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Detection of Mycoplasma gallisepticum Infection in Day-Old Chicks Using Molecular Characterization
Detection of Mycoplasma gallisepticum Infection in Day-Old Chicks Using Molecular Characterization
Eissa, S.I.*, El-Shater, S.A.*, Dardeer, M.A.*, Abd ElAziz, E. E.*, Hanaa, A. Ahmed. *and Anne,V. Gautier-Bouchardon **
*Mycoplasma Research Department, Animal Health Research Institute, Dokki, Egypt.
**Agence Française de Sécurité Sanitaire des Aliments, Unité de Mycoplasmologie-Bactériologie, Zoopôle Les Croix, BP 53, 22440 Ploufragan, France.
Summary
The present study was conducted for the detection of Mycoplasma gallisepticum (MG) infection in day-old chicks. Therefore, a total of 150 day-old chicks were taken from 15 different flocks from different breeds. Trachea, lung and air sacs samples were examined using the conventional isolation and Polymerase Chain Reaction (PCR) methods. All flocks positive by isolation were positive by PCR (3 positive flocks, 20%) while PCR was more sensitive as it gave 5 positive flocks (33.3%).
Five isolates were subjected to Arbitrarily Primed- Polymerase Chain Reaction (AP-PCR) and were found to share two common fragments of 325 and 370 bp with the used MG reference strains. Differences in DNA pattern between isolates may be attributed to different sources of samples.
Sequencing of the 16S rRNA gene was performed to confirm the infection with MG (vertical transmission) using three field isolates, and the F and 6/85 MG vaccinal strains. The obtained sequences were compared with the R (low) and A (5969) reference strains from Gene Bank And a high percentage of homogeneity was found between strains(98- 100%). In conclusion, M. gallisepticum (or MG if you prefer) infection in day-old chicks can be diagnosed by culture, PCR, AP-PCR and confirmed by sequencing of the 16S rRNA gene.
Introduction
Mycoplasma gallisepticum affects chickens and turkeys and is the most important of the pathogenic avian mycoplasmas. It is especially serious in broiler chickens in which it often acts synergistically with other agents, such as respiratory viruses or pathogenic strains of Escherichia coli to provoke chronic respiratory disease. In laying hens, it may also cause loss of egg production (Bradbury, 1998). Control of M. gallisepticum has generally been based on eradication of the organism from breeder flocks and maintenance of a mycoplasma-free status in the breeders and their progeny by implementation of biosecurity measures.
The rapid and widespread expansion of poultry in restricted geographical areas and the consequent reemergence of mycoplasma infections have necessitated a reevaluation of the strategies used to control mycoplasma infections of poultry. In endemic areas, vaccination with live vaccines is used as an alternative control strategy (Whithear, 1996; Kleven, 1997). Consequently, with the increased use of vaccination, more powerful tools are required to trace the source of contamination and to differentiate vaccine strains from circulating field isolates to aid better understanding of the epidemiology of the disease and to improve control strategies (Ferguson et al., 2005)
Diagnosis of M. gallisepticum infection in chickens has long been dependent on serological assays and/or isolation and identification of the organism (Anonymous, 1996). These serologic tests are useful for examining flocks but they sometimes lack of specificity (Glisson et al., 1984) and sensitivity (Avakian et al., 1988). Problems can arise with the use of rapid plate agglutination (RPA) test, especially when undiluted sera are tested, with non specific reactions due to bad quality or freezing of sera, or properties of the antigen. Moreover, serology is not a valid screening method if any commercial M. gallisepticum or M. synoviae vaccines have been used, because current serological tests can not differentiate between responses to vaccine or field strains.
Cultivation techniques are laborious and expensive and require awareness of any recent antibiotic treatment since this can inhibit isolation of the organisms. other problems experienced with culture include overgrowth by faster growing mycoplasma species or other bacteria (Garcia et al., 1995).
Furthermore neither PCR nor cultures are suitable for determining the mycoplasma status of a flock that has been vaccinated with a live vaccine, unless a method that differentiates between field and vaccine strains is used. Culture combined with identification by immunological methods or PCR is still considered to be the gold standard but validation should be carried out according to OIE method, and quality control of medium is essential (OIE, 2008).
In a 2005 meeting, European Mycoplasma Specialists discussed the Mycoplasma diagnostic problems and particularly those associated with the testing of day-old birds and agreed that no validated serological test or PCR could be used in day-old birds for accurate detection of Mycoplasma infections.
So the aim of this study is:
1- Comparing M. gallisepticum infection in day-old chicks by conventional culture methods and PCR in local and imported breeds.
2- Study the genetic variability between MG isolates by random amplified polymorphic DNA (AP-PCR).
3- Sequencing of PCR products of the obtained isolates and comparing them with the mostly used vaccinal and reference strains.
Material and Methods
1. Samples: 150 day-old chicks were taken from 15 different farms with different breeds (Saso, Avian 48, Hubbard, Ross and Baladi breeds)from Dakhahlia Governorate (day-old chicks from different sources) from Jan 2008 to July 2008. Ten samples at 40 days of age were also performed from one of the Saso farms. Tracheal, lung and air sacs samples were taken from these birds. All tissue samples were propagated in Frey’s broth (Frey et al., 1968) and the samples were then divided into two groups. The first one was propagated by classical methods (isolation, Digitonin sensitivity and biochemical identification) using the methods described by Erno and Stipkovits (1973). The second group was subjected to DNA extraction and PCR following the Fan et al. (1995)method.
