Isolation of
Listeria monocytogenes
and anti-listeriolysin O
detection in sheep and goats
S.B. Barbuddhe
a,*, S.V.S. Malik
a, K.N. Bhilegaonkar
a,
Prahlad Kumar
a, L.K. Gupta
baDivision of Veterinary Public Health, Indian Veterinary Research Institute, Izatnagar 243122, India bImmunology Section, Indian Veterinary Research Institute, Izatnagar 243122, India
Received 5 March 1999; accepted 13 April 2000
Abstract
Studies were undertaken to compare the detection of anti-listeriolysin O antibodies (ALLO) and isolation of Listeria monocytogenesfrom meat and milk samples of sheep and goats. Out of 201 samples (87 of milk and 114 of meat) tested, 17.64% yieldedListeriaspecies isolates.L. monocytogeneswas isolated from 6.66 and 1.56% of meat (60) and (64) milk samples of goats, respectively, and 7.4% of sheep meat (54) samples. All the samples of ewes' milk (23) were negative forL. monocytogenes. Seropositivity for ALLO was observed in 41.13 and 33.76% of goats and sheep, respectively. The culture positivity forL. monocytogenesand detection of ALLO did not show any agreement (k<0.202).#2000 Elsevier Science B.V.
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Keywords:Detection;Listeria monocytogenes; Listeriolysin O; Goats; Sheep
1. Introduction
Listeriosis is an important bacterial zoonosis caused by an intracellular pathogen Ð Listeria monocyto-genes. Listeric infections occur in a variety of animals including man. Cases of listeriosis arise mainly from the ingestion of contaminated food and the disease is particularly common in ruminants fed on silage (Low and Donachie, 1997). Listeriosis may occur in several forms, including encephalitis, abortion and septicae-mia.L. monocytogenes, has been implicated in several
foodborne outbreaks and has been isolated from dairy products, meat, poultry, ®sh, vegetables and food processing plants (Gelline and Broome, 1989).
Listeriolysin O (LLO), an extracellular 58 kDa haemolysin, is a major virulence factor of L. mono-cytogenes(Gaillard et al., 1986) and is produced by all the pathogenic strains (Geoffroy et al., 1989). LLO has been identi®ed as a candidate antigen for a serological assay (Low and Donachie, 1991), and could be a dominant antigen target of anti-listerial immunity (Bouwer et al., 1992). Antibodies to LLO (ALLO) were shown to be reliable indicators for serodiagnosis of listeric infections in sheep by immunoblotting (Low and Donachie, 1991) and by ELISA (Low et al., 1992). This paper reports the use of LLO based ELISA to detect L. monocytogenes infection in apparently healthy sheep and goats.
*Corresponding author. Present address: ICAR Research Com-plex for Goa, Ela, Old Goa 403 402, India. Tel.:91-832-286-181; fax:91-832-286-249.
E-mail address: [email protected] (S.B. Barbuddhe).
2. Materials and methods
2.1. Bacteria
Listeria monocytogenes MTCC 1143 (NCTC 11994) was obtained from the Institute of Microbial Technology, Chandigarh, India. The organism was maintained on tryptose phosphate agar slopes at 48C and used for preparation of LLO.
2.2. Samples
A total of 402 samples comprising 201 serum samples (124 from goats and 77 from sheep) and corresponding 201 samples either of meat (from 60 goats and 54 sheep) or milk (from 64 goats and 23 sheep) from the same animal case were collected randomly at slaughter houses of Delhi and Bareilly (India). The meat samples were collected mainly from male animals.
The serum samples were collected in sterile vials, transported on ice from the place of their collection and stored atÿ208C until tested. The milk/meat samples were collected in sterile test tubes/UV sterilised poly-ethylene sachets, transported on ice and stored at 48C till processed for microbiological analysis.
2.3. Enrichment and isolation of Listeria
Isolation of Listeria was attempted from the col-lected meat/milk samples as per the USDA method described by McClain and Lee (1988) after some modi®cations.
Approximately 5±10 ml of each of milk sample was directly inoculated into 50 ml University of Vermont Medium (UVM1), and incubated overnight at 308C. Meat samples (10 gm each) were instead placed in a
sterile polyethylene sachet containing 90 ml UVM1 and mixed thoroughly in Stomacher (Model BA 6021, Seward Laboratory, London) for 5 min and incubated overnight at 308C. Then, 0.1 ml of the enriched inocu-lum from UVM1 was transferred to UVM2 and again incubated overnight at 308C. The enriched inoculum from UVM2 was streaked onto Dominguez±Rodri-guez isolation agar (DRIA) and plates were incubated at 308C for 48 h.
