- Listeria was first isolated in 1926 from infected rabbits and named Bacterium monocytogenes. It was later renamed Listeria in 1940. - Listeria monocytogenes is a pathogenic bacteria that can cause listeriosis in humans and animals through the consumption of contaminated food. It is particularly associated with consumption of contaminated silage by cattle and sheep. - Symptoms of listeriosis in animals include dullness, circling behavior, head tilt, facial paralysis, and in some cases abortion. It primarily infects the central nervous system.

1. Genus Listeria
Dr Ravi Kant Agrawal, MVSc, PhD
Senior Scientist (Veterinary Microbiology)
Food Microbiology Laboratory
Division of Livestock Products Technology
ICAR-Indian Veterinary Research Institute
Izatnagar 243122 (UP) India

2. • First isolated by Murray et al. 1926 from laboratory rabbits with
septicaemia and monocytosis named Bacterium monocytogenes
• Pirie in 1927 isolated from Gerbil liver (Listerella hepatolytica) – in
honour of Surgeon Lord Lister
• 1940 - Finally named as Listeria (McLauchlin, 1980).
• The genus Listeria currently contains 17 species: L. aquatica, L. booriae,
L. cornellensis, L. fleischmannii, L. floridensis, L. grandensis, L. grayi, L.
innocua, L. ivanovii, L. marthii, L. monocytogenes, L. newyorkensis, L.
riparia, L. rocourtiae, L. seeligeri, L. weihenstephanensis, and L.
welshimeri.
• First outbreak in humans (Nova Scotia, Canada in 1981)
• First reported outbreak in the US (California in 1985)
• Most lethal outbreak 142 people were affected and 48 deaths.
• Involved coleslaw (cabbage salad)
• Pathogens was detected in unopened packages
• 34 pregnant woman and 7 adults were affected
 L. monocytogenes accounts for 98% of human cases
 Identified as human pathogen in 1929, but recognized in 1986 by WHO
 Foodborne transmission appears to be the major means of zoonotic
transmission (Rebagliati et al., 2009)
History

3. Classification of listeria

4. Most Listeria species are small, Gram-positive, coccobacillary
rods, up to 2.0 um in length
They are catalase-positive, oxidase-negative
Motile (multi-flagellar)
Non-spore forming
Facultative anaerobes
Can grow at 32-113 f (0-450
C)
Optimum growth temperature 86-98F (30-37)
Tolerates high salt concentration 10-12% Nacl
It can survive at a pH range of 5.5 - 9.6
The genus is composed of six species, three of which are
pathogenic.
L. monocytogenes; L. ivanovii; L. innocua; L. seelegeri, L.
welshimeri and L. grayi
Listeria monocytogenes, the most important of these pathogens,
has been implicated worldwide in diseases of many animal
species and humans.
The other two pathogens, L, ivanovii and L, innocua, are less
frequently implicated in diseases of animals.
Difficulty in its isolation from foods
Pathogen characteristics

5.  Listeria monocytogenes is a small, motile, Gram-positive, non-
sporeforming, extremely resistant, diphtheroid coccobacillus that
grows under a wide temperature range 4°–44°C (39°– 111°F).
 Its ability to grow at 4°C is an important diagnostic aid (the “cold
enrichment” method) for isolation of the organism from brain tissue
but not from placental or fetal tissues.
 Primary isolation is enhanced under microaerophilic conditions. It is a
ubiquitous saprophyte that lives in a plant-soil environment and has
been isolated from ~42 species of domestic and wild mammals and 22
species of birds, as well as fish, crustaceans, insects, sewage, water,
silage and other feedstuffs, milk, cheese, meconium, feces, and soil.
 The natural reservoirs of L monocytogenes appear to be soil and
mammalian GI tracts, both of which contaminate vegetation. Grazing
animals ingest the organism and further contaminate vegetation and
soil. Animal-to-animal transmission occurs via the fecal-oral route
 Listeriosis is primarily a winter-spring disease of feedlot or housed
ruminants. The less acidic pH of spoiled silage enhances multiplication
of L monocytogenes. Outbreaks typically occur ≥10 days after feeding
poor-quality silage.
 Removal or change of silage in the ration often stops the spread of
listeriosis; feeding the same silage months later may result in new
cases
Etiology and Epidemiology

