Prevalence of Listeria species including L . monocytogenes from apparently healthy animals at Baroda Zoo , Gujarat State , India

1 Assistant Professor, Mahatma Phule Krishi Vidyapeeth, Rahuri, Maharashtra 413722, India 2, Professor, 3 M.V.Sc Scholar, Department of Veterinary Microbiology, 4 Professor, Department of Veterinary Medicine, College of Veterinary Science & Animal Husbandry, Anand, Gujarat 388001, India Email: 1 drmahendrayadav@rediffmail.com (corresponding author), 2 aroyvet@yahoo.co.in, 3 bbbhanderi@yahoo.co.in, 4 rgjani@aau.in


IntroductIon
Listeriosis is an important bacterial disease of animals and a zoonosis with a broad distribution; Listeria monocytogenes is the major pathogen causing listeriosis and is of significant economic and health concern as it causes disease in a wide variety of animals including sheep, goats, cattle, buffaloes, dogs, horses, chickens, rabbits and also human beings (Katiyar 1960).
Information on the serovar allows discrimination between isolates belonging to an outbreak and those that are not part of the outbreak.All major outbreaks of listeriosis are caused by serovar 4b, which is primarily responsible for ruminant listeriosis (Rocourt & Seeliger 1985;Radostits et al. 1994).This strain is infrequent in foods compared to 1/2a strains (Buchrieser et al. 1993;Farber & Peterkin 1991).The procedure adopted to investigate outbreaks relies on serovar characterization for isolated strains.
In Gujarat State, India, there has been no report of Listeria sp. from wild animals; although L. monocytogenes has been isolated from wild animals in Nagpur City in the neighbouring Maharasthra by Kalorey et al. (2006).It is very important for public health to understand the prevalence of Listeria in wild animals, since wild animals whether captive or free-ranging may act as a reservoir for the disease.The purpose of this study was to determine the prevalence of Listeria sp. in apparently healthy zoo animals and to characterize the isolates phenotypically and genotypically.samples: A total of 56 faecal samples were collected from mammals (9) and birds (47) of Baroda Zoo.The animals screened were Sambar (2), Chital (3), Black Buck (3) Nilgai (1) and that of birds were Love birds (5), Cockatoo (5), Rosella (2), Macaw (3), Dove (10), Emu (5), Conur (4), Koel (2), Cockatiel (5), Lori (6).

Bacteria
Isolation of Listeria: Isolation of listeriae from the faecal samples of the animals followed the method of the US Department of Agriculture (USDA) described by McClain & Lee (1998) with some modifications.
Samples were enriched by two-step enrichment in University of Vermont (UVM) medium -I and II.Each of the faecal swabs was aseptically transferred into 10ml UVM-I.In UVM-I medium the samples were incubated at 30 o C for 24hr.Then the samples were incubated in the UVM -II medium at 30 0 C for up to seven days.After 24hr, 48hr and seven days of incubation samples were simultaneously streaked onto Dominguez-Rodriguez isolation agar (DRIA), PALCAM agar, and Oxford agar.

Phenotypic characterization
Haemolysis on sheep blood agar (SBA): All the Listeria isolates were tested for the type (α or β) and the degree (narrow or wider) of hemolysis on SBA.The isolates were streaked onto 7% SBA plates and incubated at 37 0 C in a humidified chamber for 24hr.After that they were examined for haemolytic zones around the colonies.Interpretation of the haemolytic reaction was based on the formation of a typical wide and clear zone of haemolysis (β-haemolysis) representing L. ivanovii and formation of a narrow zone of haemolysis (α-haemolysis) representing L. monocytogenes or L. seeligeri.
Christie, Atkins, Munch-Petersen (CAMP) test: The standard strains of Staphylococcus aureus and Rhodococcus equi were grown overnight on SBA plates at 37 0 C in a humidified chamber.The colonies were then also streaked onto freshly prepared 7 % SBA plates in a manner that the streaks were wide apart and parallel to each other.In between the parallel streaks of S. aureus and R. equi the Listeria isolates were streaked at 90 0 angles and 3mm apart before incubating them at 37 0 C for 24hr.In case of a CAMP positive reaction the synergistic effect of the haemolysins would lead to a wider zone of complete haemolysis between a Listeria strain and the S. aureus or R. equi strain.The Listeria isolates with CAMP-positivity against S. aureus were characterized as L. monocytogenes and those with CAMP positivity against R. equi were characterized as L. ivanovii .
