Introduction

Respiratory infections are among the important health problems of sheep of all ages throughout the world. Concerning Iraq, a study performed in Mosul city, northern part of the country, found that respiratory infections were more common in sheep following gastrointestinal disturbances (Dahl et al. 2021). These infections, either acute or chronic, have economic impacts on the performance of flocks (Thompson 2019). Respiratory diseases may affect individual animals or groups, leading to weak gain of live weight with high mortality rate (Kumar et al. 2000). This causes large economic losses to owners due to reduced production of milk, meat, and wool as well as decreased offspring numbers (Chakraborty et al. 2014). Small ruminants are extremely vulnerable to pulmonary infections, which cause death in roughly 50% of them (Kumar et al. 2014).

Various bacteria can infect the upper and lower respiratory tract and frequently lead to the development of bronchitis, pneumonia or other respiratory disorders. Many bacterial species either single or mixed with other types have been isolated from sheep respiratory infections (Chakraborty et al. 2014). Using conventional diagnostic methods based on phenotypic tests often need longer time to generate and analyze the results, and in most cases, extra tests are essential (Gandra et al. 2016). Furthermore, phenotypic tests can be associated with false-negative results, indicating the impact of environmental factors on gene expression (Downes and Ito 2001), adding to the relatively lower specificity and sensitivity of these tests than molecular assays (Chakraborty et al. 2014). Thus, a reliable diagnostic tool can reduce the problems of diagnosis and the possible economic losses resulting from respiratory diseases (Scott 2011).

Therefore, early, fast, and precise detection of such infections is of significant value for the small ruminants (Chakraborty et al. 2014; Kumar et al. 2014). Molecular techniques based on PCR have been proved to be valuable for the diagnostic and epidemiological studies (Yang et al. 2007; Kumar et al. 2014). However, molecular detection of the respiratory infections in these animals is considered as a challenge in Southeast Asian and African countries because of their inadequate laboratory resources. This situation makes large numbers of these animals to be slaughtered due to respiratory infections outbreaks (Daniel et al. 2006; Scott 2011).

Mixed infections have been observed for many times by researchers. Isolation and identification of these infections could be boring and time consuming (Kumar et al. 2012; Saleh and Allam 2014). Therefore, improvement of molecular diagnostic tools, such as multiplex PCR, has been validated to be useful for the detection and characterization of the causative agents of such complex infections (Dhama et al. 2014). Multiplex PCR has helped diagnosticians to decrease confusion in cases of mixed infection because such techniques are able to discriminate between different species in the same genus (Chakraborty et al. 2014). Moreover, this technique has been used successfully for detecting multiple pathogens simultaneously in the same reaction tube with high sensitivity, specificity, throughput, with low cost (Datukishvili et al. 2015; Hu et al. 2020). The main advantages of using multiplex PCR over traditional PCR is its cost-effectiveness. Multiplexing decreases the amounts of reagents, e.g., DNA polymerase, applied for each reaction. In addition, it does not require more time for preparation and analysis of results than using multiple tubes for simplex PCR (Phuektes et al. 2001). Therefore, this study was designed in an attempt to develop multiplex-PCR for the cost-effective, rapid, and accurate identification of three bacteria causing respiratory tract infections in sheep, including Staphylococcus aureus, Pseudomonas aeruginosa and K. pneumoniae.

Materials and methods

Bacterial isolates

Nine bacterial isolates were used in this study, including: 4 K. pneumoniae, 3 S. aureus, and 2 P. aeruginosa. These bacteria were obtained from the study of Aziz and Lafta (2021) performed on sheep with respiratory tract infections and reared in different farms in Baghdad, Iraq. The bacteria were already identified by the corresponding analytical profile index (API staph, API 20NE or API 20E, as well as the automated Vitek2 system (data not shown).

Molecular diagnosis

Primers design and preparation

Molecular identification of S. aureus, P. aeruginosa, and K. pneumoniae was done by partial amplification of their nuc, rpoS and gapA genes, respectively, by using specific primers designed in this study through the use of Primer3Plus software tool. The primers were checked by in silico PCR amplification as well as the Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information (NCBI) before running the conventional PCR.

To validate the primers before being used in the multiplex PCR experiments, the MFEprimer program was used to check the specificity of the primers against the background DNA that can act as a competitor to the target DNA. Here, the background DNA was genomic DNA extracted from the bacteria under study. Furthermore, the major purpose of using this program was to evaluate the candidate primers, to examine the formation of primer dimers to discard the inappropriate primers, and finally to determine the optimal primer set combination for multiplex PCR by providing a virtual electrophoresis to assist users choose the best combination of primer sets (Qu and Zhang 2015).

Prior to their use, the lyophilized primers (Macrogen, Korea) were dissolved in a suitable volume of nuclease free water to give a final concentration of 100 pmol/μL as a stock solution, yet their working concentration was 10 pmol/μL. The accession numbers of the genes used to design the primers as well as the amplicon sizes are mentioned in the Table 1 below.

