Prevalence of Listeria spp . in Animals and Associated Environment

Received: Revised: Accepted: 2013–12–21 2013–12–3

The listeriosis is a rare but serious food-borne disease as it exhibits 20-30% mortality, 91% hospitalization and 50% neonatal death rates (Low and Donachie, 1997;Kathariou, 2002).In human, the early stage of infection by L. monocytogenes generally displays initial flu-like symptoms such as chilling, nausea, fever, gastroenteritis etc. Untreated cases may lead to septicemia, meningitis, encephalitis, abortion and occasionally death (Barbuddhe et al., 2008).Listeria infection in animal shows broad range of symptoms from asymptomatic infection to uncommon cutaneous lesions or various focal infections such as conjunctivitis, urethritis, endocarditis, and severe disturbance of the gait, followed by death.The L. monocytogenes is a well-recognized cause of mastitis, abortion, repeat breeding, infertility, encephalitis, and septicemia in cattle (Barbuddhe et al., 2008;Deb et al. 2013).
The infection by L. monocytogenes is generally transmitted through contaminated food (Farber and Peterkin, 1991).Generally industrially processed foods such as raw meat, fish, milk, milk-related products has been linked for the listeriosis (Rocourt, 2000;Lyytikäinen et al.,2006;Kvistholm Jensen et al., 2010;Goulet et al., 2012;Dhama et al., 2013).. Heavy use of antibiotics as a growth promoter for farm animals and injudicious use of antibiotics accelerated evolution of bacteria towards antibiotic resistance.Since bacteria have the remarkable ability to develop resistance against antibiotics, bacterial species such as Listeria has also evolved towards multiple antibiotics resistance (Charpentier and Courvalin, 1999).
In India, the occurrence of listeriosis is poorly studied.Lack of awareness makes L. monocytogenes underdiagnosed therefore incidences in humans and animals are underestimated.Several researchers have explored incidence of listerial spp. in India from different sources (Jallewar et al., 2007;Yadav et al., 2011;Manoj et al., 1991;Dhanashree et al., 2003;Moharem et al., 2007;Kalorey et al., 2008).Also, L. monocytogenes cases have been reported

Prevalence of Listeria spp. in Animals and Associated Environment
ISSN: 2307-8316 (Online); ISSN: 2309-3331 (Print) sporadically in humans and animals (Mokta et al., 2010;Adhikary and Joshi, 2010).The present epidemiological data is insufficient and not conclusive (Barbuddhe et al., 2012).The objective of the study is to determine the prevalence of Listeria spp. in Konkan region of Maharashtra, India.

Sample Collection
A total of 215 samples were collected from Konkan region of Maharashtra, India (Table 1).Of these, 182 samples (vaginal swabs, blood, milk and faeces) were collected from domestic animals such as sheep, goat, bovines and pig, while, 33 samples were collected from cattle and pig farms surroundings (soil and floor swabs).The samples were collected in sterile containers, transported to laboratory on ice and processed for isolation of listerial spp.

Isolation of Listeria Species
Isolation of Listeria spp. was attempted as per USDA-FSIS method (USDA, 2013).The samples (blood, milk, faeces and soil) were inoculated (approx.5 ml/5 g) into 45 ml of University of Vermont Medium (UVM)-1 supplemented with acriflavin and nalidixic acid, and incubated at 37 o C for 18-24 h.Vaginal and floor swabs were directly inoculated in 10 ml UVM broth.Further enrichment of the samples was carried by inoculating 0.1 ml of UVM-1 to 10 ml of UVM-2 broth.Inoculated UVM-2 broth was incubated further for 24 h at 37 0 C. A loopful of enriched UVM-2 broth was streaked directly on PALCAM agar for selective isolation of listerial colonies.The inoculated agar plates were incubated at 37 o C for 48 h.The isolated pinpoint grayish-green colonies surrounded by black zone of esculin hydrolysis were presumed as Listeria.These colonies were further purified on PALCAM agar and stored in refrigerated conditions in BHI broth.

Biochemical Characterization and Identification of Isolates
A single isolated colony from PALCAM agar was inoculated in fresh BHI broth and incubated at 37 o C for 18 h.The freshly grown culture was then studied for their morphological and biochemical characters.Morphology was observed under light microscope while, Listeria specific

