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

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1 J Vet iagn Invest 11: (1999) Application of a nested polymerase chain reaction assay to detect Mycoplasma hyopneumoniae from nasal swabs Maria Calsamiglia, Carlos Pijoan, Alicia Trigo Abstract. The porcine respiratory disease complex (PRC) 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 PRC. 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 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 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 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. The porcine respiratory disease complex (PRC) is an increasingly important cause of decreased swine productivity and is characterized by slow growth, decreased feed efficiency, anorexia, fever, cough, and dyspnea. The effects of PRC are particularly severe in herds using segregated early weaning because the onset of disease is delayed until finishing, a more costly stage of production. Even though many farms have adopted multiple-site high health programs, the prevalence of pneumonia on many of these farms has not declined markedly and instead may be increasing on some farms. Mycoplasma hyopneumoniae is among the most prevalent and important infectious agents associated with PRC. 6 Mycoplasma hyopneumoniae infection alone causes pneumonia characterized by perivascular, peribronchial, and peribronchiolar lymphocytic cuffings, pneumocyte hypertrophy, and alveolar inflammation predominated by macrophages, plasma cells, and neutrophils. The airway damage caused by mycoplasmas often leads to secondary infections by opportunistic bacterial pathogens such as Actinobacillus From the epartment of Clinical and Population Sciences, College of Veterinary Medicine, University of Minnesota, St. Paul, MN (Calsamiglia, Pijoan), and Bayer Animal Health, Worthington, MN (Trigo). Received for publication February 3, pleuropneumoniae, Haemophilus parasuis, and Pasteurella multocida. Knowledge of mycoplasmal pneumonia has been hindered by inadequate diagnostic methods. Many of the currently available laboratory tests are relatively insensitive or nonspecific when used in a diagnostic laboratory setting or are too costly for routine diagnostic use. For instance, isolation of is seldom attempted in diagnostic laboratories because the organism is very fastidious. The technique is also rather insensitive: to culture the organism a minimum of microorganisms/ml are required, 7 and even then cultures are often overgrown by Mycoplasma hyorhinis, a common isolate from porcine lungs. Gross and microscopic lesions have been considered sufficient evidence for the diagnosis of mycoplasmal pneumonia. Reliance on these lesions, however, may also be misleading. In some studies, 19% of culturepositive lungs did not have gross or microscopic lesions. 2 In addition, macroscopic lesions indistinguishable from those caused by have been produced by coinfecting pigs with pseudorabies virus and Pasteurella multocida, 10 suggesting that gross lesions thought to be specific for mycoplasma infection may be due to a variety of respiratory pathogens. Immunofluorescent microscopy and immunohistochemistry are specific diagnostic methods but suffer 246

2 Nested PCR for 247 Table 1. Mycoplasma and bacterial strains used in this study. Species Strain Source Mycoplasma hyopneumoniae M. iowae M. iowae M. malleagridis M. gallisepticum M. synoviae M. hyorhinis M. flocculare Actinobacillus pleuropneumoniae Streptococcus suis serotype 2 Pasteurella multocida Actinomyces pyogenes Haemophilus parasuis Staphylococcus aureus Erysipelothrix rhusopathiae J 232 WS 5722 WS 232 WS 8689 K4297(1)3P K3862(384-76)10PT K3121(7248C)12PT K4914(1)5PT K4419(7)4P ATCC Cowan R. Ross, Iowa State University H. Joo, University of Minnesota A. Trigo, Oxford Laboratories A. Trigo, Oxford Laboratories A. Trigo, Oxford Laboratories R. Ross, Iowa State University R. Ross, Iowa State University from lack of sensitivity, especially when applied to postmortem specimens. 1,7 These tests are effective only when adequate quantities of antigen are present in the early stages of infection. In late stages of infection, the number of mycoplasmal organisms decreases 1 and the test is then frequently negative. Immunohistochemistry has additional disadvantages; it is time consuming and expensive and the reagents are not readily available commercially (J. E. Collins, personal communication). Serology is the only diagnostic method routinely used to detect infections in live animals. From the existing serologic tests, complement fixation (CF), indirect hemagglutination, and enzymelinked immunosorbent assay (ELISA), the ELISA is the most sensitive, although CF has been shown to detect antibodies earlier (2 weeks after infection). 