Phosphatidylinositol-specific phospholipase C (PI-PLC) assay: All the phenotypically characterized Listeria isolates were assayed for PI-PLC activity following the method of Leclercq (2004) with certain modifications.
Listeria isolates were incubated overnight on 7% SBA plates at 37 0 C in a humidified chamber.All L. monocytogenes isolates were streaked on L. mono differential agar (Hi Media Ltd, Mumbai, India) in order to assess PI-PLC activity.The inoculated plates were incubated at 37 0 C in a humidified chamber for 24hr.Light blue colonies showing formation of a halo around the inoculation site were considered positive.
Phosphatidylcholine-specific phospholipase C (PC-PLC) Assay: an egg-yolk opacity test was conducted to examine the PC-PLC activity of the isolates.Tryptic soy agar (Hi Media Ltd.Mumbai, India) plates were prepared with 2.5% egg-yolk emulsion (Hi Media Ltd.Mumbai, India) and 2.5 % NaCl and a pH of 6.5-7.Listeria isolates were streaked onto the agar surfaces and incubated at 37 0 C for 36-72 hr.Formation of opaque zones surrounding the colonies was considered positive (Coffey et al. 1996).

Genotypic Characterization
Polymerase chain reaction (PCR) based detection of virulence-associated genes: The standard strain of L. monocytogenes (MTCC 1143) was incubated overnight in brain heart infusion at 37 0 C.About 0.5ml of the culture was then centrifuged in a microcentrifuge (Sigma, USA) at 6000×g for 10 min.The recovered pellet was resuspended in 100μl of sterilized DNAse and RNAse-free milliQ water (Millipore, USA), heated in a boiling water bath for 10 min and then shock chilled in crushed ice.The obtained lysate (5μl) was used as a DNA template in PCR reaction mixture.For the amplification of the virulence associated genes of L. monocytogenes, hemolysin gene (hlyA), regulatory gene (prfA), Phosphatidylinositol phospholipase C gene (plcA), Actin gene (actA) and p60 gene (iap), primers synthesized by Sigma Aldrich, USA were used.Details about primers and PCR product sizes are shown in Table 1.PCR conditions followed methods described by multiple authors (Furrer et al. 1991;Notermans et al. 1991a;Paziak-Domanska et al. 1999;Suarez & Vazquez-Boland 2001) with some modifications.Conditions affecting the sensitivity and specificity of the reaction were optimized.Such conditions are for example the concentrations of MgCl 2 , primers and Taq DNA polymerase, the annealing temperatures (50-60 0 C) and the number of cycles for amplification of the target gene.
Based on optimization trials, the standard PCR protocol for a 50μl reaction mixture included 5.0μl of 10×PCR buffer (100mm Tris-HCl buffer with pH 8.3 containing 500mM KCl, 15 mM MgCl 2 and 0.01% gelatin), 1μl of 10mM dNTP mix (a final concentration of 0.2mM; Sigma, USA), 4μl of 25mM MgCl 2 (a final concentration of 2 mM) and 10μM of a primer set containing forward and reverse primers at a concentration of 0.1μM of each primer, 1 U of Taq DNA polymerase (Sigma, USA), 5μl of cell lysate and sterilized milliQ water to make up the reaction volume.
The 0.2ml PCR tube containing the reaction mixture was tapped thoroughly with a finger and then flash spun in a micro centrifuge.The DNA amplification was performed in a Master Cycler Gradient Thermocycler with a preheated lid (Eppendorf, Hamburg, Germany).The cycling conditions for PCR included an initial denaturation at 95 0 C for two minutes followed by 35 cycles each of 15s denaturation at 95 0 C, 30s annealing at 60 0 C and 90s extension at 72 0 C, followed by a final extension of 10min at 72 0 C and kept at 4 0 C.All the five sets of primers for virulence-associated genes  were amplified under similar PCR conditions and amplification cycles.The resulting PCR products were analyzed by agarose gel electrophoresis (1.5%; low melting temperature agarose L), stained with ethidium bromide (0.5μg/ml) and visualized by a UV transilluminator (UVP Gel Seq Software, England).

