Abstract: Background and Objective: Bacteriological isolation and identification of Mycoplasma species is difficult and time-consuming, therefore, molecular identification of Mycoplasma using PCR targeting specific genes is considered a specific and sensitive method for identification. The aim of current study was to isolate, characterize Mycoplasma infection in dromedary camels in Saudi Arabia. Materials and Methods: Nasal swabs were randomly collected from 93 camels and tested for Mycoplasma by sequencing of their 16S rRNA genes using universal primers. Results: The 93 samples, 24 were positive for Mycoplasma. However, no positive results were obtained using species-specific primers for Mycoplasma arginine, M. bovis or M. mycoides subsp. mycoides, thus, 16S rDNA sequencing methods and semi-nested PCR were employed. Sequences were matched to those in GenBank and phylogenetic analysis was performed. Mycoplasma edwardii (77-84% similarity with Mycoplasma edwardii ATCC 23462) and one isolate of Mycoplasma yeastsii (100% similarity with M. yeastsii GM274B) were identified. Further, some Mycoplasma species were identified as previously uncultured. The incidence of Mycoplasma infection in camels in Taif city, Saudi Arabia, was approximately 26%. Conclusion: This study provides insights into the accuracy and efficiency of PCR and universal primers for the detection and identification of Mycoplasma, thereby circumventing conventional culturing methods that require several days to complete and exhibit low accuracy.
INTRODUCTION
Dromedary camels are one of the primary sources of income for a significant proportion of the population living in desert areas in the Kingdom of Saudi Arabia. Moreover, the meat and milk sourced from camels supplies approximately 30% of the total demand in the Kingdom of Saudi Arabia1. Therefore, improving the overall health of domestic camels would help producers meet the recent increasing demand for high-quality camel meat and milk2. Camels were previously considered to be resistant to most known animal diseases; however, recent studies reported that camels are susceptible to a variety of pathogens3.
Many advances have been made in the diagnosis and treatment of bacterial infections in camels over the past 2 decades, but the understanding of Mycoplasma infections in camels remains inadequate. The absence of information regarding the role of Mycoplasma as a causative agent in camel disease is primarily related to the scarcity of mollicute studies, including those on Mycoplasma, Ureaplasma or Acholeplasma in camels4,5. Moreover, the primary habitat of Mycoplasma in clinically healthy camels remains unknown6,7.
Mycoplasma belong to the class Mollicutes, which consist of wall-less prokaryotes that are among the smallest known self-replicating organisms8,9. The absence of the bacterial cell wall in Mycoplasma species makes their infection difficult to diagnose and treat10,11. Mycoplasma-related diseases are undetectable when diagnosis is based solely on clinical indicators such as pathological lesions or serological testing primarily because of the close taxonomic association of many Mycoplasma species. Classical methods used to detect and identify infection by Mycoplasma are time-consuming and can be complicated by serological cross-reactions between closely related taxa. The introduction of molecular diagnoses, including PCR methods have greatly improved the detection and identification of Mycoplasma infections11,12. This study was conducted to determine the nature and type of Mycoplasma infection common in the upper respiratory tract of dromedary camels in Taif city, Mecca province, Kingdom of Saudi Arabia, using sequencing of Mycoplasma 16S rDNA, together with semi-nested PCR.
MATERIALS AND METHODS
Isolation of Mycoplasma: This study protocol was approved by the Taif University Medical Ethics Review Board and performed in accordance with the guidelines for the protection of human and animal subjects. Nasal swabs and lung tissues were randomly collected from a total of 93 camels in a slaughter house in Taif, Saudi Arabia from October, 2017 to April, 2019, with a total number of 186 samples. The 93 swabs were cultured in pleuropneumonia-like organisms (PPLO) broth media at 37°C for 24 h and then inoculated on PPLO media agar plates at 37°C for 1 week, to evaluate the presence or absence of Mycoplasma colonies13. Lung tissue showing consolidation and grey and red hepatization were considered as abnormal. Lung tissues collected from slaughtered camels were packed in individual sterile plastic bags. Samples were transported in an icebox to the laboratory for storage at -20°C until analysis.
DNA extraction: Genomic DNA was extracted from the collected lung tissue using a Tissue DNA extraction kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The DNA samples were stored at -20°C until further use.
PCR-based detection of Mycoplasma and amplification of the 16S rRNA gene: Paired primers were used for the molecular detection of Mycoplasma from nasal swab cultures. Three species-specific primers suitable for detecting three mollicute species and universal 16S rDNA primers capable of detecting all Mycoplasma species were employed (Table 1) according to Kazemiha et al.14. The universal 16S rRNA primer was designed as described by Lauerman et al.15 and was approximately 425 bp in length. These primers were used to target the conserved regions within 16S rDNA intergenic spacer regions. Thermal conditions were adjusted as described16. The semi-nested PCR primers descried previously by Pettersson et al.17.
