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Phenotypic and genotypic characterizationof Listeria spp. isolated from

spontaneous abortions in humans and animals

SIMRANPREET KAUR, S.V.S. Malik, V.M. Vaidya and S.B. Barbuddhe*

Indian Indian VeterinaryVeterinary Research Institute,Research Institute,IzatnagarIzatnagar 243 122 243 122 India India

*ICAR Research *ICAR Research ComplexComplex forfor GoaGoa, , Old Old GoaGoa 403 402 403 402 IndiaIndia

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The DiseaseThe Disease

Important bacterial infection categorized under List C of OIE diseases

An emerging food borne disease An emerging food borne disease (WHO,1986)(WHO,1986)

Sporadic incidence with a CFR of 20Sporadic incidence with a CFR of 20-- 30%30%

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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 population12 / 100,000 (17- fold) in pregnant women

(Southwick and Purich, 1996)

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Maharashtra cervix of 14% of 150 patients

past history of abortion, miscarriage, stillbirth or neonatal deaths

Krishna and co-workers, 1966

Indian scenario: Human listeriosisHuman listeriosis

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

State Prevalence/cases Remarks Ref

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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Barbuddhe et al.,(Unpublished data)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 , 199149.2% among 238

Seropositivity, occupatinally exposed persons

Barbuddhe et al., 1999

State Prevalence/cases Remarks Ref

Indian scenario: Human listeriosisHuman listeriosis

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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)

Disease in animals in India

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Isolation Isolation from foods from foods in in IndiaIndia

Milk Milk 11 to 23 %to 23 % Barbuddhe et al., 2000, 2002; Kalorey et al., 2007;Parihar et al., 2007

Beef, Beef, chevonchevon 22 to 5 %to 5 % Barbuddhe et al., 2002; Bhanu Rekha et al., 2006

Poultry meatPoultry meat 77 to 35 %to 35 % Barbuddhe et al., 2004; Gunjal, 2006

Fresh Fresh water water 13 %13 % Jallewar et al., 2007

fishfish

Seafood Seafood 0 to 17%0 to 17% Karunasagar and Karunasagar, 1992; Jeyasekaran et al., 1996; Parihar et al., 2007

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ObjectiveObjective

To To study the role study the role of of listeric infections listeric infections inin causation causation of of reproductive disorders reproductive disorders inin humanhuman and and animals animals in in IndiaIndia

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Materials and MethodsMaterials and Methods

PlacePlace of of studystudy : Division of VeterinaryPublic Health, Indian VeterinaryResearch Institute, Izatnagar, India

DurationDuration:: August 2005 to July 2006

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No. of No. of samplessamples

Total samples : 688688

Spontaneous abortion cases in human (61): 355355(Vaginal swabs, faecal swabs, placental tissue, urine, blood & serum)

Spontaneous abortion cases in animals (cattle, buffaloes, sheep and goat, 111) : 333333(Vaginal swabs, faecal swabs & blood)

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MethodsMethodsIsolation of Isolation of ListeriaListeria spp. by USDA method spp. by USDA method (McClain and

Lee, 1988) with certain modificationswith certain modifications

Enrichment: 2 step procedure: UVM 1, followed by Enrichment: 2 step procedure: UVM 1, followed by

UVM 2UVM 2

Selective Plating DRIA and PALCAMSelective Plating DRIA and PALCAM

Morphological and biochemical confirmationMorphological and biochemical confirmation

Presumptive Presumptive Listeria Listeria spp. were differentiated by DLspp. were differentiated by DL--

alaninealanine ββ napthalaminenapthalamine (DLABN) test(DLABN) test

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Haemolysis on Sheep Blood Agar (SBA)Haemolysis on Sheep Blood Agar (SBA)

Agar Agar ListeriaListeria according to Ottaviani and Agosti (ALOA)according to Ottaviani and Agosti (ALOA)(Ottaviani, (Ottaviani, et alet al., 1997)., 1997)

Christie, Atkins and MunchChristie, Atkins and Munch--Peterson (CAMP) testPeterson (CAMP) test((McKellarMcKellar, 1994), 1994)