1,4 The appearance of antibodies to, however, can be delayed by as much as 8 weeks after infection, making serologic interpretation less useful for the detection of recent infections in young pigs. 16 In late infections, it is common to find clinically affected pigs that reach slaughter while still seronegative. Also, the ELISA results may be confounded by occasional cross-reaction with Mycoplasma flocculare, a ubiquitous commensal organism of swine. Polymerase chain reaction (PCR) technology is ideally suited for diagnosis because it is rapid and specific, does not depend on viable bacteria, may be done on live or dead animals, and may be automated. Several PCR tests to specifically detect have been described. 3,5,12,15 A1- step PCR technique has been described that detects M. hyopneumoniae from lung samples and nasal swabs. 15 Here, a nested PCR assay is described and compared with a previously described 1-step PCR technique. 15 Materials and methods Organisms and growth conditions All strains used in this study are described in Table 1. Mycoplasma hyopneumoniae, M. hyorhinis, and M. flocculare were grown in liquid medium as previously described. 9 The other mycoplasmal organisms were grown in conventional mycoplasma medium (Frey). a Actinobacillus pleuropneumoniae and Haemophilus parasuis were grown in pleuropneumonia-like organism medium. Erysipelothrix rhusiopatihae was cultured on a blood agar plate, and bacteria were collected in phosphate-buffered saline (PBS). Streptococcus suis was grown in Todd Hewitt a broth, and the rest of microorganisms were grown in brain-heart infusion a broth. extraction Cultured microorganisms (1.5 ml) were centrifuged at 14,000 g for 30 min, and the pellets were resuspended in 250 l of PBS. Nasal swabs were resuspended in 300 l of PBS. Bacterial suspensions were boiled for 5 min, and was extracted with phenol : chloroform and precipitated with ethanol and sodium acetate (ph 5.2, final concentration of 0.5 M). was resuspended in 40 l of double-distilled water. Primers The first PCR was performed using the primers previously described, 16 which amplified a 649-bp region of the 16S ribosomal R (rr) gene. The primers for the nested PCR were designed by aligning b the 16S rr genes from different Mycoplasma species and other bacteria commonly isolated in the respiratory tract of swine. A species-specific region was chosen, and primers were designed using computer

3 248 Calsamiglia, Pijoan, Trigo software. c The primers amplified a 352-bp fragment that was in the 649-bp region amplified in the first reaction. The forward primer was 5-ACT AGA TAG GAA ATG CTC TAG T-3 (nucleotide positions ), and the reverse primer was 5-GTG GAC TAC CAG GGT ATC T-3 (nucleotide positions ). PCR Five microliters of the preparation was used as PCR templates in the first reaction, and 0.5 l of the product was used for the second reaction. The amplifications were performed in a 25-l reaction mixture containing 0.2 mm concentration of each primer, d 20 pmol of each nucleotide, 1 PCR buffer, e 5% glycerol, 3 mm MgCl 2, e and1uoftaq polymerase. e Both reactions were run in a thermocycler and required the same conditions: 30 cycles of denaturation at 95 C for 30 sec, primer annealing at 60 C for 45 sec, and extension at 72 C for 30 sec. Analysis of the amplified samples Seven-microliter aliquots of the amplified samples were analyzed by electrophoresis in a 1% agarose gel with 0.5 g/ml of ethidium bromide and then visualized, photographed, g and processed. h Products from both reactions were visualized to compare the sensitivity of conventional PCR versus the nested PCR. Sensitivity and specificity assays was extracted from pure cultures of bacteria (Table 1) using the same procedure described for the swab samples. To determine the sensitivity of the nested PCR, concentration of 10-fold dilutions of the was measured with a spectrophotometer. Five microliters of each dilution was then used in the first PCR, 0.5 ml of the resulting product was used for the second reaction. For gel visualization, 7 l of the 1-step and nested PCR products was used. In the specificity studies, the microorganisms listed in Table 1 were tested independently with both sets of primers. of those