Phenotypic and genotypic characterization of Listeria spp. isolated from spontaneous abortions in humans and animals SIMRANPREET KAUR, S.V.S. Malik, V.M. Vaidya and S.B. Barbuddhe* Indian Veterinary Research Institute, Izatnagar 243 122 India *ICAR Research Complex for Goa, Old Goa 403 402 India 1
The Disease Important bacterial infection categorized under List C of OIE diseases An emerging food borne disease (WHO,1986) Sporadic incidence with a CFR of 20-30% 2
Groups at Risk for Listeriosis Immunocompromised persons Cancer patients Patients with kidney disease Elderly persons HIV patients Pregnant women Healthy pregnant women show a unique predilection - Pregnancy increases risk of listeriosis - Placenta provides a protective niche for pathogen Incidence: 0.7 / 100,000 in general population 12 / 100,000 (17- fold) in pregnant women (Southwick and Purich, 1996) 3
Indian scenario: Human listeriosis State Prevalence/cases Remarks Ref Maharashtra cervix of 14% of 150 patients past history of abortion, miscarriage, stillbirth or neonatal deaths Krishna and coworkers, 1966 Delhi 3 of 100 bad obstetric history Bhujwala et al., 1973 1 case aborted foetus, blood of mother Bhujwala et al., 1973 9 of 670 women bad obstetric history Bhujwala & Hingorani, 1975 Karnataka 4 of 40 women history of abortion Stephen et al., 1978 Karnataka 2 cases history of abortion Dhanashree et al., 2003 4
Indian scenario: Human listeriosis State Prevalence/cases Remarks Ref Chandigarh 1 case History of abortion Gupta et al., 2003 Maharashtra and Madhya Pradesh 6 cases Miscarriage, kidney disease, immunocompromised Barbuddhe et al., (Unpublished data) 2 cases Rocourt, 1991 49.2% among 238 Seropositivity, occupatinally exposed persons Barbuddhe et al., 1999 5
Disease in animals in India Abortion in sheep and cattle (Chand and Sadana, 1998; Thakur, 1998) Abortion in goats (Elezebeth et al., 2007) Bovines reproductive disorders (Shakuntala et al., 2006) Sub clinical mastitis (Rawool et al., 2007) Fatal neurological disease with CFR of 70% or higher in pigs (Rahman et al., 1985; Dash et al., 1998) 6
Isolation from foods in India Milk 1 to 23 % Barbuddhe et al., 2000, 2002; Kalorey et al., 2007; Beef, chevon 2 to 5 % Poultry meat 7 to 35 % Fresh water 13 % fish Parihar et al., 2007 to 5 % Barbuddhe et al., 2002; Bhanu Rekha et al., 2006 to 35 % Barbuddhe et al., 2004; Gunjal, 2006 13 % Jallewar et al., 2007 Seafood 0 to 17% 0 to 17% Karunasagar and Karunasagar, 1992; Jeyasekaran et al., 1996; Parihar et al., 2007 7
Objective To study the role of listeric infections in causation of reproductive disorders in human and animals in India 8
Materials and Methods Place of study : Division of Veterinary Public Health, Indian Veterinary Research Institute, Izatnagar, India Duration: August 2005 to July 2006 9
No. of samples Total samples : 688 Spontaneous abortion cases in human (61): 355 (Vaginal swabs, faecal swabs, placental tissue, urine, blood & serum) Spontaneous abortion cases in animals (cattle, buffaloes, sheep and goat, 111) : 333 (Vaginal swabs, faecal swabs & blood) 10
Methods Isolation of Listeria spp. by USDA method (McClain and Lee, 1988) with certain modifications Enrichment: 2 step procedure: UVM 1, followed by UVM 2 Selective Plating DRIA and PALCAM Morphological and biochemical confirmation Presumptive Listeria spp. were differentiated by DL- alanine β napthalamine (DLABN) test 11
