Molecular Identification of Mycoplasma agalactiae in Iran Based on P30 Gene

Document Type : Original Articles

Authors

1 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran

2 Mycoplasma Reference Laboratory, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran

3 Central Laboratory Department, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran

Abstract

Mycoplasma agalactiae (M. agalactiae) is known as the main etiological agent of contagious agalactia (CA). The CA is a disease affecting dairy sheep and goats, the main characteristics of which include keratoconjunctivitis, arthritis, and mastitis. This pathogen results in milk production reduction and suppression, thereby leading to serious economic loss. In the present study, 125 sheep and goat samples were collected from 15 provinces of Iran. Cultural and molecular methods were used for sample characterization. After extracting genomic DNAs using the phenol/chloroform method, the PCR technique was employed to detect Mycoplasma genus in 163bp fragment of 16S rRNA gene (M-PCR) and M. agalactiae in 800bp fragment of conserve and specific P30 lipoprotein gene (P30-PCR) in cultural and clinical samples. Finally, to validate the experimental approach, a 375 bp amplicon of P80 lipoprotein was amplified using the MA-PCR. Out of 125 samples under investigation, 43 cases were positive, and Mycoplasma colonies were observed in the pleuropneumonia-like organisms agar culture. Based on the results of the M-PCR method, 61 specimens (out of 125 samples) were scored positive for Mycoplasma presence. Furthermore, 20 samples were positive according to the P30-PCR data. It should be mentioned that the MA-PCR was performed based on the P80 gene on 125 total samples to furtherverify the results for M.agalactiae detection. Based on the obtained data, P30 and P80 genes were presented and amplified in all Iranian M. agalactiae isolates (n=20). Our results indicated that the P30 gene was conserved and specific to all Iranian M. agalactiae isolates and this new P30-PCR method (as an MA-PCR technique) might be useful in the detection of this pathogen.

Keywords

Article Title [French]

Identification moléculaire de Mycoplasma agalactiae en Iran sur la base du gène P30

Abstract [French]

Mycoplasma agalactiae (M. agalactiae) est connu comme le principal agent étiologique de l'agalactie contagieuse (AC). L'AC est une maladie affectant les ovins et caprins laitiers, dont les principales caractéristiques comprennent la kératoconjonctivite, l'arthrite et la mammite. Cet agent pathogène entraîne une réduction et une suppression de la production laitière, entraînant ainsi de graves pertes économiques. Dans la présente étude, 125 échantillons de moutons et de chèvres ont été collectés dans 15 provinces d'Iran. Des méthodes culturales et moléculaires ont été utilisées pour la caractérisation des échantillons. Après avoir extrait les ADNs génomiques à l'aide de la méthode phénol/chloroforme, la technique de PCR a été utilisée pour détecter le genre Mycoplasma dans un fragment de 163pb du gène de l'ARNr 16S (M-PCR) et M. agalactiae dans un fragment de 800pb de la conservation et le gène de la lipoprotéine P30 spécifique (P30- PCR) dans des échantillons culturels et cliniques. Enfin, pour valider l'approche expérimentale, un amplicon de 375 pb de lipoprotéine P80 a été amplifié à l'aide de la MA-PCR. Sur 125 échantillons étudiés, 43 cas étaient positifs et des colonies de Mycoplasmes ont été observées dans la culture sur gélose d'organismes de type pleuropneumonie. Sur la base des résultats de la méthode M-PCR, 61 échantillons (sur 125 échantillons) ont été marqués positifs pour la présence de mycoplasmes. De plus, 20 échantillons étaient positifs selon les données P30-PCR. Il convient de mentionner que la MA-PCR a été réalisée sur la base du gène P80 sur 125 échantillons au total pour vérifier davantage les résultats de détection de M.agalactiae. Sur la base des données obtenues, les gènes P30 et P80 ont été présentés et amplifiés dans tous les isolats iraniens de M. agalactiae (n=20). Nos résultats ont indiqué que le gène P30 était conservé et spécifique de tous les isolats iraniens de M. agalactiae et que cette nouvelle méthode P30-PCR (en tant que technique MA-PCR) pourrait être utile dans la détection de ce pathogène.