Multiplex PCR based serotype detection of Listeria monocytogenes isolates:
The multiplex PCR assay was standardized for the detection of serovars 1/2a, 1/2b and 4b of L. monocytogenes following the methodology described by Doumith et al. (2004).The primers for detection of L. monocytogenes 0737 gene (lmo0737), transcriptional regulator gene (ORF2819), secreted protein gene (ORF2110) and phosphoribosyl pyrophosphate synthetase gene (prs) of L. monocytogenes used in this study were synthesized from Sigma Aldrich.Details of the primer sequences are shown in Table 2.
The PCR was set for 50µl reaction volume.For the detection of L. monocytogenes serotypes concentrations of molecular biologicals, annealing temperature and number of cycles for amplification of the target genes were varied until optimal conditions were determined.The optimal reaction mixture for PCR contained 5.0µl of 10x PCR buffer (), 1.5µl dNTP mix, 4µl of 25mM MgCl 2 and 100µM of forward and reverse primers for each serovar 1/2a, 1/2b and 4b (final concentration 0.1µM each) and 10µM of forward and reverse primers for each Listeria spp.(final concentration 0.1µM each), 2 units of Taq DNA Polymerase, 5µl of cell lysate and sterilized milliQ water to make up the reaction volume.
The 0.2ml PCR tube containing the reaction mixture was flash spun in a micro centrifuge.The reaction was performed in a Px2 Thermal cycler with a pre-heated lid (Thermo electronic corporation, USA).The cycling conditions included an initial denaturation for five minutes at 94 0 C followed by 35 cycles of denaturation for 30 seconds at 94 0 C, 75 seconds of annealing at 54 0 C and 75 seconds of extension at 72 0 C. It was followed by 10 minutes of extension at 72 0 C and was finally held for 30 minutes at 4 0 C.After the reaction, PCR products were kept at -20 0 C until further analysis by agarose gel electrophoresis.
Phenotypic characters: One of the Listeria isolates was CAMP positive and showed the characteristic enhancement of haemolytic zone with S. aureus.Two of the isolates did not show enhancement of the hemolytic zone either with S. aureus or R. equi.The CAMP positive isolate was also found to be positive for PI-PLC and PC-PLC assay and was confirmed to be L. monocytogenes.
Genotypic characters: The standardized PCR allowed amplification of virulence associated genes of L. monocytogenes plcA, prfA, actA, hlyA and iap to their respective base pairs, 1484 bp, 1060 bp, 839 bp, 456 bp and 131 bp, and allowed visualization of each virulence associated gene, each gene represented by a single band in the corresponding region of the DNA ladder.The primers used in the PCR were specific to the target genes and all the five genes were detected in standard strains of L. monocytogenes, whereas none All three Listeria isolates were subjected to standardized PCR for detection of five virulenceassociated genes.Only one isolate, which has been confirmed as L. monocytogenes, showed amplification for four of the virulence-associated genes (hlyA, plcA, prfA and actA), the fifth gene (iap) was not detected.The isolates confirmed as L. innocua failed to amplify any of the five virulence-associated genes.
The multiplex PCR was standardized for detection of three major serotypes of L. monocytogenes 1/2a, 1/2b and 4b by targeting various genes like Imo0737, ORF2819, ORF2110 and prs which were coding unknown protein, putative transcriptional regulator, putative secreted protein and putative phosphoribosyl pyrophosphate.The isolate showed amplification of three molecular size bands 471 bp, 597 bp and 370 bp corresponding to the genes, ORF2819, ORF2110 and prs, respectively.
All the isolates biochemically identified as Listeria, including the two isolates identified as L. inocua, amplified 370 bp product corresponding to gene prs.This was used as an internal amplification control.
