Validation and application of qPCR test kit for detection of Mycoplasma dispar DNA

INTRODUCTION

Mycoplasma dispar is a globally prevalent pathogen responsible for respiratory diseases in cattle, particularly in calves. M. dispar infection typically manifests as mucopurulent inflammation of the upper respiratory tract with frequent lung involvement, resulting in substantial economic losses in cattle production [1][2][3].

Mycoplasma dispar, a member of the genus Mycoplasma (class Mollicutes), lacks a cell wall, is pleomorphic, and has a compact 803 kb genome with a low G + C composition (28.5–29.3 mol%) [4][5].

Mycoplasma dispar requires a specific medium to enable growth and its colonies do not show – especially during the early passages – the typical “fried-egg” appearance. In addition, M. dispar are not always inhibited by hyperimmune serum, making conventional identification difficult in the early stages [5]. M. dispar glucose fermentation aligns with its phylogenetic cluster (hominis group), but unlike some mycoplasmas, it lacks arginine hydrolysis. Tetrazolium reduction occurs under both oxygen conditions. The bacterium has no phosphatase activity [6][7][8]. M. dispar produces an outer capsule consisting of a polysaccharide identified as a polymer of galacturonic acid [9]. The capsule appears to be produced during infection in response to mammalian host cells, and may exert an inhibitory effect on the activity of bovine alveolar macrophages and prevent their activation [10].

It has been reported that mycoplasma can be inhibited by alveolar macrophages if anti-capsular polysaccharide antibodies are present [9].

Mycoplasma dispar was first isolated from the lungs of pneumonic calves in England in 1969. Then these mycoplasmas were reported from Denmark, Belgium, the Netherlands, France, Australia, the USA, Canada, Korea and Japan [8]. In Europe, M. dispar infection was reported from the UK, and recently from Brazil and Italy [11][12][13][14][15]. In the Russian Federation, testing of 1,186 biological samples collected from cattle with respiratory and/or reproductive diseases from 34 different regions during 2015–2018 revealed M. dispar DNA in 37.15% of samples by agarose gel electrophoresis of polymerase chain reaction (PCR) products [16].

Mycoplasma dispar is transmitted between animals through respiratory secretions and can be detected in the respiratory tracts of both healthy calves and those with pneumonia [3]. Mycoplasma infections, particularly when combined with other respiratory pathogens under suboptimal conditions, represent a significant threat to animal health and productivity in high-density livestock operations [17][18]. In the Netherlands, M. dispar was isolated from 92% of pneumonic calves and from only 40% of healthy calves [7][19]. In Denmark M. dispar was found in over half of calf lungs showing either fibrino-necrotising or suppurative bronchopneumonia [20]. Other bacterial pathogens, including Histophilus somni, Pasteurella multocida, Arcanobacterium pyogenes, and Mannheimia haemolytica, were co-detected with mycoplasmas. M. dispar is detected frequently from pneumonic calves in the UK, and is believed to be the cause of a severe pleuropneumonia similar to the WOAH (World Organization for Animal Health) listed contagious bovine pleuropneumonia [21].

Few detailed studies have been carried out to establish the exact role of M. dispar in bovine respiratory disease (BRD) which is a chronic condition affecting beef cattle in feedlots and dairy calves. It is estimated to cost the USA cattle industry alone over US $4 billion in production losses, treatment and prevention [21].

Calves affected by BRD are usually treated with antimicrobials even though some of these are not effective against mycoplasmas [22]. Consequently, there is a real danger of the emergence of resistance as a result of chronic therapies with multiple antimicrobials, which is already observed for the other BRD bacterial pathogens [23].

The pathogenicity mechanisms have been identified in M. dispar and include its ability to produce hydrogen peroxide and biofilm, both well-known virulence factors. Studies have shown that M. dispar is able to colonize the epithelial cells of the respiratory tract exerting cytostatic and even cytopathic effects on bronchial and bronchiole cells, potentially impairing the clearance of bacteria [9][21][24]. Furthermore, M. dispar has been shown to be immunosuppressive in the host [25]. For these reasons M. dispar is included with M. bovis amongst the agents that cause or exacerbate BRD [8][21]. M. dispar is causally associated with exudative bronchitis and interstitial pneumonia in calves, with transmission occurring primarily via airborne respiratory droplets and sustained close contact [26].