Keywords [French]

  • Agalactie Contagieuse
  • Culture
  • Mycoplasma agalactiae
  • PCR
  • gène P30

Introduction

Mycoplasma agalactiae has been recognized as the main agent of contagious agalactia (CA) disease, which affects dairy sheep and goats; moreover, it is mainly identified by keratoconjunctivitis, arthritis, and mastitis (Office International des Epizooties, 2008). This pathogen results in milk yield reduction and suppression, and consequently, serious economic loss ( Oravcova et al., 2009 ). This syndrome is produced by Mycoplasma agalactiae subsp. agalactiae though other Mycoplasmas (e.g., M. capricolum subsp. capricolum, M. mycoides subsp. capri, M. mycoides subsp. Mycoides, and M. putrefaciens) have been also reported to cause this disease ( Oravcova et al., 2009 ).

Contagious agalactia has been observed in several countries and is considered an endemic disease in the majority of Mediterranean countries ( Bergonier et al., 1997 ; Zendulkova et al., 2004 ). Surface lipoprotein phase variation is a major mechanism in Mycoplasmas and is considered an important adaptive strategy in this family of pathogens ( Nouvel et al., 2009 ). It probably allows Mycoplasmas to evade their host immune response and cause chronic infection ( Browning et al., 2011 ). Recent studies on genomic comparison of different pathogenic Mycoplasmas have provided new insights into the role of lipoproteins in pathogenicity ( Browning et al., 2011 ).

According to the results of immunoblotting analysis utilizing a monospecific and polyclonal anti- P30-His serum, P30 has been considered a specific, stable, and highly immunogenic M. agalactiae antigen, and therefore, can be used to detect, identify, and subtype M. agalactiae ( Fleury et al., 2002 ). Furthermore, based on the outcomes of Southern blot analysis and PCR technique using P30 specific primers, this gene is present in all M. agalactiae strains, while being absent in other species of Mycoplasma ( Fleury et al., 2002 ).

Several molecular studies have been previously performed on the isolation, detection, and identification of M.agalactiae by the amplification of 375 bp of P80 lipoprotein gene (the studies conducted by Khezri et al. [2012] on 69 sheep samples in Kurdistan province, Kheirkhah et al. [2011] on 57 samples of Iranian goats, Ashtari et al. [2015] on 91 sheep samples affected by contagious agalactia in Khuzestan province, as well as Momen and Roshdi Maleki on 367 samples in Hamadan province).

This study aimed to detect and identify M. agalactiae using new specific primers based on conserve and specific P30 gene.

Material and Methods

Cultural Method and DNA Extraction. In total, 125 samples were collected from the eye, ear, joint exudate, and milk secretions of sheep and goats exhibiting clinical signs of probable infection with Mycoplasma. It should be noted that the samples were obtained from 15 Provinces of Iran (East Azerbaijan, West Azerbaijan, Ardabil, Isfahan, Kerman, Qom, Khuzestan, Semnan, Kermanshah, Golestan, Gilan, Yazd, Sistan and Baluchestan, Kurdistan, and Ilam). After transferring the samples to the medium and incubating them on ice, they were taken to the Mycoplasma Reference Laboratory of Razi Vaccine and Serum Research Institute, Karaj, Iran, for subsequent studies.

All samples were attenuated, filtered into fresh pleuropneumonia-like organisms (PPLO) broth, and then inoculated into PPLO agar (BBL, Becton, Dickinson and Company, Cockeysville, Sparks, MD). The inoculated agar and broth were incubated at 37°C, 5% Co2, and 98% humidity. The broths and plates were daily observed for the signs of growth and typical Mycoplasma colonies, respectively. To extract DNA, the phenol-chloroform method previously described by Tola et al. was used in this study (1997).

Detection of Mycoplasma Genus. In this study, a previously published primer set was utilized for specific detection of Mycoplasma genus (i.e. M1F: 5' GCTGCGGTGAATACGTTCT-3', M3R: 5'- TCCCCACGTTCTCGTAGGG-3') ( van Kuppeveld et al., 1992 ). Following that, the PCR was performed using 25 μl samples, each containing 2.5 μl of 10 X PCR buffer (CinnaGen, Iran), 2 mM MgCl2, 5 mM dNTPs, 2 μl of each primer (10 pm), and 0.5 U Taq DNA polymerase (CinnaGen, Iran). Eventually, 15.3 μl of deionized distilled water and 50 ng of extracted DNA as the template were added. The PCR was carried out using a Gradient Master cycler instrument (Eppendorf, Germany) according to the following protocol ( Zendulkova et al., 2007 ): 7.5 min at 94°C, followed by 30 cycles of 30 sec at 94°C, 30 sec at 56°C, and 1 min at 72°C with a final extension cycle for 5 min at 72°C. The M-PCR assay was performed for Mycoplasma genus detection in 163bp fragment of 16S rRNA gene.