The single L. monocytogenes isolate was serotyped as 4b by multiplex PCR assay.dIscussIon Prevalence of Listeria spp.and L. monocytogenes from zoo animals: In a zoo setting infectious diseases are the main cause for animal losses as well as a concern for public health.Infectious diseases are caused by bacteria, fungi, parasites, or viruses.Control of such infections is difficult because of the design and purpose of the zoo itself.In a modern zoo we strive to show the animal under natural conditions and it is impossible to provide pathogen free soil, water or air.
However, infectious diseases can be minimized with appropriate recognition of the problem and training of personnel to limit the spread of infectious organisms.
In the present study 5.36% of the animals checked were found positive for Listeria spp, of which 1.79% was L. monocytogenes and 3.57% were L. innocua.This is a fairly low infection rate compared to the results of Arumugaswamy & Gibson (1999), who reported 18.6% of the animals excreting L. monocytogenes in Taronga Zoological Garden, New South Wales, Australia, and Bauwens et al. (2003), who reported 7.5% of all wild animals in zoos were carrying pathogenic Listeria.Faeces of healthy animals have often been reported to contain L. monocytogenes (Skovgaard & Morgen, 1988).In India, Kalorey et al. (2006) isolated L. monocytogenes from eight (16.0%) of 50 faecal samples of healthy captive wild animals.
The presence of Listeria sp.including L. monocytogenes without clinical signs in zoo animals may reflect that these animals were in the incubation period of a disease or they are not susceptible to the organism.Listeria sp. are ubiquitous in nature and are commonly found in the intestines of animals and humans without necessarily causing disease.
In a zoo setting, the keeping of many species of animals in a restricted area could lead to an increased number of clinically healthy carriers posing an infection risk to susceptible animals, personnel and visitors.
In domestic cattle the reported rate of Listeria spp.identified in the faeces was in the range of 3.1-45.8% (Gronstel 1979;Loken et al. 1982).In comparison to farm animals the prevalence of Listeria sp. and L. monocytogenes in zoo animals found in our study is very low.This might be a result of individual care, isolation and better hygienic measures.
For domestic cattle the usual route of infection is via contaminated food.However, silage was not fed to the herbivores at the zoo.All Listeria in our study were found in samples from carnivores and here the raw meat products fed to the animals may be the source of infection.
The rate of isolation of Listeria sp., L. monocytogenes and L. innocua in the present study, was very low; this was probably due to several factors that were compounded by an already low incidence of the organism.One of the major factors was the extremely high microbial load of feces.Our findings thus were consistent with that of Siragusa et al. (1993) who reported that L. innocua was the most frequent species of Listeria isolated from cattle.
The PCR assay in for detection of virulence associated genes, the iap gene from the L. monocytogenes isolate was not amplified whereas the other four virulence-associated genes showed specific amplification.The iap gene might be absent or there is mutation in primer binding region.However, this did not reflect in any of the biochemical or phenotypical reactions of the isolate.Nishibori et al. (1995) has shown that the PCR detection of only one virulence associated gene is not always sufficient to identify L. monocytogenes.We have shown that at least one of the virulence associated genes can miss without an alteration of the isolate.The minimal number of virulence associated genes, which allow for exact identification of L. monocytogenes, must therefore still be determined.Geographic differences in the global distribution of serotypes apparently exist, but our data is not sufficient to allow for major conclusions.Serotype 4b however, the serotype identified in our study, has been found to be the serotype predominantly responsible for the animal listeriosis and Listeria associated foodborne outbreaks.So this result is of critical importance for the further epidemiological investigations.
In conclusion, it is clear that wild animals can act as reservoirs or carriers of L. monocytogenes.Listeria species were detected in wild animals that moved broadly and that had homeranges overlapping with areas inhabited by humans.
For public health, it is important to clarify the epidemiological relationship between L. monocytogenes in wild animals and L. monocytogenes in human cases of listeriosis and food contamination.In a zoo setting, infection risks need to be considered not only among the various zoo animal populations, but also with regard to human contacts.