The authors have declared that no competing interests exist.
In Pakistan, production of poultry has been evolved as a good alternate of beef and mutton. In this study multiplex PCR approaches were adopted to differentiate and detect avian mycoplasma in a single PCR reaction as a step forward in the economization of PCR detection. From the total of 25 samples, 25 (100%) had a positive Mycoplasma isolation result. The biochemical test yielded the highest number of isolations, with MG accounting for 10/25. Commercial DNA-based PCR kits have been developed as a result of recent advancements in diagnostic techniques for Mycoplasma infections. Tetracyclines, fluoroquinolones, tilmicosin, tylosin, spiramycin Infections should be reduced as much as possible.
In Pakistan production of poultry has been evolved as a good alternate of beef and mutton {Hussain, 2015 #132}. Government is facilitating the investors to develop it at an accelerate pace by virtue of which it has become the second largest business in Pakistan
The avian Mycoplasmosis is diagnosed by serological and isolation, while its time consuming and difficult process
The improvement of the diagnosis of the infection of avian Mycoplasma has been recently developed by PCR. The duplex PCR assay targeting the heme–agglutinin multigenes families e.g vlhA for
The important avian pathogens are
25 sick bird samples were collected from infected farm environment with the symptoms of respiratory disorder’s including air sacs exudates, trachea, and oral swabs.
In the brain heart infusion samples were inoculated directly and incubated for 6-14 days at 37 °C. the infusion colour changes was indication of positive reaction and the color of phenol change red to yellow. (
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Difco ( bacto-brain heart infusion ) | 3.7g |
dH2O | 90ml |
Horse serum | 10ml |
Thallium acetate (1%) | 1.0ml |
L-cysteine hydrochloride (10%) | 1.5ml |
Phenol red (10%) | 1.5ml |
The isolation was identified and analyzed by serological, chemical, biochemical, and morphological parameters (Kreig and Holt, 1984). under light microscope using the low magnification (4X and 10X) colony were observed on plates by applying low intensity light.
The biochemical tests were designed to detect the specific enzymatic and nutritional activity of Mycoplasmas that was relatively small.
The Brain heart infusion broth containing 10% inactivated horse serum ,1% glucose, and 0.005% phenol red. Dispense aseptically in 5 ml amount into the tube, 0.1 ml diluted culture added into the test tube
The inoculated test culture plates containing 0.1% sodium phenolphthalein di-phosphate and plates were incubated at 37°C. the plates were flooded with 5 M NaOH after the growth of mycoplasma. The reaction positive indication through color changes, after 30s the pink color appear in the colonies area.
Extraction of DNA from culture and s swab samples were conducted through non-phenolic method which involve the step of boiling, freezing and centrifugation, the supernatant used as a reaction mixture.
The conditions for the multiplex PCR reaction were followed. The primer pairs sequence used for the PCR protocol (
Primers |
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MG Forward primer | 5’CTTTCCCATCTCGACCAGGAGAAA-3 | 732bp |
MG reverse primer | 3’GGATCAATCAGTGAGTAACGATGA-5 | 732bp |
Each PCR mixture contained 5 µl of 500m mol KCL, 5 µl of 100m mol Tris HCL, 4 µl of 2.5m mol MgCl₂, 2 µl (each) DATP, DCTP, DGTP, DTTP, 1 µl of each primer, 2.5 U of ampliTaq DNA polymerase, 62.5 µl of ultra pure water and template Mycoplasma DNA in 5 µl volumes were then added to a mixture. All of the DNA amplifications were carried out in the thermal cycler (model Peqlab 2s primus, Germany). The optimum conditions were programmed in thermal cycler. (Wang
Amplified product of multiplex PCR were electrophoresis on the 1.5% agarose gel and visualize under gel documentation system. (Waltec, USA)
Out of the 25, 10 samples were of trachea, 6 of air sac, 4 of oral swab and 5 of lungs tissues. These characterize on the basis of biochemical and cultural studies.
The biochemical profile of
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The positive (+) and negative (-) sign show the band intensity.
Firstly, optimizing of annealing temperature of the thermal cycler ranging from 48°C to 58°C and its effect on the amplicon was noted. In electrophoresis the intensity of the band revelead its significance. At 48°C, no amplification was observed, at 50°C amplification was observed in
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48°C | 50°C | 52°C | 54°C | ||
1 |
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- | ++ | + | +++ |
At 48°C band intensity was negative (-), no bands will appear.