In vitro amplification of 16SrRNA gene by semi-nested PCR: The 16S rDNA genes from Mycoplasma were amplified by semi-nested PCR according to the methods of Pettersson et al.17. The semi-nested PCR primers were previous reported17. The PCR was performed using 2X PCR Go Taq® Green Master Mix (Promega). Each 25 μL reaction mixture contained all necessary reagents (200 nm of each dNTP and 0.6 units of Taq DNA polymerase). Fifty nanograms of genomic DNA and 10 pmol of each primer were added to the mix and amplification was performed as described by Persson et al.18. The presence of PCR products was confirmed by agarose (1.5% w/v) gel electrophoresis conducted at 100 V for 1 h.
| Table 1: | Oligonucleotide primer sequences used in the detection of Mycoplasma spp. |
Sequencing of 16S rRNA genes: The 16S rDNA fragments measuring approximately 425 bp were purified using the QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions and sequenced using the same primers and the gene analyzer 3121 sequencer. This step was performed by Macrogen Co. (Seoul, Korea). The Mycoplasma 16S rDNA obtained sequences were then aligned with known 16S rDNA sequences deposited in GenBank using the basic local alignment search tool of the National Center for Biotechnology Information and percent homology scores were generated to identify Mycoplasma species. The identified sequences were aligned using Molecular Evolutionary Genetics Analysis (MEGA) version 7.10 software and a phylogenetic tree was produced19.
RESULTS
Identification by PCR analysis: Ninety three samples were tested for Mycoplasma infection using specific primers for (Mycoplasma arginine, M. bovis and M. mycoides subsp. mycoides), but, not obtained positive results for each species. Then, 24 mycoplasma isolates were obtained by PCR detection using universal 16S rDNA and sequenced. This resulted in the identification of specific isolates, including M. edwardii and M. yeastsii. Specifically, isolates Myco 1, 3, 4, 6, 9, 10, 11, 12, 13, 14 and 15 were identified as Mycoplasma edwardii, exhibiting 77-84% similarity with Mycoplasma edwardii ATCC 23462. Similarly, isolate Myco 16 was identified as M. yeastsii, exhibiting 100% similarity to M. yeastsii GM274B. In contrast, other isolates were identified as uncultured Mycoplasma in the GenBank database (Fig. 1).
In vitro semi-nested PCR: To perform semi-nested PCR using U1/U8 degenerate primers, 1.5 kb fragment representing the 16S rDNA from Mycoplasma isolates was amplified using U1 and U8. The PCR yielded unsatisfactory results, therefore, PCR temperature profiles in addition to the amount and concentration of U1 and U8 primers were adjusted accordingly. The amount and concentration of U1 and U8 primers. This non-specific melting curve reflects the formation of primer dimers in the absence of target DNA, likely because of both the high primer concentrations and their degeneracy. While primer dimers were detected in association with amplicons in most Mycoplasma strain samples and in the positive control (as indicated by a sharp band identical to that observed in the gel doc measuring 1500 bp), this band was not observed in lane 2 (Fig. 2). Two other samples did not yield amplicons. As the semi-nested PCR assay was performed using the recommended set of U1/U8 primers for Mycoplasma identification, the relative sensitivity of the PCR and the semi-nested PCR assay when using the optimized PCR parameters was evaluated, by testing the serial dilution of the supernatant from a cell contaminated with a known Mycoplasma. The method was validated by PCR and visualization of the resulting PCR product was accomplished by electrophoresis in an agarose gel (Fig. 3). The semi-nested PCR assay was performed using the U2/U5 primer set showing a sharp band that was identical to those observed in the positive control (900 bp) (Fig. 4, lane 2). Accordingly, in this method, the lowest dilution of genomic DNA detectable was successfully estimated to accurately produce amplicons. The 16S rDNA sequences of mobile Mycoplasma strains deposited in GenBank were identical to those found in the current study. Ribosomal operons are essential in understating the evolution of Mycoplasma. Additionally, 16S rDNA sequences has been extensively used to reconstruct phylogenetic relationships among different Mycoplasma species. Upon using a semi-nested PCR assay utilizing the new combination of U2/U8, a sharp band was obtained which is identical to the positive control of 1250 bp. In this assay a primer pair that is complementary to the universal regions U2 and U8, yielded 900 and 1250 bp PCR products, respectively (Fig. 5) with an overlapping region of approximately 650 bp was used.
