCR amplification.     

Influence of swab material on the detection of Mycoplasma gallisepticum and Mycoplasma synoviae by real-time PCR

Recent reports have shown an increased recovery of cells from flocked nylon swabs which may improve the specimen quality and the real sensitivity of diagnostic tests in a clinical setting. In this study, the detection of Mycoplasma gallisepticum (MG) and M. synoviae (MS), using dry swabs of different materials (nylon flocked, cotton, and polyester), was investigated using real-time TaqMan PCR protocols. Different types of samples, including dilutions of pure broth cultures of MG and MS as well as swabs from tracheas of experimentally infected chickens and field cases of infection, were analyzed. There were no statistical differences in real-time PCR results among the different swab types (P < 0.05), indicating that this is not likely to be a significant factor in MG and MS detection by this method.