cultures was also extracted as described above for the swab samples. Challenge experiment Challenge inoculate. The challenge inoculate consisted of a mixture of 3 cultures containing strains WS 5722, WS 232, and MS 8689 in equal volume, after adjusting for opacity, corresponding to color-changing units/ml. Pig challenge. Ten 6 8-wk old specific-pathogen-free, M. hyopneumoniae-free pigs were challenged intratracheally with 10 ml of the challenge inoculate. Before challenge, nasal swabs were taken from those pigs to confirm their negative status for infection. The animals were housed at a research farm throughout the experiment. The temperature was maintained at 70 F, and feed and water were available to the animals ad libitum. Sampling. Intranasal swabs were taken from both nostrils prior to challenge (negative controls) and at 4 days and 1, 2, 3, 4, and 5 wk postchallenge. The swabs were placed in 1.0 ml of sterile PBS. Pigs were euthanized at different time intervals: 2 pigs at 3 wk postchallenge, 3 pigs at 4 wk postchallenge, and 5 pigs at 5 or 6 wk postchallenge, depending on the ELISA optical density reading of their sera. Lung tissues were collected and swabbed for PCR analysis. Samples of lung tissues that had been found negative in previous studies were used as negative controls. Extracted from a pure culture of was used as positive control. Farm trial Farm. A 300-sow farrow-to-finish enterprise, with a multiple site production system and in the process of expansion, was chosen for the study. New finishing barns were being built, and a delay in the building construction caused overcrowding and a break in the all-in/all-out protocols. The grow-finishing pigs at that farm then experienced an outbreak of respiratory disease, and was identified. Two finishing barns were visited. Barn A had pigs ranging from 16 to 21 wk of age. Barn B had a more homogeneous group of pigs, all of which were market weight. The observed clinical signs during the visit comprised coughing, and a relatively high number of pigs had shortened and deviated noses, suggesting atrophic rhinitis. Sampling. Three sets of pigs were sampled. Set 1 comprised 8 pigs of the youngest animals from barn A, set 2 comprised 18 pigs of the oldest animals from barn A, and set 3 comprised 14 market-weight pigs from barn B. These different sets were sampled to optimize the detection of microorganisms. The swabs were introduced into the nostrils and resuspended in PBS in the laboratory, followed by the extraction and PCR as described above. extracted from a pure culture of was used as positive control, and double-distilled water was the negative control. Identification of the nested PCR product. Four of the positive nested PCR products from these field cases were purified using a 30-k cutoff membrane ultrafiltration filter. The resulting sequences j were aligned and compared with that of 16S ribosomal. Results Specificity and sensitivity. Neither the outer nor the inner primers cross-reacted with any of the microorganisms tested, other than with the different strains used in this study (data not shown). Sensitivity results are shown in Fig. 1. The conventional PCR detected mycoplasma to a dilution of 10 4, corresponding to 0.1 ng of. Because the genome of is approximately 1,140 kilobase pairs, which is equivalent to 1.2 fg of, 10 5 fg (0.1 ng) represents approximately organisms. Conversely, the nested PCR detected to a dilution of 10 7, approximately 80 mycoplasmal organisms. Challenge experiment. All lungs from the challenged pigs gave a positive result with conventional PCR. However, only 2 of the nasal swabs from these same animals gave a positive result with the single

4 Nested PCR for 249 Table 2. Results* from nasal swabs from pigs challenged at different times after challenge with. Pig no. 1 day 4 day 1 wk 2 wk 3 wk 4 wk 5 wk * not available; died. Figure 1. Conventional PCR and nested PCR sensitivity. Tenfold dilutions of the extracted Mycoplasma hyopneumoniae (200 ng/l) from 10 1 to 10 8 were used; M. hyorhinis was the negative control (N) and yielded no product. M molecular mass marker, 100-bp ladder. k A. Conventional PCR. Product was detected up to the 10 4 dilution, which corresponds to organisms. B. Nested PCR. Product was detected up to the dilution 10 7, which corresponds approximately to 80 microorganisms. reaction. With the nested PCR, 61% of the nasal swab samples taken after challenge gave a positive reaction, as did all the lung samples. Results of individual shedding throughout