At 50°C band intensity was intermediate (++).
At 52°C band intensity was very low (+).
At 54°C band intensity was very high (+++).
Secondly, the effect of decreasing and increasing the concentration of MgCl₂ was observed and optimized at better result. A total of 25 samples were processed through multiplex polymerase chain reaction. These samples were categorized into 5 groups including 5 samples each. The concentration of MgCl₂ lies between 1.5 m Mol gave 2+ results, 2.5 m Mol gave 3+ results and 3.0 m Mol gave better results. From the above data it was concluded that the concentration of 2.5 m Mol gave better results and the concentration of better results of MgCl₂ was optimized at 2. 5 m Mol.
Thirdly, optimizing of multiplex PCR was carried out by using different amount of template DNA or target DNA after visualizing in gel documentation system. Three different quantities of 5 µl, 8 µl and 10 µl of the template DNA were used and the band intensity was noted.
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1 | MG | 5µl | + |
2 | MG | 8µl | +++ |
3 | MG | 10µl | ++ |
4 | MG | 12µl | + |
MG= Mycoplasma gallisepticum
Band intensity was very low (+)
Band intensity was intermediate (++).
Band intensity was very high (+++).
In Pakistan, poultry has emerged as a viable alternative to beef and mutton. A drop in egg production of 10-20%, an increase in embryo mortality and chick mortality of 5-10%, and a fall in weight gain and feed conversion efficiency of 10-20% are all losses caused by Mycoplasma infection. Only four of the 22 identified species recovered from avian sources were established pathogens for domestic poultry, namely Mycoplasma Gallisepticum (Ley and Yoder, 1997)
Chronic Respiratory Condition (CRD) is the name for the disease caused by Mycoplasma. Mycoplasma gallisepticum is the most important pathogenic and commercial pathogen, and the disease caused by it has been declared as notifiable by the Office International des Epizooties (OIE). It is a big concern in the poultry business around the world, because sinusitis causes infection in turkeys. Losses related to Mycoplasmosis, specifically MG infection, include decreased egg productivity and egg quality, poor hatchability (high rate of embryonic mortality and culling of day-old birds), low feed efficiency, increased mortality, and carcass condemnation, in addition to medicine costs
Various specimen samples were collected from clinically chronic respiratory disease cases of broiler and layer chickens, including trachea, air sac swab, oral swab, and lungs tissues, for the successful isolation of Mycoplasma species, but the trachea and air sac samples were the sample of choice because it is the predilection site of manipulation of Mycoplasma species. Beef heart infusion, peptones, yeast extract, and serum are the most important nutrients for the growth of mycoplasma spp. Along with NAD and cysteine, serum is a critical need for Mycoplasma growth. Additionally, because Mycoplasmas are absolute resistant to these medications, penicillin and relative insusceptible thallium acetate are utilized to produce selective
Mycoplasma species obtained in Brain Heart Infusion broth had a morphology that was tiny, smooth, circular, and had a fried egg look with a central opaque and outer translucent area. Several researchers have observed similar traits in mycoplasma species colonies.
From the total of 25 samples, 25 (100%) had a positive Mycoplasma isolation result. The biochemical test yielded the highest number of isolations, with MG accounting for 10/25. Commercial DNA-based PCR kits have been developed as a result of recent advancements in diagnostic techniques for Mycoplasma infections. However, the reaction cost for clinical samples increased by a factor of two. The multi-species PCR allowed for the detection of multiple Mycoplasma in a single sample. The methods for detecting pathogenic avian mycoplasma using multiplex PCR described above are specific, sensitive, and consistent. It assisted us in enhancing the specific and accurate diagnosis of Mycoplasmosis in our commercial poultry sector by employing specific primer sequences and contributed to the elimination and reduction of losses due to Mycoplasmosis
Tetracyclines, fluoroquinolones, tilmicosin, tylosin, spiramycin Infections should be reduced as much as possible.
MG infection status is crucial for business in birds, hatching eggs, and chicks because eradication of this virus has been a primary goal of official poultry health programmes in most countries. These programmes rely on uninfected chick purchases, all-in/all-out production, biosecurity, and regular serological monitoring. Preventative medication of known infectious flocks may be beneficial in some cases.