6. Listeria species can replicate in the environment.
They are widely distributed and can be recovered from herbage,
faeces of healthy animals, sewage effluent and bodies of fresh
water.
They have been isolated from soil, organic matter, residual
waters, animals feed, fresh and frozen chicken, processed
foods, cheese, raw milk, water and gastrointestinal tract of
asymptomatic humans and animals
L. monocytogenes has been isolated from several mammal,
birds, fishes and insect species.
Nevertheless its principal habitat is the soil and decomposing
organic matter
Habitat

7. • Cottage and cheddar cheese with pH of 5.0
• Probability in raw milk of 4.1%
• Probability in pasteurized milk of 0.4%
• Ready to eat foods
• Those of highest risk are deli-meats (sandwich meat)
• Due to its wide distribution, this organism can contaminate
food during different steps along the food chains.
• Foods are the most frequent source of infection in humans.
Association with foods

8. Antigenic structure
• Four major serogroups (1-4) based on O antigen
• Serotypes based on H antigen
• Type 1b accounts for most infections through one
may also find 1a and 4b in significant amount

9. Listeria: Virulence Factors
• Internalin an invasion factor controlled by the inl A gene
• The organisms are taken into the cell by forced phagocytosis,
become enclosed in a vesicle and rupture it.
• Listeriolysin - alpha hemolysis hly gene.
• Lecithinase - hemolysin Phospholipase
• Listera is a facultative interacellular organism

10. • Growth on Mullar Hinton agar with sheep blood as enrichment
• Aerobic and facultative anaerobe
• Grow on nutrient agar: a very light growth and give bluish coloration ,
reflects blue light
• Specimens are enriched if the tissues are kept at 40
C and plated on the
media
• Hemolytic activity on blood agar has been used as a marker to
distinguish L. monocytogenes from other Listeriaspecies, but it is not
an absolutely definitive criterion.
Cultural and growth characteristics
Listeria colonies are typically black
with a black halo (esculin positive)

11. FDA, USDA, ISO methods
Enrichment: Cold enrichment,
UVM I & II (University of Vermont Modification
Medium (UVM) )
Fraser’s broth
Tryptose phosphate broth with polymixin
Selective media : McBride
PALCAM
Oxford agar,
DRIA,
LiCl-ceftazidime agar, modified
Trypaflavine Nalidixic acid-serum agar
L. monocytogenes hydrolyzes esculin to esculetin and dextrose.
Esculetin reacts with ferric ions and produces black zones
around the colonies.
Polymyxin- Acriflavin- Lithium chloride-Ceftazidime - Aesculin-
Mannitol (PALCAM) Agar was formulated by Van Netten et al (1)
and is recommended for the isolation of L. monocytogenes
from foods.
Media used for isolation

12. • Bacteria are faculative anaerobic micobes
• Catalase + / oxidase -
• Motile
• Flagella are produced at room temperature, but not at 37 °C.
• Listeria produce acid and not gas in various sugar fermentation
tests.
• CAMP-positive Listeria monocyogenes inoculated at right angles
to β-hemolytic Staphylococcus aureus.
• Note the arrow shaped zone of weak enhancement hemolysis
indicating a positive CAMP test.
Biochemical Reactions
Catalase positive“umbrella motility” at 25°C

13. Listeria organisms that are ingested or inhaled tend to cause
septicemia, abortion, and latent infection.
Those that gain entry to tissues have a predilection to localize in
the intestinal wall, medulla oblongata, and placenta or to cause
encephalitis via minute wounds in buccal mucosa.
Infection with L. monocyiogenes usually follows ingestion of
contaminated feed and may result in septicaemia, encephalitis
or abortion.
Organisms probably penetrate the M cells in Peyer's patches in
the intestine.
Spread occurs via lymph and blood to various tissues.
In pregnant animals, infection results in transplacental
transmission.
There is evidence that the organism can invade through breaks
in the oral or nasal mucosa. From this site, migration in cranial
nerves is thought to be the main route of infection in neural
listeriosis.
Lesions in the brain stem, often unilateral, are composed of
microabscesses and perivascular lymphocytic cuffs.
Pathogenesis

14. Pathogenesis and pathogenicity
Listeria monocytogenes has the ability to invade both phagocytic
and non-phagocytic cells, to survive and replicate intracellularly
and to transfer from cell-to-cell without exposure to humoral
defence mechanisms.
Specific surface proteins, internalins, fascilitate both the
adherence of organisms to host membranes and their
subsequent uptake.
Virulent strains also possess a cytolytic toxin, listeriolysin (LLO;
from hly gene), which destroys the membranes of phagocytic
vacuoles allowing listeria to escape into cytoplasm.
In the cytoplasm, the organisms utilize cellular microfilaments to
generate tail-like structures which confer motility.
The motile listeria contact the internal surface of the cytoplasmic
membrane and induce pseudopod-like projections. These
projections containing the bacteria are taken up by adjacent
cells. The entire process is then repeated following replication of
listeria in newly-infected cells.