Mycoplasma dispar causes purple to red consolidation mainly in the cranioventral areas of the lung [27]. M. dispar were shown to be capable of causing a mild sub-clinical bronchiolitis with lymphoid cuffing in gnotobiotic calves [10]; occasional cases of mastitis were also reported [28]. Other authors report that M. dispar is frequently related to alveolitis, in which neutrophils, macrophages, and edema liquid aggregate in the alveolar wall and spaces [26][29]. Field cases of subclinical pneumonia from which M. dispar was isolated had similar lesions [30].

The genomic information of M. dispar in publicly available databases is limited. The genome succession of M. dispar reference strain ATCC 27140 was delivered in the GenBank in 2015. Based on the phylogenetic analysis of 16S rRNA gene sequence M. dispar was clustered with M. ovipneumoniae, M. flocculare and M. hyopneumoniae, isolated from sheep and goats [31].

Controlling mycoplasmosis effectively requires a multi-faceted approach that includes minimizing environmental stress, ensuring proper animal husbandry, and maintaining good air circulation within facilities. Measures to prevent infection of calves from adult animals are required [8].

Timely diagnosis is one of the key components in bovine mycoplasmosis control. Laboratory confirmation of M. dispar infection is clinically important, as this globally distributed pathogen significantly impacts livestock health and production [18][32][33][34].

Conventional identifying bovine mycoplasmas through isolation on nutrient media is a crucial first step, as it allows for detailed examination of their cultural, morphological, and biological characteristics. This method also enables the establishment of a clinical isolate bank, which can support future development of improved prevention, control, and eradication strategies for mycoplasmosis, as well as facilitate antimicrobial resistance monitoring [8][18][33][34]. However, it should also be noted that this method is laborious and requires 7–10 days for diagnosis [3][18][34].

While not widely used, serological tests like radial hemolysis, ELISA [35], and passive hemagglutination have been reported for detecting M. dispar antibodies [36][37]. The authors described that weak antibody presence against M. dispar in cattle, despite its surface-lung location, might be due to the relatively low sensitivity of the serological tests used, rather than the animal’s immune system failing to mount an adequate response [35].

While PCR has significantly improved mycoplasma detection, it was not until 2004 that a PCR method specific to the M. dispar genome was reported [38]. Both specific and universal oligonucleotides were utilized to detect single nucleotide polymorphisms in the 16S rRNA gene sequence. The PCR/DGGE technique (denaturing gradient gel electrophoresis) was also described, which can detect and identify more than 70 different mycoplasmas, including M. dispar [39][40].

Contemporary diagnostics can achieve high efficiency through real-time PCR (qPCR), enabling rapid and precise detection of specific mycoplasma genomic loci in biological samples [18][32][34][41].

The use of fluorescently labeled TaqMan probes that bind to a specific sequence within the amplified DNA region (the interprimer segment) significantly enhances the specificity of PCR-based assays. Real-time PCR, unlike traditional PCR, doesn’t require post-amplification handling of the sample, reducing the risk of contamination and leading to faster, more efficient analysis.

The incorporation of an internal control sample (ICS) in commercial qPCR assays ensures result reliability by detecting PCR inhibition, thereby preventing false-negative interpretations [42]. An important need in qPCR test kit development is incorporating ICSs to monitor both nucleic acid extraction and the subsequent amplification process [43][44][45][46].

It is also worth noting that, in accordance with the WOAH requirements, it is recommended to include ICS into each PCR test for quality control [47].

Tools for the molecular identification of mycoplasma genomes, including of M. dispar, enable not only to monitor the animal disease situation in domestic farms, but also to control both the import of animals and their use for production purposes [34][48].

Currently, there are no commercially available domestic qPCR test kits to detect M. dispar DNA in the Russian Federation, and therefore the development of a specific qPCR diagnostic test kit and its introduction into veterinary practice is an urgent task [34].

The purpose of this work was to determine the main validation characteristics of the qPCR test kit for detection of M. dispar DNA developed by the Federal Centre for Animal Health, and to introduce it into veterinary practice.