Detection of Mycoplasma agalactiae Based on P30 Gene. One pair of primers (i.e. P30F: 5'-GCA GTT TTA AAT AAC ACA GG-3' and P30R: 5' AAA TCT TGC GCG CAG CAA GA-3') was designed in the open reading frame region of P30 gene by Oligo 5.0 software. The P30 coding sequence was amplified via the PCR technique using 50 μl samples, each containing 5 μl of 10 X PCR buffer (CinnaGen, Iran), 2 mM MgCl2, 10mM dNTPs, 2 μl of each primer (10 pm), 0.2 μl (1 unit) of Taq DNA polymerase (CinnaGen, Iran), 37.8 μl of deionized distilled water, and finally, 100 ng of extracted DNA as the template. The PCR was performed in a Gradient Master cycler (Eppendorff, Germany) based on the following protocol: 3 min at 95°C, followed by 35 cycles of 1 min duration at 93°C, 30 sec at 54°C, and 40 sec at 72°C with a final 5-min extension cycle at 72°C. In the P30-PCR assay, 800bp fragment of P30 membrane lipoprotein gene was amplified to detect M. agalactiae species.

Detection of Mycoplasma agalactiae based on P80 Lipoprotein. The following M. agalactiae - primer set was used for the specific detection of M. agalactiae species: FS1: 5'AAA GGT GCT TGA GAA ATG GC-3' and FS2: 5'-GTT GGC AGA AGA AAG TCC AAT CA-3', as suggested in a previous study ( Tola et al., 1997 ). The prepared 25 μl samples for PCR assay contained 2.5 μl of 10 X PCR buffer (CinnaGen, Iran), 2 mM MgCl2, 5 mM dNTPs, 2μl of each primer (10 pm), 0.5 U Taq DNA polymerase (CinnaGen, Iran), 15.3 μl of deionized distilled water, and 50 ng of extracted DNA as the template. The PCR was conducted in a Gradient Master cycler (Eppendorf, Germany) based on Tola et al. (1997) reported protocol with some modification: 5 min at 95°C, followed by 34 cycles of 1 min duration at 94°C, 1 min at 50°C, and 1 min at 72°C with a final 5-min extension cycle at 72°C. In MA-PCR, a fragment of the P80 lipoprotein gene with 375 base pair length was amplified to detect M. agalactiae species.

Visualization of amplified products (M-PCR, MA-PCR, and P30-PCR) was performed through ultraviolet illumination following electrophoresis (using agarose gel (1%] in TAE buffer) and SYBR Safe staining ( Tola et al., 1997 ).

Results

According to the cultural method, typical Mycoplasma colonies were observed in 43 (34.4%) sheep and goat samples (out of 125 samples), while 82 (65.6%) samples were negative for this pathogen. The positive Mycoplasma samples in the PPLO broth media were also cultured on PPLO agar media, and the resulting Mycoplasma colonies exhibited distinctive "fried egg" morphology under a light microscope.

Out of 125 total samples, 61 (48.8%) and 64 (51.2%) positive and negative samples for Mycoplasma presence were identified by Mycoplasma genus PCR (M-PCR) assay, respectively. For the detection of Mycoplasma genus, a DNA fragment with approximately 163 bp from 16SrRNA was amplified successfully in all Mycoplasma culture-positive samples and 18 other specimens (Figure 1).

Figure 1.Mycoplasma genus PCR (M-PCR): PCR electrophoresis analysis in %1 gel agarose. M: Marker (100bp DNA ladder). Lane C+: Positive control (163bp band, Mycoplasma genus), Lane C-: Negative control (uncultured PPLO broth), and Lane 1 to 4 are the Mycoplasma isolates in this study.

In the P30-PCR method, 20 (16%) and 105 (84%) samples were positive and negative, respectively. The complete coding sequence of the P30 gene with 800bp length was successfully amplified in all 20 isolates of M. agalactiae (Figure 2).

Figure 2. P30-PCR: PCR electrophoresis analysis in %1 gel agarose. M: Marker (100bp DNA ladder). Lane C-: Negative control (uncultured PPLO broth), Lane C+: Positive control (800bp band, P30 gene of Mycoplasma agalactiae), and Lane 1 to 4 are the Mycoplasma isolates in this study.

