Development of a specific and sensitive method for detection of Salmonella in Chickens using Polymerase chain reaction technique
Date
2012-10-08
Authors
Tuchili, Lawrence Musonda
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Abstract
Conventional microbiological techniques used in the detection and identification of Salmonella infections in chicken samples are relatively lengthy. In Zambia today, the situation has even been made worse by the indiscriminate use of antibiotics. Detection of Salmonella from such treated birds has recently been very poor and hence the need to develop other quick and reliable systems in the identification of Salmonella infections in poultry in Zambia.DNA detection with polymerase chain reaction (PCR) as a means of identifying Salmonella infection in chickens has been developed. Amplification of Salmonella was evaluated using the known nucleotide sequences within the InvA and phoE genes of Salmonella typhimurium. Targeted genes of Salmonella were successfully amplified fi-om a single colony of the bacteria by applying the PCR technique. With the two pairs of primers {InvA, phoE), a 284-base pair (bp) and 365-bp fragments, respectively, from Salmonella gene could be amplified specifically. Specificity and selectivity of the primers {InvA and phoE) was tested on both Salmonella gallinarum and Salmonella typhimurium as well as on other 10 Salmonella DNA. It was interesting to note that both Salmonella gallinarum and Salmonella typhimurium and other 10 Salmonella strains which were tested for specificity in PCR produced specific amplified bands whose molecular weight was same as that of S. typhimurium band obtained with either the InvA or phoE gene primers. On the other hand, other bacteria other than Salmonella bacteria did not generate any specific bands when applied in PCR using the InvA and phoE gene primers. The amplification of both the InvA and phoE genes of Salmonella by the single colony method confirmed that Salmonella did contain genes which were potential targets for PCR.
Once the specificity and applicability of the InvA and phoE gene primers had been confirmed, the InvA gene primers were then successfully used to detect both S. gallinarum and S. typhimurium DNA from experimentally infected chicks. Using the InvA primers, it was possible to detect S. gallinarum and S. typhimurium in 15 out of 20 (75 per cent) and in 16 out of 20 (80 per cent) organ samples respectively, barely 21 hours after experimental infection. PCR was therefore shown to be more sensitive in the detection of Salmonella than the conventional isolation methods as it was possible to detect in total 50 out of 150 (33.3 per cent) organ samples compared to 24 out of 150 (16 per cent) by bacterial isolation methods. Detection by PCR still appeared more sensitive even in late stages of infection as S. gallinarum DNA could be detected from samples which were negative for bacterial isolation on three, seven and 14 days after infection. The size of the amplified band was a 284-bp fragment.
Having succeeded in amplifying S. gallinarum from experimental infection, the PCR was then used to detect Salmonella in naturally infected chickens using the phoE gene primers. Results obtained show that the PCR was more sensitive in detecting Salmonella DNA from clinical samples as it was possible to detect Salmonella in 10 out of 17 (58.8 per cent) spleen samples compared to 7 out of 17 (41.1 per cent) by isolation in enriched media. The size of the amplified band was a 365-bp fragment.
PCR technique was also used for detection of Salmonella in chicken pen samples. When compared with conventional isolation methods, PCR was shown to be more sensitive as it was possible to detect Salmonella in 13 out of 48 (27.1 per cent) faeces compared with 9 out of 48 (18 per cent) by conventional bacterial isolation methods.
In order to enable detection of Salmonella organisms directly from chicken eggs, an attempt was made to inoculate the egg samples directly into the PCR mixture. By direct PCR amplification method, DNA could be amplified from 20 out of 45 (44.5 per cent) samples from chicken yolks. Detection by bacteria isolation methods on the other hand, was only possible in 6 out of 45 (13.3 per cent). The PCR was once again shown to be more sensitive when compared with conventional bacterial isolation methods. The size of the amplified band was a 365-bp DNA fragment. Direct PCR amplification of Salmonella gene from the yolk of chicken eggs and embryos is a new finding. Detection of Salmonella directly from chicken yolk samples is a great scientific contribution as the need for DNA isolation and purification was completely eliminated. The chicken yolk samples were found free of PCR inhibitors, a fact which makes detection of Salmonella by PCR from these samples more efficient and quicker. The total time required for the detection of Salmonella organisms from eggs using PCR technique was only 6 hours compared to 3 days by conventional bacterial isolation procedures.
The complete sequence of the InvA and phoE genes of Salmonella were firstly elucidated and compared with those of known InvA and phoE genes of S. typhimurium. Except for one base pair at position 259, all the other bases showed greater homology to the InvA genes of S. typhimurium indicating that the amplified band was a Salmonella gene. Similarly, the sequence of the phoE gene showed great homology to that of S. typhimurium. In the case of phoE gene, all base pairs were identical except for five at position No. 211, 212, 213, 214, and 235.
From the results obtained in this study, it is clear that:
1.A sensitive and faster method for the detection of Salmonella from
poultry has been developed. The culturing of bacteria which normally
takes 3 days was eliminated and the period required for identification of
Salmonella infection by PCR was only a day.
2.There was no need for primary bacterial isolation from clinical
samples in order to run the PCR test as DNA for PCR was obtained
directly from infected chicken organs. Extraction of Salmonella DNA
directly from infected chicken organs is also a new finding.
3.Direct PCR amplification of Salmonella from the yolk of chicken eggs and embryos is a new finding requiring further exploitation.
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Keywords
Salmonellosis in poultry , Bacteria diseases in chicken--diagnonsis