MATERIALS AND METHODS

Bacteria and viruses. The M. dispar reference strain (ATCC No. 27140), delivered from the collection of strains of microorganisms of the Federal Centre for Animal Health, and biological samples collected from cattle of various age groups (stabilized blood; nasal and tracheal swabs; pieces of lungs, trachea and lymph nodes; pleural fluid) were used.

To assess the analytical specificity of the developed test kit, the following bacterial strains were used: M. bovis ATCC No. 25523, M. bovigenitalium ATCC No. 19852, M. bovis Kaluga 2020, Mycoplasma mycoides subsp. mycoides SC (MmmSC) Madugri-8 DNA (Federal Research Center of Virology and Microbiology, Russia), Mycoplasma mycoides subsp. mycoides SC (MmmSC) “T1/44/ARRIAH”, and other bacterial and viral agents responsible for analogous diseases in cattle: Escherichia coli “EC-21”, Mannheimia haemolytica “No. 1412”, Pasteurella multocida “No. 1414”, M. bovigenitalium isolate, M. dispar isolate, M. bovis isolate, bovine parainfluenza 3 virus “VGNKI-4”, bovine respiratory syncytial virus “Vologda/2020”, bovine viral diarrhea virus “NADL-ARRIAH”.

Mycoplasma dispar culture. The M. dispar reference strain (ATCC No. 27140) was cultured on the 1699 Revised Mycoplasma Medium nutrient medium recommended by the American Type Culture Collection, which contained 7.5 g of brain heart infusion broth; 40.0 µL of 10× Hank’s Balanced Salts Solution; 10.0 µL of 0.25% phenol red solution; 200.0 µL of heat inactivated porcine serum; 100.0 µL of 5% lactalbumin hydrolysate in 1× phosphate buffer solution; 20.0 µL of yeast extract and 660.0 µL of distilled water. The bacteria were cultured in an incubator at (37 ± 0.5) °C and 5% CO₂ for 5 days. After incubation, turbidity in test tubes and discoloration of the medium (yellowing) were observed, M. dispar-like colonies formed on a solid nutrient medium; M. dispar biological activity was determined by colony-forming unit counting (CFU/µL) [49][50].

DNA was extracted using a commercial Ampli Prime Ribosorb kit (Central Research Institute of Epidemiology, Rospotrebnadzor, Russia) in accordance with the manufacturer’s instruction.

Real-time PCR protocol. The reaction mixture for amplification per one reaction contained the following components: PCR-buffer-B for Taq DNA polymerase 10× (Syntol, Russia); 5 U/µL of SynTaq DNA polymerase with enzyme-inhibiting antibodies (Syntol, Russia); 25 mM of aqueous solution of magnesium chloride MgCl₂ (Syntol, Russia); 100 mM of aqueous solutions of four deoxynucleoside triphosphates (dNTP): dATP, dGTP, dTTP, dCTP (a common mixture of dNTP is prepared and diluted with nuclease-free water to a concentration of 10 mM of each dNTP; Fermentas, Lithuania); direct primer (100 pmol/µL), reverse primer (100 pmol/µL) and a TaqMan probe (100 pmol/µL) to detect a region of the gene encoding M. dispar16S rRNA (Syntol, Russia); direct primer (100 pmol/µL), reverse primer (100 pmol/µL) and TaqMan probe (100 pmol/µL) for detection of artificially synthesized ICS (Syntol, Russia) [51]. The resulting volume of the reaction mixture was adjusted to 15 µL with deionized, nuclease-free water (Eurogen, Russia). After that, 15 µL of the reaction mixture and 10 µL of the DNA matrix of the tested samples were added to the prepared tubes. Amplification was performed in a Rotor-Gene real-time PCR cycler (QIAGEN, Germany).

A plasmid construct containing the oligonucleotide sequence of the genome region (with an initial concentration of 2 × 10⁷ copies/µL) and M. dispar genome specific region (target fragment) was used as a positive control sample.

Deionized, nuclease-free water (Eurogen, Russia) was used as a negative control sample (NCS) and as a negative PCR control.

