Molecular Epidemiology of Salmonella of Poultry Origin and Development of Rapid Diagnostic Kit
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Date
2012
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Publisher
TANUVAS, Chennai
Abstract
A study was undertaken on molecular epidemiology of Salmonella organisms
isolated from poultry, poultry related products and environmental samples and to
develop a rapid diagnostic kit for early detection of Salmonella from samples. A total
of 1215 samples were collected from 154 farms and screened for the presence of
Salmonella organisms. The collected specimens comprised of liver, spleen, ovary,
intestinal contents, pooled organs and yolk from poultry, meat, egg, feed, fishmeal,
meat and bone meal of poultry products and environmental samples such as water,
drag swab, boot swab, muconium and fecal samples. The collected samples were
subjected to conventional isolation method involving pre-enrichment in buffered
peptone water, selective enrichment in tetrathionate broth and selective plating in
brilliant green agar. Pink colour colonies developed in BGA plates were further
subjected to biochemical tests such as urease activity, indole production, MR- VP
test and lactose fermentation. Twenty one isolates were confirmed as Salmonella
enterica subsp enterica from the total specimens screened. The Salmonella isolates
were designated as S2, S44, S87, S99, S100, S102, S184, S185, S186, S255,
S380, S381, S585, S586, S711, S714, S759, S791, S828, S931 and S932. The
isolates which were confirmed as Salmonella enterica subsp enterica by
conventional method were subjected to molecular identification and further typing.
The overall isolation of Salmonella in this study was 1.73 per cent (21/1215).
Tissue samples showed highest isolation of 3.06 per cent (12/392) followed by
poultry related products 1.52 per cent (7/460) and environmental samples 0.55 per
cent (2/363). The incidence of Salmonella isolation was high in winter (2.8%) as
compared to summer (0.66%). Among the tissue samples collected from affected
birds, the isolation was high in chicks below 3 weeks of age (7.27%) followed by
layer chicken below 40 weeks (3.6%) and layer chicken above 40 weeks of age
(1.78%).
Electron microscopic study of two isolates (S184 and S585) showed the
presence of rod shaped morphology with microstructure such as peritrichous
flagella and pili external to the cell wall. Antibiotic sensitivity of all the isolates
showed cent per cent sensitivity to ciprofloxacin and none of the isolates were
sensitive to oxytetracycline.
The molecular tests viz., multiplex PCR, alllele specific PCR, real time PCR
were used for the detection of Salmonella organisms and ERIC PCR, rRNA spacer
region polymorphism, ribotyping and pulse field gel electrophoresis were performed
for typing the Salmonella isolates.
Multiplex PCR was used to differentiate the Salmonella at serotype level by
amplifying the fimbrial virulence gene pefA and restriction enzyme gene kpnl.
Salmonella Typhimurium produced two amplification fragments at 363 bp and 497
bp and Salmonella Enteritidis produced one amplification fragment at 497 bp level.
Out of 21 isolates, 16 isolates (S2, S44, S87, S99, S100, S102, S184, S185, S186,
S255, S380, S381, S711, S714, S931 and S932.) were identified as Salmonella
Typhimurium and five isolates (S585, S586, S759, S791 and S828) as Salmonella
Enteritidis.
Allele specific PCR was carried out for differentiating group D Salmonella
from other Salmonella as well as from non–Salmonella organisms based on
amplification of rfbS gene which is present in serogroup D Salmonella only. Out of
21 isolates four (S586, S759, S791 and S828) were identified as Group D
Salmonella in allele specific PCR which produced amplification fragment of 720 bp.
The Real time PCR assay was carried out for the specific detection of
Salmonella targeting the gene invA. This assay incorporated both primers and
hybridization probes based on the sequence of the Salmonella invA gene. All the
Salmonella isolates produced amplification curve by detecting the invA gene with
Ct value ranged from 23.11 + 0.104 to 38.61 + 0.316.
To identify the variation within the isolates, ERIC PCR was carried out by
using the ERIC primers. All the isolates produced different banding pattern ranging
from two to six bands with the fragments ranging from 150 bp to 2000 bp. In this
study six ERIC profiles were noticed (ERIC 1 to 8).
The variation in the length of the internal transcribed spacer (ITS) region
between the 16S and 23S rRNA genes of the Salmonella isolates was analyzed by
PCR amplification. All the 21 isolates produced two to three fragments with the size
ranging from 500 bp to 700 bp.
Ribotyping was used to sequence the Salmonella isolates. The 16S rRNA
gene of five selected Salmonella isolates (S87, S100, S184, S585 and S759) was
amplified with universal primers and sequenced. All the five isolates produced
amplification of 1500 bp for 16S rRNA gene. The nucleotide sequencing of S87,
S100 and S184 had a homology pattern of 93 per cent, 93 per cent and 99 per cent
with Salmonella Typhimurium and remaining two isolates (S585 and S759) had a
homology of 96 per cent and 95 per cent with Salmonella Enteritidis respectively.
Pulse Field Gel Electrophoresis was used to study the genetic relationship
between the Salmonella strains. Out of twenty one isolates, 19 isolates (S2, S44,
S87, S99, S184, S185, S186, S255, S585, S586, S380, S381, S711, S714, S759,
S791, S828, S931 and S932) generated restriction fragments in PFGE with Xba1
enzyme and two isolates (S100 and S102) were untypable. Nineteen Salmonella
isolates generated up to 13 restriction fragments with molecular size ranging from 40
to 700 kb fragments with 11 PFGE patterns (PFGE1 to PFGE11). Among these 11
different PFGE patterns, there were five PFGE patterns (PFGE1, PFGE 2 PFGE 3,
PFGE 4 and PFGE 5) represented by two isolates (10.5%) each, PFGE 6 was
represented by four isolates (21%) and remaining 5 PFGE patterns represented by
single isolate.
Based on the molecular tests carried out in the present study, a rapid
diagnostic kit was developed with multiplex PCR for early detection of Salmonella
organisms using Salmonella specific primers in poultry, poultry related products and
environmental samples.
Description
TNV_THE_Y2012_DPV08010
Keywords
null, Veterinary Microbiology