Genotypic Characterization
Biochemically confirmed L. monocytogenes isolates were subjected to serotyping by multiplex PCR method as described by Doumith et al. (2004).The genomic DNA was extracted using DNA isolation kit (Chromos Biotech, India).Oligonucleotide primers (Doumith et al., 2004)  PCR amplification of the hlyA gene was performed as described by (Paziak-Domańska et al., 1999).The 50 µl PCR mixture contained 2.5μl of 10x PCR buffer (100 mM Tris-HCl, pH 8.3 at 25°C; 500 mM KCl; 15 mM MgCl 2 ; 0.01% gelatin), 2 mM dNTP mix, 2 mM MgCl 2 and 0.3 μM of forward and reverse primers, 1.5 units of Taq DNA Polymerase and 50ng of DNA template.The final volume was adjusted by sterilized deionised water.The reaction mixture was subjected to an initial denaturation at 95 0 C for 2 min followed by 35 cycles each of 15 s denaturation at 95 0 C, 30 s annealing at 60 0 C and 1 min 30 s extensions at 72 0 C. It was followed by final extension of 10 min at 72 0 C and held at 4 0 C. The PCR products were tested as described above.

Antibiotic Sensitivity of Isolates
Disk diffusion susceptibility tests were performed according to the standard reference procedure of the Clinical and Laboratory Standards Institute (Anon, 2006;Altuntas et al., 2012).A single well-isolated colony of L. monocytogenes was transferred into 10 ml BHI broth, incubated at 37°C for 24 h, diluted 1:10 in 9 ml 0.1% peptone water to a turbidity equivalent to 0.5 McFarland standard, and spread on surface of Mueller-Hinton Agar (MHA) plate.Antibiotic discs of vancomycin (VA10), ciprofloxacin (Cf5), erythromycin (E15), gentamycin (G10), penicillin-G (P10), sulphazidine (Sz 30), amphicilin (A10), oxytetracycline (O30), chloramphenicol (C30) and doxycycline (D30) were placed on the surface of each inoculated MHA plate.After incubation for 24 h at 37°C, the diameter (in mm) of the zone around each disk was measured and interpreted in ISSN: 2307-8316 (Online); ISSN: 2309-3331 (Print) accordance with the Clinical and Laboratory Standards Institute Standards guidelines (Anon et al., 2006) to classify the antibiotic sensitivity of each isolate.Staphylococcus aureus ATCC 6538 was used as standard strain.

Isolation and Identification of Listeria
Out of 215 samples (clinical and farm environment) collected, 27(12.55%)samples were found positive for Listeria spp.After further characterization, 11 (5.11%) isolates were confirmed as L. monocytogenes, 2 (0.93%), L. ivanovii, 11 (5.11%),L. innocua, 1 (0.46%), L. welshimeri and 2 (0.93%) as L. seeligeri (Table 1).Except clinical samples collected from cattle, all other types of samples showed presence of Listeria spp.Four L monocytogenes were isolated from clinical samples collected from sheep, five from goats, one each from pig and pig-associated environment.The L. ivanovii was isolated from a clinical case in pig.No L. monocytogenes or L. ivanovii could be isolated from cattle and associated environment.
Biochemically identified L. monocytogenes and L. ivanovii spp.were further characterized by phenotypic assays.All the L. monocytogenes and L. ivanovii exhibited weak hemolysis.In CAMP test, L. monocytogenes and L. ivanovii showed increased zone of hemolysis toward Staphylococcus aureus (MTCC 1144) and Rhodococcus equi (MTCC 1135), respectively.All the Listeria spp.were tested on ALOA medium to determine the ß-glucosidase and ability to produce the phospholipase enzymes.All the isolates showed blue-green color zone confirming the Listeria spp.while in addition, L. monocytogenes and L. ivanovii exhibited an opaque white halo of hydrolysis of phosphotidyliositol or lethicin in the medium due to phospholipases.Presence of phospholipase activity was considered as indicator the pathogenicity.L. monocytogenes isolates were characterized for the presence of the hemolysin gene and by multiplex PCR basedserotyping.Multiplex PCR based serotyping revealed 5 isolates as serotype 4b while remaining 6 were of serotype 1/2b (Table 2, Figure 1).All the L. monocytogenes isolates were found to possess hlyA gene, which is responsible for hemolysin.In case of L. ivanovii genus, specific prs gene was amplified.

Antibiotic Sensitivity Testing
L. monocytogenes isolates were checked for their susceptibility towards the commonly prescribed antibiotics.All isolates were sensitive towards, erythromycin, oxytetracycline, ampicillin, doxycycline and ciprofloxacin and showed intermediate resistances towards the chloramphenicol, penicillin, gentamicin and vancomycin.However, a single L. monocytogenes serotype 4b isolate from pig placental tissue exhibited multi-antibiotic resistance.All 10 L. monocytogenes isolates shown resistance to sulphazidine.