Confirmation Haemolysis on Sheep Blood Agar (SBA) Agar Listeria according to Ottaviani and Agosti (ALOA) (Ottaviani, et al., 1997) Christie, Atkins and Munch-Peterson (CAMP) test (McKellar,, 1994) Phosphatidylinositol- specific phospholipase C (PI-PLC) PLC) assay (Notermans et al., 1991) PCR for detection of virulence associated genes i.e. prfa, hlya, plca, acta A and iap iap (Notermans et al et al., 1991 and Kotlowski et al., 1996) 12
Pathogenicity testing Mice inoculation test Chick embryo inoculation test (Menudier et al., 1991) (Terplan and Steinmeyer, 1989) 13
Serological Studies LLO - based indirect ELISA (Barbuddhe et al., 1999) Adsorption with SLO (60 µg/ ml) as per the method described by Berche et al., 1990 14
Antibiogram Antibiogram of confirmed pathogenic Listeria isolates (Bauer et al., 1966) 15
Results Isolation of Listeria spp. from humans (61) Listeria spp. No. of isolates Percent L. monocytogenes 3 4.92 L. seeligeri 5 8.19 L. welshimeri 1 1.64 16
Isolation of Listeria spp. from animals Species Sheep (18) Goat (23) Cattle (50) Buffalo (20) L. ivanovii 1 - - - L. seeligeri 7 2 5 1 L. welshimeri - 3 1 - L. innocua - 1-1 17
Fig: Christie, Atkins, Munch-Petersen (CAMP) test 1. Standard Listeria ivanovii showing synergetic hemolytic activity with Rhodococcus equi 2. Standard Listeria monocytogenes showing synergetic hemolytic activity with Staphylococcus aureus 3. Isolates recovered from abortion cases 18
Fig: Colony characteristics on ALOA medium (i) Pathogenic Listeria showing blue green colonies with halo (MTCC1143, NCTC7973, MSV6, PHP7 and NHU9) (ii) Non-pathogenic Listeria showing blue green colonies without halo (PHP25 and PHV25) (iii) Non-Listeria showing white colonies 19
plca prfa acta hlya iap Fig: Multiplex PCR of virulence associated genes of standard L. monocytogenes (MTCC 1143) Lane M : PCR marker (100 bp to 3000 bp) Lane 1 : Amplified products of the 4 genes i.e. plca 1484 bp, acta 839 bp, hlya 456 bp and iap 131 bp Lane 2 : Amplified products of the 4 genes i.e. prfa 1060 bp, acta 839 bp, hlya 456 bp and iap 131 bp 20
Virulence associated genes of Listeria ivanovii plca prfa acta 21
PCR Profile of Listeria isolates from human with spontaneous abortions S.No. Isolate Code iap Virulence associated genes hlya acta prfa 01 Lm + + + + + 02 Ls - - - - 03 Ls - - - - plca - - 04 Ls - - - - 05 Lm + + + - 06 Lw - - - - 07 Ls - - - - 08 Lm + + + + 09 Ls - - - - 10 Atypical - - - - - - - - + - - 22
Multiplex PCR profile of virulence associated genes of L. monocytogenes isolates recovered from human abortion cases plca acta hlya iap Lane M : PCR marker (100 bp to 3000 bp) Lane 1 : Standard L. monocytogenes (MTCC 1143) Lane 2 : Negative control Lane 3 : Amplified products of 4 virulence genes of L. monocytogenes isolate (PHP7) Lane 4 : Amplified products of 4 virulence genes of L. monocytogenes isolate (NHU9) 23
Multiplex PCR profile of virulence associated genes of L. monocytogenes isolates recovered from human abortion cases prfa acta hlya iap Lane M : PCR marker (100 bp to 3000 bp) Lane 1 : Standard L. monocytogenes (MTCC1143) Lane 2 : Amplified products of the 4 virulence genes of L. monocytogenes isolate (PHP7) Lane 3 : Amplified products of the 4 virulence genes of L. monocytogenes isolate (NHU9) Lane 4 : Negative control 24
