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Isolation and Seroprevalence Studies on Listeria Monocytogenes using Anti-Listeriolysin O ( Allo) based indirect Elisa in Slaughtered Buffaloes

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Title Isolation and Seroprevalence Studies on Listeria Monocytogenes using Anti-Listeriolysin O ( Allo) based indirect Elisa in Slaughtered Buffaloes
 
Creator Bharate, S. J.
 
Contributor Zade, N. N.
 
Subject Veterinary Public Health
Isolation and Seroprevalence Studies on Listeria Monocytogenes using Anti-Listeriolysin O ( Allo) based indirect Elisa in Slaughtered Buffaloes
 
Description A systematic study was carried out for the isolation of pathogenic Listeria
monocytogenes from meat and blood samples vis-a-vis detection of antilisteriolysin
O antibodies (ALLO) among the buffaloes slaughtered in Nagpur
region. Isolation of the organism was attempted from these samples by selective
enrichment in University of Vermont medium (UVM) broth and subsequent plating
onto Polymixin Acriflavin Lithium chloride Ceftazidime Aesculin Mannitol
(PALCAM) agar. The further confirmation of isolates was done by employing biochemical and sugar fermentation patterns of D- xylose, L- Rhamnose and
α-Methyl-d- Mannoside. Out of 200 samples analysed (100 each of meat and
blood) L. monocytogenes were isolated from six per cent (4% and 2%) from meat
and blood respectively; L. seeligeri was isolated from three (1% and 2%) of meat
and blood samples respectively; while two (2%) L. welshimeri were isolated from
meat samples. Thus the overall prevalence reported to be highest of
L. monocytogenes (6%), followed by L. seeligeri (3%) and L. welshimeri (2%)
among the slaughtered buffaloes.
All the listerial isolates were screened by employing three in-vitro
pathogenicity tests viz. haemolysis on 7% sheep blood agar (SBA), Christie
Atkins Munch Petersen (CAMP) test and Phosphatidylinositol-specific
phospholipase (PI-PLC) assay. All the six L. monocytogenes isolates turned
pathogenic in all these in-vitro tests except one isolate deviated in PI-PLC test,
All three L. seeligeri showed weak haemolysin on SBA, weak CAMP positivity
against S. aureus and PI- PLC negativity, while one isolate of L. welshimeri
exhibited PI- PLC positivity.
Study on antibiogram of Listeria spp. to 12 different antibiotics commonly
used in field revealed a multiple drug resistance patterns. The maximum degree
of resistance was observed against nalidixic acid, kanamycin and trimethoprim
(100% each), followed by cloxacillin (36.36%), rifampicin (27.27%), penicillin-G
and norfloxacin (18.18% each) and gentamicin, tetracycline and erythromycin
(9.09% each). The highest degree of sensitivity was observed towards ampicillin
(100%); followed by gentamicin (90.90%), penicillin-G (81.81%), erythromycin
and norfloxacin (72.72% each), rifampicin (63.63%), tetracycline and
cephotaxime (54.54 each %), cloxacillin (27.27%). The observations revealed
multidrug resistant isolates of Listeria. However; ampicillin, gentamicin and
penicillin can remain the treatment of choice.
In order to screen serum samples for serodiagnosis; purification and
characterization of Listeriolysin-O (LLO) a major virulence factor from standard
strain of L.monocytogenes (MTCC1143) was done by using DEAE- cellulose ion
exchange chromatography. The subsequent characterization of purified LLO
revealed homogenous protein of 58.85 kDa by SDS-PAGE and evaluation of
haemolytic activity by SRBCs could register it to the tune of 250 HU/mg. The
antibodies were raised against LLO (ALLO) into two healthy rabbits by injecting100 μg of purified LLO each. The rabbits were test bled after 14 days to
achieve a satisfactory titre of anti-LLO serum. Finally indirect ELISA was
standardized by checker board analysis using LLO as a candidate antigen which
was further employed for screening of buffalo sera samples.
The screening of 100 serum samples collected from slaughtered
buffaloes revealed six per cent positivity. The correlation study among the
seropositivity and cultural positivity showed that the seropositive animals were
negative in cultural positivity thus confirming the role of ALLO in elimination of
pathogen among the slaughtered buffaloes. While the seropositivity and
simultaneous culturally positivity as observed in a single case in the present
study might be due to the recent infection in this animal which has yet to elicit an
immune response or positive cross contamination of the samples as
environmental contamination.
Analysis of polypeptide profile of L. monocytogenes isolates and positivity
in haemolysin production could be correlated with the expression of haemolysin
(LLO) in the range of 50- 60 kDa among the isolates. Moreover the PI-PLC
activity as detected in the isolates could also be correlated with its parallel
production of protein in the range of 29-33 kDa as demonstrated by SDS-PAGE
analysis. However, the antigenicity of these proteins for their contribution in
virulence needs further immunoblotting study.
As recorded in the present investigation prevalence of six per cent with
respect to highly virulent L. monocytogenes pathogen with multidrug resistance
vis-a-vis seropositivity of six per cent against ALLO among the buffaloes
slaughtered in Nagpur stands to be significant findings from food safety and
public health point of view.
 
Date 2017-01-04T16:00:30Z
2017-01-04T16:00:30Z
2011-09-15
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/94541
 
Language en
 
Format application/pdf
 
Publisher MAFSU