PhosphatidylinositolPhosphatidylinositol-- specific phospholipase C (PIspecific phospholipase C (PI--PLC) PLC) assayassay ((NotermansNotermans et al., et al., 1991)1991)

PCR for detection of virulence associated genes i.e. PCR for detection of virulence associated genes i.e. prfprfAA,,hlyhlyAA, , plcplcA, A, actactA and A and iapiap

((NotermansNotermans et alet al., 1991 and ., 1991 and KotlowskiKotlowski et alet al., 1996)., 1996)

Confirmation

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Mice inoculation testMice inoculation test(Menudier (Menudier et alet al., 1991)., 1991)

Chick embryo inoculation testChick embryo inoculation test(Terplan and Steinmeyer, 1989)(Terplan and Steinmeyer, 1989)

PathogenicityPathogenicity testingtesting

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LLO - based indirect ELISA (Barbuddhe et al., 1999)

Adsorption with SLO (60 µg/ ml) as per the method described by Berche et al., 1990

Serological StudiesSerological Studies

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AntibiogramAntibiogram

Antibiogram of confirmed pathogenic Listeria

isolates (Bauer et al., 1966)

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ResultsResults

ListeriaListeria sppspp.. No.No. of of isolatesisolates

Percent Percent

L. L. monocytogenesmonocytogenes 33 4.924.92

L. L. seeligeriseeligeri 55 8.198.19

L. L. welshimeriwelshimeri 11 1.641.64

Isolation of Isolation of ListeriaListeria spp. from humans (61)spp. from humans (61)

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Isolation of Isolation of Listeria Listeria sppspp. . from animalsfrom animals

SpeciesSpecies Sheep Sheep ((18)18)

Goat Goat ((23)23)

Cattle Cattle ((50)50)

Buffalo Buffalo (20)(20)

L. L. ivanoviiivanovii 11 -- -- --

L. L. seeligeriseeligeri 77 22 55 11

L. L. welshimeriwelshimeri -- 33 11 --

L. L. innocuainnocua -- 11 -- 11

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Fig: Christie, Atkins, Munch-Petersen (CAMP) test1. Standard Listeria ivanovii showing synergetic hemolytic activity with

Rhodococcus equi2. Standard Listeria monocytogenes showing synergetic hemolytic activity

with Staphylococcus aureus3. Isolates recovered from abortion cases

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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

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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 bpLane 2 : Amplified products of the 4 genes i.e. prfA 1060 bp, actA 839 bp,

hlyA 456 bp and iap 131 bp

plcA

actA

hlyA

prfA

iap

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Virulence associated genes of Virulence associated genes of ListeriaListeria ivanoviiivanovii

plcplcAAprfAactA

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PCR Profile of Listeria isolates from human with spontaneous abortions

Virulence associated genesVirulence associated genesS.No.S.No. Isolate CodeIsolate Codeiapiap hlyhlyAA actactAA prfprfAA plcplcAA

0101 Lm ++ ++ ++ ++ ++----

--------++----

0202 Ls -- -- -- --0303 Ls -- -- -- --

0404 Ls -- -- -- --0505 Lm ++ ++ ++ --0606 Lw -- -- -- --0707 Ls -- -- -- --0808 Lm ++ ++ ++ ++0909 Ls -- -- -- --1010 Atypical -- -- -- --

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Multiplex PCR profile of virulence associated genes of L. monocytogenes isolates recovered from human abortion cases

Lane M : PCR marker (100 bp to 3000 bp)Lane 1 : Standard L. monocytogenes (MTCC 1143)Lane 2 : Negative controlLane 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)

plcA

actA

hlyA

iap

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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

prfA

hlyA

actA

iap

Multiplex PCR profile of virulence associated genes of L. monocytogenesisolates recovered from human abortion cases

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Sera tested for ALLO No. of positive sera

% Seropositivity

Before adsorption with SLO

24 48

After adsorption with SLO

8 16

(No. of samples: 50)