Table 1 The genes and their accession numbers used to design the specific primers and sizes of the PCR amplicons
Full size table

DNA extraction and quantification

Genomic DNA was isolated from the bacterial growth according to the protocol of ABIOpure Extraction. Then, Quantus Fluorometer was used to detect the DNA concentration of the samples.

Uniplex and multiplex PCR optimization

To examine the optimum annealing temperature of the primers, uniplex PCR was optimized initially by amplifying one DNA template with the corresponding primer pair at different annealing temperatures of 50, 52, 55, and 58 °C. The PCR cycling conditions involved the use of the following program: denaturation at 95 °C for 5 min, followed by 30 cycles of denaturation at 95 °C for 30 s; annealing at one of the four temperatures mentioned above for 30 s; and extension at 72 °C for 30 s. A final extension step was done for 7 min at 72 °C, followed by 10 min incubation at 4 °C to stop the reactions. The PCR reaction mixture composed of 20 μL total volume containing 10 μL GoTaq Green Master Mix (2×); 1 μL of each primer (10 pmol/μL); 6 μl nuclease free water, and 2 μL of template DNA.

Following the optimization of the uniplex PCR conditions, multiplex PCR was run where the reaction mixture composed of the same components and volumes mentioned above for the uniplex PCR, except that the template DNA amount was increased to 3 μL and the nuclease free water volume was decreased. Concerning the PCR program, an annealing temperature of 58 °C was used for all the primers, and temperatures of the other steps were the same.

Results

Validation of the primers

The MFEprimer software was successful in designing the multiplex-PCR experiments and determining the optimal primer set combinations. Briefly, it stated melting temperatures (Tm) of each primer, primer binding numbers, and the amount of energy required (delta G measured by Kcal/mol). Importantly, the report showed absence of hairpins and primer dimers. Amplicon details were also described in the report for each primer set combination, such as the expected size of the amplicon, GC contents, and delta G of each primer (data not shown).

Optimum conditions for PCR

Fig. 1 shows amplification of partial regions of the nuc, gapA, and rpoS genes of S. aureus, Klebsiella pneumoniae, and P. aeruginosa, respectively using conventional PCR. It is clear from the image that each of the temperatures 50, 52, 55 and 58 °C was able to efficiently amplify the aforementioned genes, despite the presence of non-specific bands that appeared at 50 and 52 °C in case of the gapA gene. Therefore, the highest annealing temperature of 58 °C was used for the next experiments.

Fig. 1
figure 1

Optimization of the PCR reaction conditions. A- Diagram showing the virtual electropherogram to choose the optimum PCR annealing temperatures for three genes. B- Agarose gel electrophoresis for the PCR products amplified under different annealing temperatures of 50, 52, 55 and 58 °C shows bands of ~506 bp representing the nuc gene of S. aureus (left panel), bands of ~391 bp representing the gapA gene of Klebsiella pneumoniae (middle panel), and ~ 236 bp for the rpoS gene of P. aeruginosa (right panel)

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Multiplex PCR

Multiplex PCR was successful in targeting one of the three microorganisms, i.e. either S. aureus, K. pneumoniae or P. aeruginosa when their primers were put together in the same PCR reaction tube (Fig. 2). Similarly, this technique efficiently detected combinations of two organisms, either S. aureus with K. pneumoniae, S. aureus with P. aeruginosa or K. pneumoniae with P. aeruginosa (Fig. 3). Importantly, multiplex PCR was also able to detect the presence of the three organisms together in the same reaction tube (Fig. 3).

Fig. 2
figure 2

Gel electrophoresis for the multiplex-PCR targeting one of three isolates in the same reaction tube. A- Diagram showing the virtual electropherogram of the multiplex PCR targeting one bacterium. B- The experimentally validated electrophoretogram for the multiplex-PCR targeting DNA of one bacterial isolate. Bands of ~506, ~391, and ~ 236 bp represent partial amplification of the nuc, gapA, and rpoS genes of S. aureus, Klebsiella pneumoniae, and P. aeruginosa, respectively

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Fig. 3
figure 3

Gel electrophoresis for the multiplex-PCR targeting two or three isolates in one reaction tube. A- Diagram showing the virtual electropherogram of the multiplex PCR targeting two or three bacterial combinations. B- Diagram showing the presence (+) or absence (−) of a certain bacterium as represented by the corresponding gene (nuc, gapA, rpoS). C- The experimentally validated electropherogram for the multiplex-PCR targeting two or three bacterial isolates. Bands of ~506, ~391, and ~ 236 bp represent partial amplification of the nuc, gapA, and rpoS genes of S. aureus, Klebsiella pneumoniae, and P. aeruginosa, respectively

Full size image

Discussion

Mixed bacterial infections are often seen in various animal disorders; thus, isolation and identification of such causative agents is always boring and wearing to carry out (Kumar et al. 2012). Due to evolution in the PCR technique, massive progress in the detection of animal respiratory infections has occurred (Munir et al. 2013). Advances in molecular biology and biotechnology have resulted in progression of different diagnostic procedures, including multiplex PCR among others (Deb and Chakraborty 2012; Dhama et al. 2013a, b).