Validation. The validation parameters of the test kit were determined according to the recommended method by S. A. Bustin et al. guidelines for publication of developed quantitative PCR protocols [52].

To assess the specificity of the developed qPCR, biological samples containing Mycoplasma DNA and viral and bacterial nucleic acids which can cause similar diseases in cattle were tested. The test kit sensitivity was assessed using known positive biological samples containing M. dispar DNA.

The limit of M. dispar DNA detection (analytical sensitivity) of the developed test kit was assessed using a positive control sample containing M. dispar DNA with an initial concentration of 2 × 10⁷ copies/µL, each dilution was tested in 5 replicates.

PCR amplification efficiency was assessed using serial 10-fold dilutions of a positive sample of biological material containing M. dispar DNA in 3 replicates and calculated according to the formula:

where slope is standard curve (plot of Ct vs. log₁₀ input template concentration).

The data were statistically analyzed using Microsoft Excel, including calculations of mean values with standard deviations (± SD), regression analysis, and coefficients of variation. The coefficient of variation (CV) for reproducibility and repeatability should not exceed 10%.

Intermediate precision (reproducibility) was assessed by testing the same sample in 5 replicates in 3 independent qPCR runs (n = 15 total replicates).

RESULTS AND DISCUSSION

The Federal Centre for Animal Health developed and validated Russia’s first qPCR test kit for Mycoplasma dispar DNA detection, incorporating an ICS.

The use of ICS improve PCR reliability and accuracy, particularly by identifying reaction inhibition. The Laboratory Technology Committee of the American Association of Veterinary Laboratory Diagnosticians (AAVLD) has recommended to its membership that all new molecular assays being validated and implemented include an inhibition monitoring strategy based on internal validation for the host, target species, and sample matrix combination being tested [53].

The initial validation step involved identifying a temperature profile that optimizes both the sensitivity and amplification efficiency of the test kit.

The qPCR temperature-time profile was optimized using pre-established reaction component concentrations to achieve consistent threshold cycle (Ct) values. The results are given in Table 1.

The following qPCR temperature-time profile was considered optimal for M. dispar DNA detection: 5 minutes at 95 °C (heating of the reaction mixture), followed by 40 PCR cycles consisting of DNA denaturation for 15 seconds at 95 °C, primer annealing and cDNA elongation for 60 seconds at 60 °C.

Validation testing confirmed that the developed kit eliminates false-positive results when challenged with DNA from bovine respiratory disease-associated bacteria. It was shown that the test kit has 100% analytical specificity for M. dispar (Table 2), which is higher than demonstrated by J. B. W. J. Cornelissen et al., in which the specificity was 98.2% [54]. The oligonucleotides were verified using the NCBI BLAST database, confirming no significant homology with other Mycoplasma species and demonstrating specificity for the M. dispar genome.

The amplification efficiency was evaluated using serial 10-fold dilutions of a positive sample containing M. dispar DNA. Based on the obtained Ct values for each dilution, the amplification efficiency (E) was 99.01%, which was more significant than the 97.49% efficiency demonstrated by J. B. W. J. Cornelissen et al. during the development of a single PCR [54]. Linear correlation value (R2) was 0.9832 (Fig. 1).

In-laboratory precision under reproducibility conditions was evaluated by examining how consistently measurements are obtained when varying personnel, time, and equipment. The tests were performed using biological material containing and not containing M. dispar DNA. Recent studies have shown that the results of M. dispar DNA detection using the developed test kit are fully consistent with the expected results and do not depend on time, personnel, or equipment.

Intermediate precision under reproducibility (repeatability) conditions was assessed by testing the same sample in five replicates in three independent qPCR runs (n = 15 total replicates).

The threshold cycle (Ct) mean value in three PCR runs ranged from 26.74 to 27.36 with a standard deviation (SD) of 0.18 to 0.34. The coefficient of variation (CV) of 0.91% is well within the acceptable range of 10%. When summing up the results of three qPCR runs, the mean Ct value and the standard deviation was 27.03 and ± 0.25, respectively (Table 3).