the experiment are shown in Table 2. Farm trial. Mycoplasma hyopneumoniae was detected in the 3 tested groups; in barn A, 3/8 young pigs and 8/18 older pigs were positive. A gel of the nested PCR samples run for some of the older pigs of barn A is shown in Fig. 2. In barn B, 9/14 market-age pigs were positive. In total, 20/40 samples were positive. Four of those nested PCR products were sequenced, and their identity was confirmed as (data not shown). iscussion In the nested PCR assay used to detect from nasal swabs and other clinical samples, the gene encoding the 16S rr served as the target sequence for amplification. Even though this gene is present as a single copy gene in, 13 it has some advantages for this assay. Ribosomes have an essential function in protein synthesis; thus, the ribosomal genes show a high degree of conservation across species. 8 Highly conserved stretches can be used to amplify ribosomal genes of various species, whereas variable fragments can be used to amplify species-specific gene fragments. Species-specific regions of the ribosomal gene are used in both reactions, which makes it a highly specific assay. In a previous study, 17 4 diagnostic techniques were compared using experimentally infected animals. PCR analysis was the most sensitive assay for detecting positive animals using nasal swabs, in spite of the relatively low detection level of the assay (10 3 organisms). Although more sensitive than other diagnostic tests available, these results suggest that the conventional 1-step PCR is not sensitive enough for accurate detection of the organism in nasal swabs. In the present study, the conventional PCR assay Figure 2. Nested PCR amplification of nasal swabs from pigs on a farm experiencing an outbreak of Mycoplasma hyopneumoniae infection. A1 A8 nested PCR products from nasal swabs from group A pigs, M molecular mass marker, 100-bp ladder k ;Ppositive control, extracted from a pure culture of ; N negative control, double-distilled water.

5 250 Calsamiglia, Pijoan, Trigo was able to detect no fewer than bacterial cells. This level of detection was lower than that for other previously described PCR tests, 15,17 probably because of the rapid extraction protocol in which may have been lost or sheared. However, was extracted, because more repeatable PCR results are obtained than those obtained by only boiling the samples. In addition, extraction also eliminates Taq polymerase inhibitors from tissue samples. 14 Also, this extraction protocol is more rapid and inexpensive, being therefore better suited for large number of samples. The conventional PCR detected from all challenged lungs but only from 2/55 of nasal samples. The nested PCR was more sensitive and detected approximately 80 microorganism from a pure culture. The nested PCR was also able to detect the organism in 61% of the nasal swabs. The assay was therefore suitable for the detection of from nasal swabs, both in experimentally challenged pigs and in pigs from a farm experiencing a disease outbreak associated with. The organism is seldom isolated from nasal cavities, 11 for perhaps several reasons. The organism may be present in the nostrils in much smaller numbers than in the lower airways. These low numbers of microorganisms are difficult or impossible to detect by traditional culture techniques, because samples from the nasal cavity are usually highly contaminated. Another possibility is that the nested PCR detects from dead or degraded organisms, which will not grow on culture medium, explaining the poor culture results. This ability could represent a problem in the interpretation of a positive nested PCR; the assay could be detecting bacterial detritus instead of active mycoplasma shedding or colonization. However, this scenario is not very likely because during both swab sampling and rapid extraction and precipitation considerable bacterial is lost. Therefore, larger quantities of bacteria would be needed to yield at least 100 fg of purified. It is more likely therefore that the detected by the nested PCR was from actual live bacteria in the nostrils. However, further research is needed to determine the significance of these positive results and to correlate them with the carrier state and transmission of the microorganism. Another problem that these highly sensitive techniques have is contamination during manipulation of the samples: sample collection, extraction, PCR setting, and gel analysis. Care must be taken during each of these steps, and negative