15. Intracellular bacteria have comet-tails of filamentous actin
Red = L. monocytogenes;
Green = filamentous actin
Actin polymers form near bacterial
surface

16. L. monocytogenes Survival is a Multi-Step Process
• Internalin =
stimulates entry
into cells
• Listeriolysin O =
pore-forming
toxin for escape
from
phagolysosome.
• ActA = actin
polymerization
for intracellular
movement.
• 2 Phospholipase
Toxins = entry into
2nd cell.
• Antibodies do not
work!
INTRACELLULAR.
1. Internalin induces uptake
2. LLO to escape
3. ActA for actin-based motility
4. Protrusion
5. LLO + two
PLCs for release

17. Intracellular life
history of
Listeria
monocytogenes LLO
ActA
PLC-A, PLC-B
LLO

18. Infection Cycle

19. Animal model of listeriosis

20. Clinical Infections
 Infections with L. monocytogenes have been recorded in more than 40
species of domestic and wild animals.
 Sporadic abortions in sheep and cattle have been attributed to
infection with L. ivanovii.
 Listeria innocua has been implicated in a case of ovine
meningoencephalitis.

21. Listeriosis, a disease of the central nervous system, is caused by
the bacterium Listeria moncytogenes.
This bacterium can live almost anywhere: in soil, manure piles,
and grass.
Listeriosis is common in cattle, sheep and goats and can occur in
pigs, dogs, and cats, some wild animals, and humans.
Animals infected with Listeria can show signs restlessness, loss
of appetite, fever and nervous system disorders.
Although not seen in every case, the most notable symptom
gives this disease its nickname, "Circling Disease“. Cattle with
listeriosis are often seen walking in circles.
Other, more subtle symptoms include uncoordinated
movements, leaning against objects, and progressive paralysis.
Death can occur within 2 to 3 days after the onset of symptoms,
but cattle can survive for up to 2 weeks with the disease.
LISTERIOSIS

22. • Listeria is a soil-borne bacterium and causes disease following
ingestion by the animal.
• It is especially associated with cattle eating mouldy silage in
winter.
• The silage harvester picks up bits of soil during harvesting,
there is usually poor fermentation around this, as well as the
presence of Listeria.
• It can also occur at grass if cattle graze close to the ground or
eat concentrates directly off the ground.

23. Listeriosis in Ruminants
Listeriosis in ruminants may present as encephalitis, abortion,
septicaemia or endophthalmitis.
Usually only one form of the disease occurs in a group of
affected animals.
Septicaemia, often encountered in newborn piglets, foals, cage
birds and poultry, can also occur in adult sheep.
Although L. monocytogenes is widely distributed in the
environment, outbreaks of listeriosis tend to be seasonal in
European countries and to affect silage-fed animals in late
pregnancy.
Listeria monocytogenes can replicate in the surface layers of
poor-quality silage with pH values above 5.5. In such
circumstances, listerial numbers may reach 1o7
cfu/kg of silage.
In good quality silage, multiplication of the organisms is
inhibited by the acid produced by fermentation.
Susceptibility to infection with L. monocytogenes has been
attributed to decreased cell-mediated immunity associated with
advanced pregnancy.

24. Clinical signs
The incubation period of neural listeriosis (circling disease)
ranges from 14 to 40 days.
Dullness, circling and tilting of the head, are common clinical
signs.
Unilateral facial paraIysis results in drooling of saliva and
drooping of the eyelid and ear.
Exposure keratitis may occur in some cases.
Body temperature may be elevated in the early stages of the
disease.
In sheep and goats, recumbency and death may follow within a
few days of the emergence of clinical signs.
The duration of illness is usually longer in cattle.
Abortion, without evidence of systemic illness may occur up to
12 days after infection.
Septicaemic listeriosis, with a short incubation period of 2 to 3
days, is most commonly encountered in lambs although it may
occur occasionally in pregnant sheep.
In cattle and sheep, keratoconjunctivitis and iritis (ocular
listeriosis) are localized, often unilateral and have been
attributed to direct contact with contaminated silage.