At the next stage, the detection limit (analytical sensitivity) was determined for M. dispar DNA isolated from serial 10-fold dilutions of an artificially synthesized DNA sequence corresponding to a specific region of the M. dispar genome, starting at an initial concentration of 2 × 10⁷ DNA copies/µL. Each dilution was tested in five replicates (Table 4).

It has been established that for the developed qPCR-based test kit, the detection limit of M. dispar DNA is 10 DNA copies/μL (100 DNA copies/reaction).

In studies conducted by L. McAuliffe et al., DGGE of the 16S ribosomal DNA PCR product was used to differentiate 32 mycoplasma species. This method is a valuable tool for quickly identifying various Mycoplasma species, especially when specific PCR tests are not available [39][40]. However, artifacts introduced during PCR or the subsequent DGGE analysis lead to skewed results when this method is used for quantitative analysis of α-diversity or relative operational taxonomic unit abundance [55].

While some researchers have used conventional PCR to detect M. dispar [31][38][56], qPCR offers faster and more accurate results without the need for gel electrophoresis [57].

Analysis and interpretation of the results. Both fluorescence channels (Green/FAM and Red/Cy5) shared identical qPCR parameters: dynamic background adjustment, slope correction, 10% emission cutoff, linear scale quantification, and a detection limit of 0.05 (Fig. 2). Results are interpreted according to whether fluorescence curves intersect the threshold line, corresponding to the presence or absence of threshold cycles (Ct) in both Green/FAM and Red/Cy5 channels. These data are displayed in the corresponding amplification plots and result tables generated by the thermocycler.

The result of qPCR is considered reliable provided that the correct results are obtained for the negative PCR control, the NCS (extraction control) and M. dispar positive control. The control requirements are described in Table 5.

The results for the tested samples should be interpreted in accordance with Table 6.

Using the developed test kit, 228 biological samples collected from cattle with respiratory clinical signs were tested, which were received by the Federal Centre for Animal Health, in 2024 from 13 regions of the Russian Federation. The results are given in Table 7.

It was established that M. dispar DNA detection rate in biological samples was 39.47%. The findings demonstrate widespread prevalence of M. dispar among cattle populations across Russian farms in 2024.

CONCLUSION

This study established the validation parameters and demonstrated the utility of the “MIC-DISPAR qPCR” kit for detecting Mycoplasma dispar DNA via real-time polymerase chain reaction. The assay exhibited high analytical specificity and sensitivity, with excellent repeatability and reproducibility across various test conditions (coefficient of variation range: 0.66–0.91%) The amplification efficiency (E) was 99.01%, and the detection limit was 100 DNA copies/reaction. These validation parameters represent essential criteria for assessing polymerase chain reaction test kit reliability.

Inclusion of an exogenous internal controls example prevents false-negative result interpretation. Using the developed test kit M. dispar DNA was detected in 39.47% of the tested samples received from different regions of the Russian Federation by the Federal Centre for Animal Health, in 2024, which indicates the relevance of M. dispar diagnostic tests. In this case, the internal controls example serves as an indicator of the extraction stage and the presence of possible inhibitors.

While the test’s validation framework could be extended to include related mycoplasmas (e.g., M. bovirhinis, M. flocculare, and M. ovipneumoniae), current data lack evidence of these pathogens circulating in cattle and small ruminants within the Russian Federation. M. ovipneumoniae is the primary cause of mycoplasmal pneumonia in sheep and goats, and the risk of its transmission to cattle is minimal [58][59][60]. The demonstrated high specificity of the oligonucleotides (confirmed by BLAST analysis) supports the test kit’s suitability for M. dispar diagnostics in cattle.

Thus, this test kit can serve as an effective tool in veterinary laboratory practice for the detection of M. dispar DNA in biological samples from cattle.

Contribution of the authors: All authors contributed equally to formulating the research objectives and aims, conducting literature reviews and data collection; designing and performing laboratory tests, analyzing and systematizing research data; interpreting results and drawing conclusions and preparing and writing the manuscript.

Вклад авторов: Авторы внесли равнозначный вклад при определении целей и задач исследования, сборе литературных данных; при разработке и проведении лабораторных исследований, анализе и систематизации полученных данных; при обобщении, интерпретации результатов и оформлении основных результатов исследования в виде статьи.