The greenish-yellow, glistening, iridescent and pointed colonies of about 0.5 mm diameter
sur-rounded by a diffuse black zone of aesculin hydrolysis were considered to be of Listeria. The presumed colonies of Listeria (at least three per plate) were further con®rmed.
2.4. Con®rmation of the isolates
Morphologically, typical colonies were veri®ed by Gram's staining, catalase reaction, tumbling motility at 20±258C, methyl red-Voges Proskauer (MR-VP) reactions, CAMP test with Staphylococcus aureus, nitrate reduction, fermentation of sugars (rhamnose, xylose, mannitol anda-methyle-D-mannopyranoside) and haemolysis on 5% sheep blood agar.
The isolates found as rhamnose and MR-VP posi-tive, and xylose and nitrate negative were tested for their pathogenicity by mice inoculation.
2.5. Mice inoculation test
Mice inoculation test was performed according to the method described by Menudier et al. (1991). Mice of either sex weighing 18±20 gm were inoculated intraperitoneally with 0.4 ml of inoculum having
107CFU of the test organism/ml. The mice were
observed for mortality over a period of 72 h.
2.6. Isolation and puri®cation of LLO
LLO was prepared and puri®ed from the cell free supernatant by ion-exchange chromatography in accordance with the method of Lhopital et al. (1993). The purity of the LLO was checked by SDS-PAGE, which showed it to be a homogeneous 58.0 kDa protein. The fractions having LLO were pooled and the protein content was estimated, and ®nally stored atÿ208C until used.
2.7. ELISA
0.05% Tween 80 (PBS-T). Each of the test serum was diluted 1:200 in PBS and added (100ml per well) to the plates. The sealed plates were incubated at 378C for 90 min and again washed as before. Subsequently, horseradish peroxidase conjugates (HRPO) diluted in PBS were added (100ml per well) to the plates. The conjugates were diluted as, rabbit anti-goat HRPO (1:6000; National Institute of Immunology, New Delhi) and rabbit anti-sheep HRPO (1:2000). The plates were again incubated at 378C for 90 min and washed as described earlier. Finally,o -phenylenedia-mine dihydrochloride (OPD) solution (1 mg/ml) in citrate buffer with 12ml/100 ml of hydrogen peroxide was added (100ml per well) as a substrate and after 15 min, the reaction was read by ELISA plate reader (Anthos Labtek) at 492 nm. Each sample was tested in duplicate. Samples showing a positive to negative optical density ratio of 2.0 or above in serum dilution of 1:200 were considered as positive.
2.8. Analysis of data
The data was analysed for test agreement (Martin et al., 1993).
3. Results
3.1. Isolation of listeriae
Listeriasp. were isolated from 21.66% of 60 meat samples and 9.37% of 64 milk samples from goats. Out of these four (6.66%) and one (1.56%) isolates were con®rmed asL. monocytogenesfrom meat and milk, respectively (Table 1).
Out of 54 meat and 23 milk samples from sheep, 18.51 and 30.43% were positive for listeriae,
respec-tively. Four (7.4%) isolates from meat samples were con®rmed asL. monocytogenes. All the samples from ewes' milk turned out to be negative for L. mono-cytogenes(Table 1). From milk and meat samples in all, nine isolates were con®rmed asL. monocytogenes.
3.2. Pathogenicity testing
All the nineL. monocytogenesisolates were patho-genic to mice. The liver and spleen of dead mice showed considerable congestion and the pathogen was re-isolated from the liver and spleen of dead mice.
3.3. Detection of anti-listeriolysin O (ALLO) antibodies
ALLO antibodies were detected in the sera of sheep and goats tested by indirect-ELISA (Table 2). Anti-LLO titres were higher in case of goats (absorbance492
>1.212).
3.4. Isolation rate and anti-listeriolysin O (ALLO) positivity
LLO based indirect-ELISA did not agree (k0.0± 0.202) with the cultural isolation ofL. monocytogenes
in cases of goats and sheep. All the ®ve L. mono-cytogenespositive goats had high ALLO titres. Of the 14 cases of goats showing cultural positivity for
Listeria sp. other than L. monocytogenes, seven (50%) demonstrated seropositivity for ALLO. How-ever, the remaining seven (50%) culture positive goats were found negative for ALLO. Out of 105 goats with cultural negativity, 39 (37.14%) were found as positive for ALLO, whereas 66 (53.22%) were negative.