DISCUSSION
Listeria spp.are ubiquitous in nature and has been isolated from wide environmental sources (Liu, 2008).The organism possesses ability to survive in harsh conditions and therefore can persist in environment.Because of such persistence Listeria spp.can easily enter in food chain (Carpentier and Cerf, 2011).Of the known Listeria spp., L. monocytogenes is pathogenic to humans and animals, while L. ivanovii is mainly an animal pathogen.Several foodborne outbreaks are attributed to L. monocytogenes (Ramaswamy et al., 2007).Therefore, food quality controlling authorities from several developed countries have enforced strict regulations over occurrence of L. monocytogenes in food and food products (CDC, 2012).However, such regulations are largely lacking in developing countries because of underestimated listerial scenario.In India, due to lack of awareness, burden of other traditional diseases, rare expertise, and poor reporting, the incidence of listeriosis is unknown (Dandona et al., 2004;Dandona et al., 2009).To understand the listeriosis in detail, there is a need of systematic and coordinated studies to estimate the prevalence of L. monocytogenes in different habitats, occurrence of listeriosis in humans as well as in animals and their concordance of occurrence and actual disease.The epidemiological studies would help in understanding of the sources of infection and persistence and their risk assessment, routes of transmission, clinical forms and allow for better management of the listerial infection.
Present study was performed to determine the prevalence of Listeria spp.from Konkan region of Maharashtra, India.The Konkan region contributes a great source of animal and crop originated food.Listeria spp.were isolated from 12.55% samples.L. monocytogenes and L. ivanovii were isolated from 5.11% and 0.93% samples, respectively.The L. monocytogenes isolates were mainly isolated from sheep and goats.A bacterial etiology studied in relation to abortions by over 203 sheep and goat flocks showed 5-6% of L. monocyotgenes infections (Sharma et al., 2008).Barbuddhe et al. (2000) have reported presence of L. monocytogenes in 6.66% and 7.4% meat samples of goats.In India, L. monocytogenes has been reported from fish (Dhanashree et al., 2003;Gawade et al., 2011;Parihar et al., 2008;Das et al., 2012), meat (Doijad et al., 2010;Bramhabhatt and Anjaria, 1993), and milk (Doijad et al., 2011;D'Costa et al., 2012).In the present study, L. monocytogenes could not be isolated from cattle.L. ivanovii was isolated from pig samples.Presence of L. monocytogenes in animal associate-environment may lead to cause infection to animals being reared in that area, however, it warrants future investigations.
There are 12 serotypes of L. monocytogenes known till date, of which serotypes 1/2a, 1/2b and 4b contribute more than 98% of outbreaks (Cheng et al., 2008).Our serotyping data confirmed 6 isolated to be serotype 1/2b and 5 as serotype 4b.L. monocytogenes serotype 4b has been considered as cosmopolitan (Herd and Kocks, 2001;Cheng et al., 2008).Presence of such outbreak-associated serotype is of public health concern.However, more studies with large number from humans and animal clinical cases may present the actual scenario.
The antibiotic resistance of the pathogen is a significant public health concern.Recent reports suggest the evolution of L. monocytogenes towards antibiotic resistance (Charpentier and Courvalin 1999;Altuntas et al., 2012;Soni et al., 2013).It is suggested that the increased use of antibiotics for therapeutic purposes in animals and humans may lead to the development of antibiotic resistance (Palumbo et al., 2010;Yan et al., 2010).Depending upon different geographical area, antibiotic resistance patterns of L. monocytogenes in food and environmental sources may change (Yan et al., 2010).We tested all L. monocytogenes isolates for their antibiotic sensitivity.A single L. monocytogenes serotype (4b) isolates from pig placental tissue exhibited multi-antibiotic resistance while remaining 10 isolates were sensitive towards the majority of the antibiotics studied.Interestingly, all the L. monocytogenes isolates were resistant to sulphadiazine.The study conducted by Dhanashree et al., (2003) found similar results wherein L. monocytogenes isolates which were sensitive towards commonly used antibiotics were reported.
The present study showed the prevalence of L. monocytogenes in clinical and environmental samples from Konkan region of Maharashtra, India.The isolates belonged to serotypes having epidemic potential.There is a possibility of transfer of this pathogen from animals to foods of animal origin and thereafter the food chain.
biochemical tests such as catalase, oxidase, characteristics tumbling motility at 20-25 C and fermentation of sugars (rhamnose, xylose, mannitol and α-methyl Dmannopyranoside) were performed.The isolates were compared with standard Listeria spp.for identification.Isolates suspected as L. monocytogenes from biochemical tests were further subjected to specific tests such as hemolysis on sheep blood agar (SBA), Christie, Atkins, Munch-Petersen (CAMP) test and phosphatidylinositol phospholipase C activity (PI-PLC)(Gorski et al., 2008).

Table 2 :
Multiplex PCR serotyping and detection of Virulence Gene (hly A) of the isolates.