The results of MA-PCR were similar to those of P30-PCR exactly. The complete coding sequence of the P80 gene with 375 bp length was amplified in 20 samples (out of 125 samples) which were positive in the P30-PCR method as shown in Figure 3. Furthermore, M. agalactiae positive samples from a total of 125 samples were successfully detected using two P30-PCR and MA-PCR M. agalactiae specific PCR methods. According to both P30-PCR and MA-PCR results, milk samples were the most affected specimens, while joint exudates and ear samples were the least influenced samples by M. agalactiae (Table 1). In addition, it was proved that 20 (16%) samples were infected with M. agalactiae based on cultural and PCR methods, and this pathogen was successfully detected using both methods.

Figure 3.Mycoplasma agalactiae PCR (MA-PCR): PCR electrophoresis analysis in %1 gel agarose. M: Marker (100bp DNA ladder). Lane C+: Positive control (375bp band, Mycoplasma agalactiae), Lane C-: Negative control (uncultured PPLO broth), and Lane 1 to 4 are the Mycoplasma isolates in this study.

Tests Result Milk sample Ear sample Eye sample joint sample Total
Culture Positive 24 3 15 1 43
Negative 30 5 36 11 82
M-PCR Positive 31 4 25 1 61
Negative 23 4 26 11 64
P30-PCR Positive 14 0 6 0 20
Negative 40 8 45 12 105
MA-PCR Positive 14 0 6 0 20
Negative 40 8 45 12 105
Table 1. Results of cultural and three PCR methods in all samples based on the clinical sampling

The results of P30-PCR and MA-PCR methods were similar exactly, and therefore, the P30-PCR method using the newly designed primers (P30-F, P30-R) can be a good alternative to the routine MA-PCR method using commonly used primers (FS1-FS2) in M. agalactiae detection (Table 1).

Discussion

The samples of the mammary gland (milk), conjunctivae (eye), external canal of ear, and joint exudate of sheep and goats for M. agalactiae detection were used in this study based on the P30 gene. All 125 samples (isolated from 15 provinces of Iran) were cultured in the PPLO broth media for the diagnosis of Mycoplasma. The M-PCR was also performed to detect Mycoplasma genus based on the 16S rRNA gene. Furthermore, the P30-PCR method was used to identify samples by M. agalactiae (Table1). In addition, it was proved that 20 (16%) samples were infected with M. agalactiae based on cultural and PCR methods, and this pathogen was successfully detected using both methods.

The results of P30-PCR and MA-PCR methods were similar exactly, and therefore, the P30-PCR method using the newly designed primers (P30-F, P30-R) can be a good alternative to the routine MA-PCR method using commonly used primers (FS1-FS2) in M. agalactiae detection (Table 1).

M. agalactiae based on P30 gene coding for a membrane and immunodominant lipoprotein. To verify our results for M. agalactiae detection by the P30-PCR method and gain a better understanding of Iranian isolates of this pathogen, the presence of the P80 lipoprotein gene was checked in all samples using the MA-PCR method. On the other hand, a comparative analysis was performed between two P30-PCR and MA-PCR methods to examine the specificity of the P30 gene for M. agalactiae isolates. According to the results, the expected 800 bp fragment belonging to the P30 gene was amplified from chromosomal DNA of all M. agalactiae isolates used in this study, while it was not observed for other tested Mycoplasma species. Therefore, it can be concluded that the P30 gene is highly specific to M. agalactiae isolates.

In order to determine the sensitivity of the P30-PCR assay, a serial dilution of extracted DNA was prepared (100ng- 10ng- 1ng- 100pg- 10pg). Finally based on the results, the sharp band belonging to the P30 gene appeared in >1ng dilution of extracted DNA from M. agalactiae isolates.

As a molecular detection method, PCR is a very sensitive and commonly used approach, which can be considered an early and rapid warning system for clinical samples. This method allows performing a full assessment in the case of positive results though negative outcomes cannot be regarded as definitive. The detection limit of the cultural method for M. agalactiae has been suggested to be 2×102 CCU*/ml ( Bergonier et al., 1997 , Tola et al., 1997 ). Our findings were in line with the results reported by Kheirkhah et al. (2011) in Iran and Amores et al. (2010) in Spain, indicating that PCR was a more reliable and successful technique for M. agalactiae detection, compared to the cultural method. Furthermore, the results of the present study were consistent with those observed by Tola et al. (1997), proving that PCR was much sensitive and faster than the cultural method and reduced the required time for detection.

