Occurrence of Listeria species in different captive wild animals of Nandankanan Zoo, Baranga, Odisha, India

Listeria species were isolated from faecal samples collected from different captive wild animals of Nandankanan Zoo, Baranga, Odisha, using selective enrichment medium. The isolates were characterized based on their cell morphology, biochemical and sugar fermentation characteristics as well as culture morphology. Further, in vitro and in vivo pathogenicity tests were carried out to assess the pathogenic potential of the isolates. Listeria were found in 24 (23.07%) of the total 104 faecal samples. Listeria were isolated from the samples of tiger, bear, hyena, leopard, zebra, elephant, jackal, lion, barking deer, porcupine, chital, monkey and wild boar. Out of the 24 Listeria isolates 11 were confirmed as L. monocytogenes. The other 13 isolates included L. innocua, L. seeligeri, L. welshimeri and L. ivanovii. The pathogenicity study revealed that only four isolates were pathogenic. Three of these were L. monocytogenes isolated from tiger, hyena and elephant and one was L. ivanovii isolated from leopard. Antibiotic sensitivity of the 24 isolates was high towards ciprofloxacin, levofloxacin, amoxicillin, azithromycin and enrofloxacin. The isolates showed resistance towards oxytetracyclin, gentamicin, cephadroxil, penicillinG and nalidixic acid.

species in faecal samples of wild animal species in the Nandankanan Zoo, Baranga, Odisha, India.

Materials and Methods
Test strains: The standard strains of Listeria monocytogenes (MTCC 1143), Listeria ivanovii (MTCC 7056), Staphylococcus aureus (MTCC 1144) and Rhodococcus equi (MTCC 1135) were obtained from the Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh, India.The strains were tested for their purity as well as for their morphological and biochemical characteristics.The L. monocytogenes strain was also tested for its pathogenicity in vitro and in vivo.All the strains were maintained by sub culturing them in Brain Heart Infusion (BHI) at 15 days interval.
Collection of samples: One hundred and four faecal samples were either collected from an individual animal, if it was kept in a separate enclosure (noted as individual samples) or from several animals if they were housed together in one enclosure (noted as pooled samples).All the samples were collected in the morning hours non invasively (Table 1).
Isolation method and media used: Listeria were isolated per USDA method described by McClain & Lee (1988) with slight modifications.Therefore, the samples were enriched in a two-step enrichment procedure and were then plated onto a selective medium as follows.
The swabs were directly transferred into sterile test tubes containing University of Vermont Medium 1 (UVM-1, Hi-Media) and were incubated at 37 0 C for 24hr.0.1ml of the enriched inoculum was then transferred to University of Vermont Medium 2 (UVM-2, Hi-Media) and incubated at 37 0 C for 48hr.The enriched medium was streaked on Polymyxin Acriflavin Lithium chloride Ceftazidime Esculin Mannitol agar (PALCAM, Hi-Media).The streaked plates were incubated at 37 0 C for 48hr.The typical grey-green colonies with black centre and a black halo around the colonies were presumptively identified to be Listeria.
Confirmation of the isolates: The isolates were characterized by cell morphology, Gram's staining, catalase test, oxidase test, tumbling motility at 20-25 0 C, MR-VP test, DLABN test and fermentation of sugar [rhamnose, xylose, maltose and mannitol] (Seeliger & Jones 1986;Low & Donachie 1997).In vitro pathogenicity was tested with blood agar and Christie, Atkin, Munch-Petersen (CAMP) test with Staphylococcus aureus and Rhodococcus equi.In vivo pathogenicity was tested by inoculating mice and chicken embryos.