controls must be included for each reaction. However, this reaction could also give false-negative results. To detect those cases where the negative result is due to a lack of amplification in that particular sample, an internal standard could be added in each reaction. In the experimental challenge trial, was detected from nasal swabs throughout the experiment. Nasal shedding was not continuous, although no conclusions can be drawn at this time on shedding dynamics, because the study involved only 10 animals. To better characterize the duration and timing of nasal shedding, further studies will be performed involving more animals and a longer observation period. In conclusion, the nested PCR described in this study was highly sensitive, specific, and better than conventional PCR for the detection of microorganisms from nasal swabs, a site where the organism is apparently present in lower quantities and that is more contaminated than the lower respiratory sites. This PCR should constitute a very valuable tool for diagnosing the disease and detecting carrier animals and should be particularly useful in matching the health status of incoming replacement animals to that of the existing swine population, a major problem in modern operations. Sources and manufacturers a. BBL Microbiology System, Cockeysville, M. b. MegAlign, STAR, Madison, WI. c. OLIGO 4.0 program, National Biosciences, Plymouth, MN. d. Ransom Hill Bioscience, Ramona, CA. e. Boehringer GmbH, Mannheim, Germany. f. Perkin-Elmer, GeneAmp PCR system 2400, Branchburg, NJ. g. Stratagene, La Jolla, CA. h. Adobe Photoshop 3.0 for the Macintosh, Adobe Systems, San Jose, CA. i. Amicon, Beverly, MA. j. ABI PRISM 377 sequencer, Foster City, CA. k. GIBCO BRL, Gaithersburg, M. References 1. Amanfu W, Weng CN, Ross RF, et al.: 1984, iagnosis of mycoplasmal pneumonia of swine: sequential study by direct immunofluorescence. Am J Vet Res 45: Armstrong CH, Scheidt AB, Thacker HL, et al.: 1984, Evaluation of criteria for the postmortem diagnosis of mycoplasmal pneumonia of swine. Can J Comp Med 48: Artiushin S, Stipkovits L, Minion FC: 1993, evelopment of polymerase chain reaction primers to detect Mycoplasma hyopneumoniae. Mol Cell Probes 7: Bereiter M, Young TF, Joo HS, et al.: 1990, Evaluation of the ELISA and comparison to the complement fixation test and radial immunodiffusion enzyme assay for detection of antibodies against Mycoplasma hyopneumoniae in swine serum. Vet Microbiol 25: Blanchard B, Kobisch M, Bove JM, et al.: 1996, Polymerase chain reaction for Mycoplasma hyopneumoniae in tracheobronchiolar washings from pigs. Mol Cell Probes 10: ee S: 1996, The porcine respiratory disease complex: are subpopulations important? Swine Health Prod 4: oster AR, Lin BC, Erickson E: 1985, Use of various labo-

6 Nested PCR for 251 ratory techniques for the diagnosis of mycoplasmal pneumonia in swine. Proc Annu Meet Am Assoc Vet Lab iagn 28: Fox GE, Stackebrandt E, Hespell RB, et al.: 1980, The phylogeny of prokaryotes. Science 209: Friis NF: 1975, Some recommendations concerning primary isolation of Mycoplasma suipneumoniae and Mycoplasma flocculare. A survey. Nord Vet Med 27: Fuentes M, Pijoan C: 1987, Pneumonia in pigs induced by intranasal challenge exposure with pseudorabies virus and Pasteurella multocida. Am J Vet Res 48: Goodwin RFW: 1972, Isolation of Mycoplasma suipneumoniae from the nasal cavities and lungs of pigs affected with enzootic pneumoniae or exposed to this infection. Res Vet Sci 13: Harasawa R, Koshimizu K, Takeda O, et al.: 1991, etection of Mycoplasma hyopneumoniae by the polymerase chain reaction. Mol Cell Probes 5: Huang Y, Stemke GW: 1992, Construction of the physical maps of Mycoplasma hyopneumoniae and Mycoplasma flocculare and the location of rr genes on these maps. Can J Microbiol 38: Luneberg E, Skovjensen J, Frosch M: 1993, etection of Mycoplasma pneumoniae by polymerase chain reaction and nonradioactive hybridization in microtiter plates. J Clin Microbiol 31: Mattsson JG, Bergstrom K, Wallgreen P, et al.: 1995, etection of Mycoplasma hyopneumoniae in nose swabs from pigs by in vitro amplification of the 16S rr gene. J Clin Microbiol 33: Sitjar M, Noyes EP, Simon X, et al.: 1996, Relationships among seroconversion to Mycoplasma hyopneumoniae, lung lesions, and production parameters in pigs. Swine Health Prod 4: Sorensen VP, Ahrens K, Barfod A, et al.: 1997, Mycoplasma hyopneumoniae infection in pigs: duration of the disease and evaluation of four diagnostic tests. Vet Microbiol 54:23 34.