Seropositivity for listeric infection by LLO based indirect ELISA Sera tested for ALLO Before adsorption with SLO After adsorption with SLO No. of positive sera % Seropositivity 24 48 8 16 (No. of samples: 50) 25
Pathogenicity, Serological and PCR profiles of Listeria monocytogenes isolates from spontaneous abortion cases in women and sheep Source of the isolate Human abortion Hemolysis on SBA + Pathogenicity profile In vitro tests Col BG ALOA Halo CA MP with S/R PI- PLC assay In vivo tests Mice lethal ity Chick embr yo lethal ity Antibod ies to LLO (ALLO) + +S + + + + + + + + + plc A PCR profile of virulence- associated genes prf A act A hly A iap Human abortion ++ BG ++ +S ++ + + + + + + + + Sheep abortion +++ BG ++ +R ++ + + ND + + + - - Human abortion + BG - +S - - - - - - + + + 26
Antibiogram of pathogenic Listeria isolates from abortion cases in women and animals Sl. No. Type of Antibiotic Disc Listeria monocytogenes isolates Listeria Iivanovii isolate PHP7 NFU9 MSV6 1 Lincomycin L 2 mcg ++ ++ + 2 Netilmicin Nt 30 mcg + ++ - 3 Ofloxacin Of 2 mcg +++ +++ + 4 Amikacin Ak 30 mcg +++ +++ - 5 Cephalothin Ch 30 mcg - - - 6 Clindamycin Cd 2 mcg ++ ++ - 7 Erythromycin E 15 mcg ++ ++ - 8 Gentamicin G 10 mcg +++ +++ ++ 9 Amoxycillin Am 10 mcg - - - 10 Tetracycline T 30 mcg +++ +++ ++ 11 Ciprofloxacin Cf 5 mcg +++ +++ ++ 12 Chloramphenicol C 30 mcg +++ +++ + 13 Cephalexin Cp 30 mcg ++ ++ ++ 14 Ampicillin A 10 mcg +++ +++ - 25 Cephotaxime Ce 30 mcg +++ ++ + 16 Norfloxacin Nx 10 mcg +++ +++ + 17 Penicillin-G P 10 units ++ +++ ++ 18 Cloxacillin Cx 5 mcg - - - 27
Conclusions (Human Abortion Cases) Isolation of pathogenic L. monocytogenes from 2 cases and detection of high titres of listerial antibodies in the SLO adsorbed sera by LLO-based ELISA in 8 out of 61 cases of spontaneous abortions in the women highlighted association of this important pathogen human abortions. L. monocytogenes isolates (hemolytic and pathogenic) recovered from human abortion cases - Two (Source: Placental tissue & Urine) Prevalence rate in humans with abortions 3.28% 28
Conclusions (Animal Abortion Cases) L. ivanovii isolates (hemolytic & pathogenic) recovered from animals (sheep) One Prevalence rate in animals with reproductive disorders 0.9 % 29
Contd Seropositivity for listeric infection in human cases 16% Reduction of seropositivity consequent to sera adsorption with SLO, from 48 % to 16 %, indicating that adsorption of the sera eliminates cross-reactions in serological tests. Variable sensitivity to different antibiotics PI-PLC assay and Multiplex PCR based on hlya, plca and prf A hold a promise as rapid and reliable in-vitro alternatives to in-vivo pathogenicity tests. 30
Need for future research on Listeria in Indian Subcontinent Screening of large number of human samples from various risk groups In view of the variable pathogenic potential of Listeria isolates, sequencing and comparison of the virulence gene locus Risk assessment of the foods Effect of various stress responses 31
Acknowledgements Director Indian Veterinary Research Institute, Izatnagar 243 122 India for facilities Dr. S.V.S. Malik Indian Veterinary Research Institute, Izatnagar, India Dr. D.R.Kalorey Dr. N.V.Kurkure Maharashtra Animal and Fishery Sciences University, Nagpur, India Dr. S.P. Chaudhari Central Agricultural University, Aizwal, India Dr. I. Shakuntala ICAR Research Complex for NEH Region, Barapani, Meghalaya Dr. D.B. Rawool Albamy Medical College, Albamy,, NY 32
Thank You 33