Of the four sheep culturally positive forL. mono-cytogenes, only two demonstrated the presence of
Table 1
Incidence ofListeriasp. andListeria monocytogenesin milk and meat samples
Animals Milk Meat
Sample no.
Listeria sp. (%)
L. monocytogenes (%)
Sample no.
Listeria sp. (%)
L. monocytogenes (%)
Goat 64 7.81 1.56 60 15.0 6.66
Sheep 23 30.43 0.00 54 11.11 7.4
ALLO. Out of 13 cases showing cultural positivity for
Listeriasp. other thanL. monocytogenes, ®ve (38.5%) demonstrated seropositivity for ALLO. However, eight (61.5%) culture positive sheep were found tive for ALLO. Out of 60 sheep with cultural nega-tivity, 19 (31.66%) were found as positive for ALLO, whereas 41 (53.24%) were negative.
4. Discussion
The percentage culture positivity of L. monocyto-genes in milk observed in the present study is in agreement with the reported incidences in milk as 2.2% (Farber and Peterkin, 1991), 3.48% (Ryser and Marth, 1991) and 5.78% (Bhilegaonkar et al., 1997). The observed positivity for the pathogen in goats' milk agrees with the reported rate of 0.8% from UK (Green-wood et al., 1991) and 7.8% from Yugoslavia (Katic and Stojanovic, 1992).
In the present study, in spite of high isolation rate of
Listeriasp. from ewes' milk (30.43%),L. monocyto-genes was not detected. This is consistent with the ®ndings that most of the sheep farms (93.38%) in Spain produced milk free from L. monocytogenes
throughout 1 year sampling period (Rodriguez et al., 1994). In the Indian context, sheep are often reared by extensive grazing where the chances of their exposure to dampness favouring growth of the pathogen are minimised.
The isolation rates ofListeriasp. andL. monocy-togenes in meat were similar to reports where the recovery rates of L. monocytogenes have been reported as 0±80% from raw meat and 7±80% from European ground meat (Ryser and Marth, 1991), and 0±100% from fresh meat samples in USA (Buchanan et al., 1989). The large variations in the reported incidence rates may be partly due to differences in methods of detection including the factors like
pro-cedures used, the sample size, the number of single colony isolates taken to con®rm the presence of haemolytic colonies and the source of samples (Farber and Peterkin, 1991).
In the present study, a high percentage of positivity for ALLO was detected by indirect-ELISA in goats and sheep. These results can be explained in the light of published reports indicating seroconversion to ALLO after oral or subcutaneous infection in sheep (Low and Donachie, 1991; Lhopital et al., 1993). The high positivity for ALLO in the absence of any clinical disease observed in our study may possibly be attrib-uted to exposure of these animals to low infective doses which has been reported to elicit persistent immune response to LLO equal to that with higher infective doses (Lhopital et al., 1993) through various established sources of infection including poor quality silage (Low et al., 1992) and vegetables (deSimon et al., 1992; Arumugaswamy et al., 1994).
The results of the present study are in accordance with the ®ndings reporting the detection of ALLO in naturally infected sheep with a history of enteritis and abortions (Low et al., 1992). In the present study, ALLO could not be detected in two cases which were culture positive forL. monocytogenes. The reason for this discrepancy was not apparent. ALLO was detected, however, in serum samples of animals cul-turally negative for L. monocytogenes. Studies on kinetics of ALLO production following experimental infection with pathogenicL. monocytogenesof sheep (Lhopital et al., 1993), goats (Miettinen et al., 1990; Banu Rekha, 1997) and buffaloes (Choudhari, 1997) showed that faecal, nasal and blood cultures became negative for the pathogen as ALLO peaked in the sera of infected animals. In these studies, despite the failure of the recovery of L. monocytogenesfrom cultures, signi®cant levels of ALLO titres were detected up to long periods.
Listeriolysin-O (LLO) has been known to be a major virulence factor produced by all pathogenic strains of L. monocytogenes (Low et al., 1992) and is known to induce T-cell recognition during the course of an acute listeric infection (Berche et al., 1987). Also speci®c antigenic epitopes in LLO have been reported (Nato et al., 1991). Culturing of all the samples for the pathogen is costly and cumbersome. Therefore, LLO as an antigen can be employed in serological assays for epidemiological surveys.
Table 2
Anti-listeriolysin O antibodies in goats and sheep detected by indirect-ELISA
Animals Samples Percent positive
Goat 124 41.13
Acknowledgements
The authors are thankful to the Director, Indian Veterinary Research Institute, Izatnagar, for providing necessary facilities. Thanks are also due to Council of Scienti®c and Industrial Research, New Delhi, for ®nancial assistance.
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