2. M. Gallisepticum specific PCR (Kempf et al., 1993): Two oligonucleotide primers were selected for the detection of MG. The primer 1 sequence was: 5‘- TAA CTA TCG CAT GAG AAT AAC-3‘. The primer 2 sequence was: 5‘- GTT ACT TAT TCA AAT GGT ACA G-3‘. Primers were prepared in Germany by the Sigma Company.
The PCR reaction mixture (total volume of 50 µl) was: 5 µl of 10 X reaction buffer (Promega), 1.5µl of 25 mM MgCl2, 1 µl of 10 mM of dNTP (Sigma), 2 µl of primer (containing 400 ng of each forward and reverse primer), 2 µl of DNA template (containing 40 ng of DNA), 0.5 µl (2 units) of taq DNA polymerase (Promega) and distilled water to complete the mixture to 50 µl . PCR was performed on a Progene programmable thermal controller (UK). The amplification was performed by heating the samples for 5 min at 97°C then, by using thirty cycles of denaturation for 1 min at 94°C, annealing for 1 min at 55°C and extension for 1 min at 72°C; a final extension step at 72°C was held for 10 min. The analysis of PCR products was performed by using 10 µl of the amplified PCR product, mixed with 2 µl loading buffer and electrophoresed through 1% agarose gel and DNA was visualized by UV flourescence after ethidium bromide staining, and then photographed.
3-Arbitrary primed PCR (AP-PCR): was performed according to the Fan et al. (1995) method using M16s PCR 5‘-AGGCAGCAGTAGGGAAT-3‘ primer.
Three cycles of 940C for 15 sec, 28 0C for 2 min and 74 0C for 3 min and 35 cycles of 94 0C for 15 sec, 45 0C for 2 min and 74 0C for 3 min were performed. Image analysis was made by Image Quant TL-2005 software (Amersham Bioscience).
4- Sequencing and sequence analysis: The 16S rRNA gene PCR product was purified using Sephaglas TM BandPrep Kit (Amersham Pharmacia Biotech).
Sequencing was conducted by the Sigma Company (Germany) and was determined for each strand of DNA. The resulting chromatograms were examined in Codon Code Aligner (Version 2.0.6; Dedham, MA). The forward and reverse complemented sequences were compared to produce a consensus sequence for each strain. All the sequences were then aligned and compared using CLUSTAL W (1.82) multiple sequence alignment. All the tested isolates were compared with M. gallisepticum R (accession number AE016969) and A5969 (accession number L08897) reference strains.
Results and Discussion
Although generally regarded as the 'gold standard' for definitive diagnosis, Mycoplasma culture has the disadvantage of being very laborious, particularly in cases of mixed infections, and it also depends on the presence of viable organisms.
While positive results may be obtained within 4 to 7 days, up to 30 days may be required for a negative result. On the other hand PCR-basedDNA probe tests take only 1 to 2 days for the detection of Mycoplasmas and are not dependent upon viability of the organisms (Kleven, 1994).
Six flocks out of the 15 tested flocks (40%) were Mycoplasma-negative by both isolation and PCR, 5 were positive by PCR (33.3%) and among them 3 were positive by isolation (20%). Two flocks were positive for Mycoplasma gallinarum and 2 were Acholeplasma-positive (13.3% each). One flock of Saso breed (with day-old chicks positive by both culture and PCR) was tested at 40 days of rearing and was positive by PCR but not by culture (Table 1 and Photo 1). No data at that age were recorded from any previous publications.
Bacterial or yeast contamination and the presence of non viable mycoplasmas could be one reason for the low number of positive cultures. Moreover, the large number of positive results according to MG-PCR could be explained by the fact that PCR detects DNA from viable and non viable mycoplasmas. However, only viable mycoplasmas should be considered as a potential source of infection (Marois et al., 2002).
| 1- 100 bp Ladder Marker (Pharmacia) | 2- R reference strain |
| 3- F reference vaccinal strain | 4- 6/85 vaccinal strain |
| 5- Field isolate (1) 1-day-old | 6- Field isolate (2) 1-day-old |
| 7- Field isolate (2) 40-day-old | |
Photo 1: Results of PCR amplification of different isolates using 16S rRNA gene
Table 1: Recovery rate and PCR results of the examined flocks:
| Breed | Culture | PCR | Diagnosis | |||
| D | G | A | F&S | |||
| Avian 48 | - | - | - | - | - | -ve |
| Saso | + | + | - | - | + (330bp) | M. gallisepticum |
| Hubbard | - | - | - | - | - | -ve |
| Saso | + | + | - | - | + (330bp) | M. gallisepticum |
| Hubbard | - | - | - | - | - | -ve |
| Hubbard | + | - | + | + | - | M. gallinarum |
| Ross | + | - | + | + | - | M. gallinarum |
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