25. Apart from dullness, which is a consistent
feature, the symptoms of listeriosis
depend on which particular nerves are
involved in the infection.
Symptoms include a head tilt and circling
(hence, it is often called ‘Circling Disease’),
as well as paralysis on one side of the face.
This manifests as drooping of the ear and
eyelid on one side, together with drooling
of saliva out of the same side of the mouth.
Occasionally, animals can have difficulty
swallowing, which dramatically diminishes
the prospects for recovery.
A high temperature is present in many
early cases, but may not be present in
advanced cases that have been missed for
a few days.
Symptoms

26. • CN V - Trigeminal - Loss of sensitivity to the face, poor jaw
tone, inability to pretend or masticate food
• CN VII - Facial - Paralysis of the facial muscles, drooping ear,
eyelids, and mouth
• CN VIII - Vestibulocochlear - Head tilt, nystagmus, circling
• CN IX - Glossopharyngeal - Difficulty swallowing
Cranial Nerves Affected by Listeria Infection

27. Human Listeriosis
In immunocomponent host it is not likely to cause infection due
to cell mediated immunity.
If normal healthy adults acquire infection, the disease usually
presents as a mild febrile illness resembling influenza.
Papular lesions on the hands and arms, principally in
veterinarians and farmers, can result from contact with
infective material.
Infection with L. monocytogenes can lead to abortion in
pregnant women and can be life-threatening in neonates, the
elderly and in immunosuppressed individuals.
Human infections usually result from consumption of
contaminated food such as raw milk, soft cheeses, coleslaw and
uncooked vegetables.
Listeria monoctogenes may survive pasteurization because of its
intracellular localization and toIerance to heat.
Direct transfer from infected animals to humans is uncommon
and is of little consequence in healthy, non-pregnant individuals.
Coleslaw: a salad of finely sliced or chopped raw cabbage,
usually moistened with a mayonnaise dressing

28. • Occur usually at third semester when cell mediated immunity
decreases
• The bacteria infection infects the fetus and 22% of these
infections results in neonatal death
• Surviving babies are often born prematurely with active
infection
• Prophylaxis: Pregnant women should avoid milk, soft cheeses,
butter and deli meals
Infection in pregnant woman

29. • Infection is acquired in utero.
• It may be contacted from an asymptomatic mother, with vaginal
colonization with Listeria during vaginal birth.
• Infection results in neonatal meningitis presenting about 2
weeks post partum.
• Listeria monocytogenes causes 20% of all nenonatal meningitis.
Infection in fetus and neonate

30. • Pregnant woman
• Newborns
• People with weakened immune system
• People who are taking immuno-suppressing medication
Who are at risk with listeriosis

31.  Characteristic neurological signs or abortion in association with silage
feeding may suggest listeriosis.
 Appropriate specimens for laboratory examination depend on the
form of the disease:
 Cerebrospinal fluid (CSF) and tissue from the medulla and pons of animals
with neurological signs should be sampled.
 Fresh tissue is required for isolation of organisms and fixed tissue for
histopathological examination.
 Cotyledons, foetal abomasal contents and uterine discharges from cases of
abortion.
 Fresh liver or spleen and blood from septicaemic cases.
 By isolation of organism in cultures obtained on blood ,CSF and other
fluids
 Smears from cotyledons or from liver lesions may reveal Gram-
positive coccobacilIary bacteria.
 Immunofluorescence using monoclonal antibodies may facilitate a
rapid diagnosis.
 Histological examination of brain tissue reveals microabscesses and
heavy perivascular mononuclear cuffing in the medulla and elsewhere
in the brain stem.
 White cell numbers exceeding 1.2 x 107L-I and a protein concentration
of greater than 0.4gL-I in CSF are found in neural listeriosis.
Diagnosis

32. Test for Listeria in Milk
 Add fluorescent-labelled antibodies specific for Listeria
monocytogenes to milk
• Pass through a flow cytometer. fluid is passed through a small
opening
• Listeria detected by a laser beam
NO CULTURE NEEDED!!!