In vitro pathogenicity tests
Haemolysis on sheep blood agar (SBA) plate: Listeria isolates were tested for the type and degree of haemolysis on SBA.Detection of haemolysin allows to discriminate between virulent and avirulent Listeria species.The isolates were streaked onto SBA plates, incubated at 37 0 C in a humidified chamber for 24hr and examined for haemolytic zones around the colonies.The characteristic wide and clear zone of β-haemolysis suggested L. ivanovii, while a narrow zone of β-haemolysis was characteristic for L. monocytogenes (McKellar, 1994).
Christie, Atkin, Munch-Petersen (CAMP) test: CAMP test was conducted following the standards of the Bureau of Indian Standards (BIS 1994) with some modifications.The standard strains of Staphylococcus aureus and Rhodococcus equi were cultured for 24hr on SBA plates at 37 0 C. The cultured inoculates of each strain were streaked onto freshly prepared 7% SBA plates in parallel streaks with considerable space between streaks.Afterwards, the Listeria isolates were streaked onto these plates at 90 0 angle and 3mm apart from S. aureus and R. equi streaks.The inoculated plates were incubated at 37 0 C for 24hr.The plates were examined for enhancement of haemolytic zone; in case of a CAMPpositive reaction the haemolytic zone between a Listeria strain and the S. aureus or R. equi strain was enhanced due to the synergistic effect of their haemolysins.All the Listeria isolates with CAMP-positivity against S. aureus or R. equi were characterized as L. monocytogenes and L. ivanovii respectively (McKellar 1994).

In vivo pathogenicity tests
Mice inoculation test: The pathogenicity of the Listeria isolates was tested by inoculating mice according to the method described by Menudier et al. (1991) with suitable modifications.The test isolates of Listeria were grown on Brain Heart Infusion (BHI) slants at 37 0 C for 24hr.The bacterial growth was harvested with sterile normal saline solution (NSS) and the opacity of solution was adjusted to McFarland Nephelometric tube number 1. Mice of either sex weighing 18-20 g were inoculated intraperitoneally with a volume of 0.4ml of the solutions containing approximately 10 7 colony forming units (cfu) of the test organism per ml.The inoculated mice were observed for mortality over a period of 72hr.
Chick embryo inoculation test: The pathogenicity of Listeria isolates was also tested by inoculating chicken embryos following the method described by Notermans et al. (1991).The blood vessel-free surface of the chorioallantoic membrane (CAM) of two precandled 10day old embryonated chicken eggs was inoculated with 0.1ml of the test culture in BHI broth.Control eggs were inoculated with 0.1ml of BHI broth.All eggs were sealed with molten paraffin and were horizontally placed at 37 0 C for 3 days.The eggs were examined twice a day by transillumination for embryo death.A test isolate causing embryo mortality after 24 hours of inoculation was considered to be pathogenic.

Results
Out of 104 examined faecal samples 24 (23.07%)samples were found to be Listeria positive.Listeria isolates were recovered from the faecal sample of tiger, bear, hyena, leopard, zebra, elephant, jackal, lion, barking deer, porcupine, chital, monkey and wild boar.Out of 24 Listeria isolates 11 were identified as L. monocytogenes, six as L. innocua, four as L. seeligeri, two as L. welshimeri and one L. ivanovii (Table 1).Only four of the Listeria isolates were found to be pathogenic.These were three isolates of L. monocytogenes which were from tiger, hyena and elephant and one isolate of L. ivanovii from leopard (Table 2).
The Listeria isolates were tested in vitro for their sensitivity to different antibiotics.