Based on the results of this study, 12% of eye samples and 26% of milk samples were positive in P30-PCR and MA-PCR, respectively. However, P30 and P80 genes were not detected in ear samples and joint exudates. Kheirkhah et al. (2011) have detected M. agalactiae in milk and joint exudates of goats and observed the highest number of Mycoplasma colonies in joint exudates. The obtained data in the present study were partially consistent with their findings since M. agalactiae was detected in the milk and eye samples; however, this pathogen was not detected in joint exudates and ear samples. The results might have been dependent on the initial number of clinical samples obtained from different sampling sites. As can be observed in Table 1, the total numbers of joint and ear samples were fewer than those of milk and eye specimens due to difficulty in sampling. The present study was conducted on suspected samples, and it was associated with more reliable results, compared to other studies ( De la Fe et al., 2007 ; Zendulkova et al., 2007 ) on samples with or without signs of CA. In the above-mentioned studies, M. agalactiae has been detected based on the DNA fragment of the P80 gene with 375bp length.

Based on previous studies, the prevalence of contagious agalactia (agalactiae) in a herd of sheep/goat in Iran has been significantly increased in recent years such that since 2011, the incidence rate of this disease has reached 33.3% ( Kheirkhah et al., 2011 ). Many variable genes in M. agalactiae play prominent roles in inducing host immunity to infection and facilitating immune evasion ( Browning et al., 2011 ). Therefore, detection and identification of conserved and specific genes in M. agalactiae are among important strategies for controlling M. agalactiae infections. One of the conserved genes in M. agalactiae is P40, which encodes an immunodominant lipoprotein that plays a role in cytoadherence and pathogenicity ( Oravcova et al., 2009 ). Mahdavi et al. (2009) have studied the P40 gene in three Iranian vaccine strains of Mycoplasma agalactiae and reported the homology percentage of this gene among these strains; however, the aforementioned study was limited to vaccine strains and involved no field isolates. Furthermore, based on a study carried out by Fleury et al. (2002), P40 is associated with a functional analogy in Mycoplasma bovis and Mycoplasma hominis species. They found that the P40 gene has no satisfactory specialty for M. agalactiae . However, according to previous studies conducted by Fleury et al. (2002), the P30 is a specific, stable, and highly immunogenic M. agalactiae antigen, inducing a strong and persistent immune response.

To analyze the specificity of the P30 gene for M. agalactiae , a primer pair (P30F and P30R) was designed that was complementary to the extremities of P30. For the first time in Iran, the expected 800bp complete coding sequence was amplified from chromosomal DNA of all M. agalactiae isolates used in this study (Figure 1); however, other Mycoplasma species were not tested in this regard. ​Based on the results, the P30 gene was present in all tested M.agalactiae, and it was specific to this pathogen species. The findings of this study were in agreement with the data reported by Fleury et al. (2002) in Switzerland, in which the P30 gene was specific to M. agalactiae species according to PCR outcomes.

According to the findings of our study, the P30 gene was specific to M. agalactiae , and no particularly similar analogs to P30 were found in related species. Moreover, it was indicated in this study that the new PCR assay designed based on the P30 gene was useful in detecting M. agalactiae as an MA-PCR approach.

A similar study is required for phylogenetic analysis of the P30 gene of M. agalactiae in field and vaccine isolates via applying this lipoprotein gene sequence in GenBank to identify the source of infection. Furthermore, it is necessary to conduct a complete P30 molecular typing study to obtain genomic diversity and genomic homogeneity in Iranian M. agalactiae isolates as candidates for the application in diagnostic ELISA kits and recombinant vaccines against contagious agalactia (agalactiae).

*Color Chaining Unite

Authors' Contribution

Study concept and design: S. A. P.

Acquisition of data: M. B.

Analysis and interpretation of data: M. E.

Drafting of the manuscript: M. B. and S. A. P.

Critical revision of the manuscript for important intellectual content: Z. N.

Statistical analysis: H. G.

Administrative, technical, and material support: RVSRI

Ethics

It is declared that all ethical standards have been respected in the preparation of the submitted article.

Grant Support

This study was financially supported by Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran (Grant Number: 2-18-18-94133).

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Archives of Razi Institute
Volume 76, Issue 1
March 2021
Pages 61-68

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