The semi-nested PCR relies on the interference of PCR with the first primer pair complementing the global regions U 1 and U 8. The resulting product is approximate 1500 bp which can be readily used in amplification of 95% of the genes.
| Fig. 1: | Phylogenetic tree based on 16S rRNA sequences of Mycoplasma species and related species |
| Fig. 2: | Amplification of PCR products from mycoplasma isolates using primers U1 and U8 |
Lane 1: Positive control and Lanes 2-25: Sample isolates, approximate size of the amplicons was 1500 bp |
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The amplicon is then diluted and amplified again in two independent semi-nested PCR cycles with one primer pair complementary to the universal regions U1 and U5 and the other complementary to the universal regions U 2 and U 8. These nested -PCR reactions produce 650, 900 and 1250 bp, respectively. Thus, it can be clarified that the three outcomes of chain polymers reactions give sequences of specific lengths using the eight primers mentioned through which the species of mycoplasma can be defined.
| Fig. 3: | Amplification of PCR products from mycoplasma isolates using primers U2 and U8 |
| Lane 1: Positive control and Lanes 2-25: PCR products, approximate size of the amplicon was 1250 bp |
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| Fig. 4: | Amplification of PCR products from mycoplasma isolates using primers U1 and U5 |
Lane 1: Positive control and Lanes 2-25: Sample isolates, approximate size of the amplicons was 900 bp |
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| Fig. 5: | Amplification of universal primer regions U1, U2, U5 and U8 of mycoplasma isolates with deferent amplicon sizes |
Lane 1: Positive control and Lanes 2-25: U1, U2, U5 and U8, approximate sizes of the amplicons were 1500, 1250, 900 and 650 bp, respectively |
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DISCUSSION
Bacteriological isolation and identification of Mycoplasma species is difficult, insensitive and costly20,21. Bacteriological isolation of 93 lung tissues collected from diseased or apparently healthy camels were unable to identify Mycoplasma isolates in any of the samples. The failure to obtain positive results using these traditional methods agreed with the findings of previous studies20,21. Those authors also found that the low isolation percentage of Mycoplasma identified was attributed to the presence of toxic or inhibitory substances in the camels’ tissue, preventing cell culture and to the fastidious nature of many pathogenic Mycoplasma species. Using PCR in tandem with universal primers, 24 of the 93 samples tested positive for Mycoplasma infection. All 24 Mycoplasma isolates were molecularly identified at the genus level using PCR by detecting the 16S rRNA gene. The 16S rDNA sequences will likely play an important role in classifying Mycoplasma as more species are described22. It was used primers specific to 3 Mycoplasma species (M. arginine, M. bovis and M. mycoides subsp. mycoides), however, no positive results were obtained. It was identified specific isolates, including M. edwardii and M. yeastsii. Specifically, isolates were identified as similar to M. edwardii ATCC 23462 and M. yeastsii GM274B. Traditional identification methods (morphological methods) provide significantly less diagnostic information compared to PCR-based methods. The number of sequences of the ribosome gene from mycoplasma deposited in GenBank is approximately one hundred genetic sequences, thus the genetic sites and genetic affinity of the new mycoplasma species can be determined quickly.
PCR primers that complement the global regions of the 16R rRNA genes can be designed from Mycoplasma and such primers can be used in the polymerase chain reaction of genes such as the mycoplasma gene9,14,23.
Then it is used to determine the sequence of such genes, which can be compared with the genetic databases of mycoplasma , and to determine the ratio of genetic affinity and the precise definition of the different genera. In addition, it can be determined and precisely whether these types of mycoplasmas have been determined by the row before or not24,25.
As is known so far , approximately 100 16 S rRNA Mycoplasma has been deposited in databases , allowing the production of genetic trees with genetic affinity that allow precise identification of the type and gender of mycoplasmas and these databases are the source and reference in the molecular definition of such genera of mycoplasma. The 16S rRNA genes sequencing provide a powerful tool in Mycoplasma classification26,27. These primers are primarily designed for the study of Mycoplasma , but can also be used to amplify and sequence 16 S rRNA genes for many bacterial species related to mycoplasma . Methods of designing a polymerase chain reaction (PCR), especially this semi-nested PCR, have been discussed in addition to the possibility of applying this unique reaction of a high -specific type and speed with many bacterial species28. The data of this study demonstrated that the incidence of mycoplasmas in camels in Taif city was approximately 26%. Further, these findings indicated that PCR sequencing using universal primers is an accurate and time-efficient method to detect and identify mycoplasmas. This method overcomes the issues commonly associated with conventional culture methods used in to identify mycoplasmas.
CONCLUSION
The current study indicates that, the importance of using nested PCR and 16S rDNA sequencing technology for diagnosis of camel’s Mycoplasma infection, the nested PCR technology is specific molecular technology for Mycoplasma identification, more sensitive test. These techniques are simple and quick methods of detection and isolate infected animals, so it is a way to reducing economic losses in animal industry.
SIGNIFICANCE STATEMENT
Camels are susceptible to various pathogens including mollicute bacteria such as Mycoplasma. Few studies have addressed Mycoplasma infection in camels and Mycoplasma are difficult to detect. Here, we identify known and previously undescribed Mycoplasma from camel nasal tracts and demonstrate an effective method for doing so.