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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%
3
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Barbuddhe et al.,(Unpublished data)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
15
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
1919
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 %
30
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