This study focused on three different bacterial species associated with sheep respiratory tract infections, including: K. pneumoniae, S. aureus, and P. aeruginosa. These bacteria are the primary pathogens associated with morbidity and mortality in less developed countries due to respiratory infections (Hu et al. 2020). Other studies stated that both S. aureus and K. pneumoniae are among the ovine pathogens that have been isolated from respiratory infections (Azizi et al. 2013; Saleh and Allam 2014). In addition, P. aeruginosa, which is also opportunistic pathogen that exists in different ecosystems, can be implemented with dangerous animal diseases (Yang et al. 2015; Bird et al. 2017; Dapgh et al. 2019). It can cause many diseases in sheep, such as respiratory illness and mainly pneumonia that is linked with physiological and physical stress resulting in high economic losses and considerable mortality rates (Bangar et al. 2016).

In the current study, the designed primer pairs were efficient in amplifying their targets in the above microorganisms when tested separately by uniplex PCR, where one amplicon only was obtained from each target. Similarly, the same amplification efficiency was obtained when a single bacterium was identified using multiplex PCR. Although Hu et al. (2020) observed that the sensitivity of detecting a single organism in multiplex PCR was 5–100 times lower than that of uniplex PCR, and the sensitivity of identifying four bacteria using multiplex PCR was 5–50 lower than that of one bacterium using the multiplex PCR. This was illustrated by the fact that using combination of primers in the same reaction tube could make specific primers less available than necessary. Likewise, competition between nucleotides and enzymes might occur if numerous targets are present in one reaction.

Here, two targets were also detected successfully and efficiently by multiplex PCR. Combinations of K. pneumoniae with S. aureus, K. pneumoniae with P. aeruginosa, or S. aureus with P. aeruginosa were detected effectively and concurrently in a single reaction by this technique. However, this assay has been commonly used by many researchers to amplify DNA targets belonging to the same species or same genus by using several pairs of primers in the same reaction (Okolie et al. 2015a, b; Gandra et al. 2016). It has been demonstrated by Datukishvili et al. (2015) that concentrations of the templates and primers are crucial factors in some multiplex PCRs reactions.

Nevertheless, a weak band of ~506 bp, which represents partial amplification of the nuc gene of S. aureus, observed in this study. This can be attributed to many factors, for instance, low concentration of DNA of S. aureus, or due to inadequate nucleotides available for amplifying a sharp band of such large size due to competition between the primers, especially the volume of the PCR reaction mixture applied in this study was 20 μL, which is less than 50 μL used by many investigators. Weak and not clear bands were suggested by Gandra et al. (2016) to occur when there is low concentration of the template DNA (~102 CFU/mL). Other authors proposed different detection limits in experiments involving multiplex PCR. These variations in the detection limit of this technique and the lack of amplification in the majority of cases when DNA concentrations less than 103 CFU/mL are used, can be linked to DNA purity and/or presence of inhibitors within the samples or microorganism culture media, which might cause false negative results (Gandra et al. 2016).

A fact already confirmed by Tamarapu et al. (2001) indicates that when the same primers are used in multiplex and uniplex PCR, in the latest case the detection limit was found to move from 102 to 101. The study of Gandra et al. (2016) showed that the detection limit was 103 CFU/mL when individual target (either S. aureus or Yersinia enterocolitica) was used. However, the detection limit became 104 CFU/mL when both bacteria were used together in multiplex PCR. The authors stated that the difference in the detection limit can be related to the reaction itself (Gandra et al. 2016). Increasing the amount of transgenic material in the samples leads to consistent increase in the DNA bands intensity (Datukishvili et al. 2015). Furthermore, Ramesh et al. (2002) confirmed the effect of the primer numbers on the detection limit. In the current study, the primers targeting the nuc gene of S. aureus were successful in amplifying this gene when only one or two bacteria were present in the same tube, yet the multiplex PCR efficiency decreased in amplifying that gene when three bacteria existed together.

Overall, the primers designed in this study worked successfully in amplifying and detecting three different targets related to three different bacteria. This might be due to using the software program MFEprimer-3.0 (Wang et al. 2019) for primers quality control. This software has been developed and used for checking the presence of primers dimers, hairpins, non-specific amplicons, single nucleotide polymorphisms (SNPs) within the binding sites, as well as other important parameters (Shen et al. 2010; Qu and Zhang 2015).

Conclusions

To conclude, the present study highlighted the importance of using the in silico software programs, such as MFEprimer-3.0, for primers quality control. Here, multiplex PCR was proved to be specific, sensitive, fast, accurate, and cost-effective molecular diagnostic method for identification and differentiation of three clinically prevalent bacteria, including K. pneumoniae, S. aureus, and P. aeruginosa associated with ovine respiratory infections. This can efficiently support control and treatment of sheep respiratory diseases. This in turn, would increase the economy of the animals’ owners and wellbeing of the animals.