33. Isolation methods:
 Specimens from cases of abortion and septicaemia can be inoculated directly
onto blood agar, selective blood agar and MacConkey agars.
 The plates are incubated aerobically at 370
C for 24 to 48 hours.
 A cold-enrichment procedure is necessary for isolating the organism from
brain tissue - Small pieces of medulla are homogenized and a 10% suspension
is made in nutrient broth. The suspension is held at 4°C in a refrigerator and
sub-cultured weekly onto blood agar for up to 12 weeks.
Identification criteria for L. monocytogenes isolates:
 Colonies are small, smooth and flat with a blue-green colour when
illuminated obliquely.
 Rough variants occur infrequently.
 Individual colonies are usualIy surrounded by a narrow zone of complete
haemolysis.
 Catalase test is positive, distinguishing this organism from streptococci and
Arcanobacterium pyogenes which have similar colonies but are catalase
negative.
 CAMP test is positive with S. aureus but not with Rhododoccus equi.
 Acsculin is hydrolysed.
 Isolates incubated in broth at 25o
C for 2 to 4 hours exhibit a characteristic
tumbling motility.
 Most isolates of animal origin are virulent, a characteristic which can be
confirmed by animal inoculation.
Anton’s Eye test: Instillation of a drop of broth culture into the eye of a
rabbit induces keratoconjunctivitis

34. Differentiation of Listeria species

35. • Early treatment with antibiotics e.g. oxytetracycline or penicillin, will
be effective in most cases.
 Ruminants in the early stages of septicemic listeriosis respond to
systemic therapy with ampicillin or amoxicillin.
• A long course of treatment is usually necessary, however, as relapses
occur, in severe cases, it is often recommended to continue treatment
for up to one week after the clinical symptoms have disappeared.
• Supportive care, including housing and the provision of plenty of
fluids, is an important part of therapy. Animals that cannot swallow
will need to be stomach-tubed with water and electrolytes to give
them more time to respond to antibiotic treatments.
• The prospects for complete recovery is fair (50% to 75%) if the affected
animal is up and able to walk and swallow.
 Response to antibiotic therapy may be poor in neural listeriosis
although prolonged high doses of ampicillin or amoxicillin combined
with an aminoglycosidc may be effective.
 Ocular listeriosis requires treatment with antibiotics and
corticosteroids injected sub-conjunctivally .
• Cephalosporins and fluroquinolones are not active against L.
monocytogens
Treatment

36. • Treatment of listeria infection involves antibiotic therapy directed against
the bacteria. Potentially successful antibiotics must be generated against
Gram positive anaerobes, and have adequate penetration of the blood
brain barrier.
• Previously successful drugs include B-lactams (penicillin, amoxicillin,
ampicilin), macrolides, tetracyclines, sulfonamides, and chloramphenicol.
It has been noted the cephalosoprins are NOT effective against listeria.
• Therapy be instituted for 2-4 weeks, and should begin at the first sign of
disease, due to the fact that if the disease progresses to recumbancy and
convulsions, the bacteria has progressed too far and the animal must be
euthanized.
• In the case of an outbreak, or for cases that do not resolve with antibiotic
therapy, submission of a sample for culture and antibiotic sensitivity
testing may be recommended, as drug-resistant strains of listeria have
been reported.
• Nonsteroidal anti-inflammatories may be added to the regime to help ease
the inflammation of the peripheral nerves and the associated clinical signs.
• Complications of listeriosis such as dry eye, acid-base disturbances, bloat,
and secondary musculoskeletal injury should be treated symptomatically.
• With timely initiation of treatment, survival rates for cattle are roughly
70%.
Treatment

37. • Due to the fact that the bacteria responsible for listeriosis is
persistent in the environment, eradication of the bug is not
practical.
• Prevention methods must be aimed at limiting growth of the
bacteria, such as properly fermenting silage to prevent
proliferation of listeria, which can thrive at pH greater than 5.0.
Prevention

38. Avoid soft cheese
Do not drink raw milk/
unpasteurized milk or foods
made from such milk
Do not eat un-heated
smoked sea foods
Completely cook all meats
and eggs
Carefully wash raw
vegetables before eating
Wash hands thoroughly
after handling raw meat
Keep raw away from raw
vegetable and prepared
foods.
After cutting raw meat
wash the cutting board
with detergent before
using it for vegetables
Ideal way to prevent listeriosis

39. Control
Poor-quality silage should not be fed to pregnant ruminants.
Silage feeding should be discontinued if an outbreak of
listeriosis is confirmed.
Feeding methods which minimize direct ocular contact with
silage should be implemented.
At pasture, the risk is reduced by feeding animals from feed
troughs rather than directly on the ground.
Vaccination with killed vaccines, which do not induce an
effective cell-mediated response, is not protective because L.
monocytogenes is an intracellular pathogen.
Live, attenuated vaccines, which are available in some countries,
are reported to reduce the prevalence of listeriosis in sheep.

40. Thanks
Acknowledgement: All the material/presentations available online on the subject
are duly acknowledged.
Disclaimer: The author bear no responsibility with regard to the source and
authenticity of the content.
Questions???