Seropositivity for listeric infection by LLO based indirect ELISA

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Pathogenicity, Serological and PCR profiles of Listeria monocytogenesisolates from spontaneous abortion cases in women and sheep

Pathogenicity profilePathogenicity profile

In vitro In vitro teststests In vivo In vivo teststests

ALOAALOA

Hemolysis Hemolysis on SBAon SBA ColCol HaloHalo

Human Human abortionabortion

++BGBG

++ +S+S ++ ++ ++ ++ ++ ++ ++ ++ ++

Human Human abortionabortion

++++BGBG

++++ +S+S ++++ ++ ++ ++ ++ ++ ++ ++ ++

Sheep Sheep abortionabortion

++++++BGBG

++++ +R+R ++++ ++ ++ NDND ++ ++ ++ -- --

Human Human abortionabortion

++BGBG

-- +S+S -- -- -- -- -- -- ++ ++ ++

CACAMP MP with with S/RS/R

PIPI--PLC PLC assayassay

Mice Mice lethallethalityity

ChickChickembrembr

yo yo lethallethal

ityity

plcplcAA

prfprfAA

actactAA

hlyhlyAA iapiap

PCR profile of virulencePCR profile of virulence--associated genes associated genes

AntibodAntibodies to ies to LLOLLO

(ALLO)(ALLO)

Source Source of the of the isolateisolate

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Antibiogram of pathogenic Listeria isolates from abortion cases in women and animals

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 - - -

Sl. No. Type of Antibiotic Disc

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Conclusions(Human Abortion Cases)

Isolation of pathogenic L. monocytogenesL. monocytogenes from 2 casesfrom 2 cases and detection of high titres of listerial antibodies in the SLO adsorbed sera by LLOLLO--based ELISA in 8 out of 61 casesbased ELISA in 8 out of 61 cases of spontaneous abortions in the women highlighted association of this important pathogen human abortionshuman abortions.

L. monocytogenes isolates (hemolytic and pathogenic)(hemolytic and pathogenic)recovered from human abortion cases - TwoTwo

(Source:(Source: Placental tissue & Urine)Placental tissue & Urine)

Prevalence rate in humans with abortions – 3.28%

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L. ivanovii isolates (hemolytic & pathogenic)(hemolytic & pathogenic)

recovered from animals (sheep) –– OneOne

Conclusions(Animal Abortion Cases)

Prevalence rate in animals with reproductive disorders– 0.9 %

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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, plcAand prf A – hold a promise as rapid and reliable in-vitroalternatives to in-vivo pathogenicity tests.

Contd…

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Need for future research Need for future research on on ListeriaListeria in Indian in Indian SubcontinentSubcontinent

Screening of large number of human samples from various risk groups

In view of the variable pathogenic potential of Listeriaisolates, sequencing and comparison of the virulence gene locus

Risk assessment of the foods

Effect of various stress responses

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AcknowledgementsAcknowledgements

DirectorDirectorIndianIndian VeterinaryVeterinary Research Institute, Research Institute, IzatnagarIzatnagar 243 122 243 122 India for facilitiesIndia for facilities

Dr. S.V.S. MalikDr. S.V.S. MalikIndian Indian VeterinaryVeterinary Research Institute, Research Institute, IzatnagarIzatnagar, , IndiaIndia

Dr. D.R.Dr. D.R.KaloreyKaloreyDr. N.V.Dr. N.V.KurkureKurkureMaharashtraMaharashtra AnimalAnimal andand FisheryFishery SciencesSciences University, University, NagpurNagpur, , IndiaIndia

Dr. S.P. Dr. S.P. ChaudhariChaudhariCentral Central AgriculturalAgricultural University, University, AizwalAizwal, , IndiaIndia

Dr. I. Dr. I. ShakuntalaShakuntalaICAR Research ICAR Research Complex for Complex for NEH Region, NEH Region, BarapaniBarapani, , MeghalayaMeghalaya

Dr. D.B. Dr. D.B. RawoolRawoolAlbamyAlbamy MedicalMedical College, College, AlbamyAlbamy, NY, NY

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Thank YouThank You