Discussion
Listeria monocytogenes is ubiquitous in nature and has been isolated from numerous sites, including soil, sewage water and decaying plant materials etc.Its viability is remarkable, with survival in soil or silage for more than two years.Studies have shown that about 50% of faecal samples collected from different animals like cattle, sheep, goat, pig and poultry contain L. monocytogenes but don't exhibit any clinical symptoms of listeriosis (Meng & Doyle 1997;Wesley 1999).Listeria species have previously been reported in Odisha from milk samples of cows and from faecal samples of various domestic animals (Sarangi et al. 2009;Sarangi & Panda 2012).In the present study Listeria species were isolated from 24 (23.07%)out of 104 faecal samples of healthy zoo animals.Listeria infection is mostly food borne  (Ryser 1999) and raw meat, vegetable and fruits used as animal feeds in zoological parks might be the major source of Listeria infections.Listeria species have been reported from a number of wild animals in the past.L. monocytogenes have been isolated from the faecal sample of foxes, Roe Deer, badgers, hares and Pine Marten (Weis 1975), African Lion (Haigh et al. 1978), Grey Fox (Black et al. 1996), primates, hippopotamus, fox and bear (Arumugaswamy & Gibson 1999), farm chinchilla (Sabocanec et al. 2000), captive antelopes (Bauwens et al. 2001), wild boars (Hayashidani et al. 2002), Colobus Monkey (Kock et al. 2003), jackal, Indian Fox, Sambar, wolf and Spotted Deer (Kalorey et al. 2006) and from other deer, raccoon and moose (Lyautey et al. 2007).Similarly, L. ivanovii has been isolated from Grauer's Gorilla and Red Ruffed Lemur (Bauwens et al. 2003) and from a septicaemic chinchilla (Kimpe et al. 2004).However, we isolated Listeria species from tiger, hyena, leopard, zebra, elephant, blue bull, barking deer and porcupine which all have to date not been reported to carry Listeria.
Our overall infection rate of 23.07% is comparable to infection rates found by other researchers.Bernagozzi et al. (1999) reported 24.1% of Listeria spp. in faeces of wild animals in National Park of Casentine forest (112 samples).Bauwens et al. (2001) isolated Listeria spp.from 22.8% of the faecal samples of Wild Animal Park, Planckendael (70 samples).Bauwens et al. (2003) also reported a Listeria occurrence of 21% in faecal samples of wild animals in Antwerp Zoo (200 samples).In India, Kalorey et al. (2006) isolated L. monocytogenes in 16% of faeces of wild animals (50 samples).A much lower infection rate was reported by Yoshida et al. (2000), who only found an infection rate of 6.1% (623 samples) in all animals tested.However in Japanese macaques the infection rate was also 20%.Recently, Yadav et al. (2011) isolated two Listeria species (L.monocytogenes and L. innocua) out of three positive isolates from 56 faecal samples collected from mammals and birds at Baroda Zoo, Vadodara, Gujarat State, India, which equals to a prevalence of only 5.3%.However as the range of species and animals examined in these studies are entirely different it is not possible to conclusively compare the infection rates found.
In the present study 11 (10.57%)L. monocytogenes were isolated which differ from the observation of Arumugaswamy & Gibson (1999) who after examination of 86 animals of Taronga Zological Garden, New South Wales, Australia, revealed 25.6% were carrier of Listeria species and 18.6% were excreting L. monocytogenes.This difference may be due to varying environmental condition between different locations, previous occurrence of the disease and hygienic measures adopted in the zoo.
Listeria spp.have in the past been reported to be sensitive to antibiotics active against gram positive bacteria (Hawkins et al. 1984), but some time later reports of resistance in Listeria spp.appeared (Abrahim et al. 1998;Walsh et al. 2001).An increase in antibiotic resistance in Listeria spp. is in line with a global pattern of an increasing antibiotic resistance, including resistances against multiple antibiotics in many groups of bacteria.The occurrence of antibiotic resistance in non-pathogen forms poses a major risk as there is possibility to transfer the resistance factor from non-pathogens to pathogenic organisms (Walsh et al. 2001).
In our study we found high to moderate sensitivity towards ciprofloxacin, levofloxacin, amoxicilin, azithromycin, enrofloxacin, chloramphenicol and amikacin and resistance towards oxytetracyclin, gentamycin, cephadroxil, penicillin-G, tobramycin, cephotaxim, cephalexin, ceftriaxone and nalidixic acid.Similar results have been reported by Lyautey et al. (2007) who found resistance against kanamycin, gentamycin, streptomycin and rifampicin.This differs from findings of Butko et al. (1972), who reported the sensitivity of Listeria spp. to erythromycin, chlortetracycline, streptomycin, levomycin, neomycin and monomycin and resistance to penicillin.This variation in antibiotic sensitivity might be due the above mentioned global increase in antibiotic resistance in the bacteria over decades.
The present study shows the occurrence of Listeria species in captive wild animals of Nandankanan Zoo, Baranga, Odisha, which may act as nidus of infection for other susceptible animals and cause death of animals.A further study investigating the role of Listeria in sick wild animals is necessary and emphasis should be given to control the food borne transmission of the diseases.