1. Genus Yersinia
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. The causative agent of plague, Yersinia pestis, was discovered by the
French/Swiss microbiologist A.E.J. Yersin and Kitasato Shibasaburō
Japanese bacteriologist in Hong Kong in 1894.
It was formerly described as Pasteurella pestis (known trivially as the
plague-bacillus by Lehmann and Neumann in 1896.
In 1944, van Loghem reclassified the species P. pestis and P.
rondentium into a new genus, Yersinia.
Ukrainian scientists D. Samoilovich, D. Zabolotny and others
contributed greatly to the study of the mechanisms of its
The French microbiologists G. Girard and T. Robic obtained a live
vaccine from the attenuated EV strain.
Bubonic plague, caused by Y. pestis, is an ancient disease that has
killed millions of people over the centuries.
It is believed to have killed more than 100 million persons in an
epidemic in the sixth century.
Another epidemic in the 14th century killed one fourth of the
The London plague in 1665 killed more than 70,000 persons.
In 1893, an epidemic began in Hong Kong and spread to India where
more than 10 million individuals died over a 20-year period.
3. Biochemical and growth characteristics
• Are Gram negative Enterobacteriaceae member
• Previously classified in Pasteurellaceae family.
• Based on DNA similarities with E. coli, Y. pestis is now part of
• Short coccobacillary, ovoid or rod shaped, pleomorphic Gram
• Exhibit bipolar staining in Geimsa stained smears from animal tissues.
• Yersinia species are non-lactose fermenters
• Motile with the exception of Y. pestis.
• Are catalase positive, oxidase negative, Indole -ve, ONPG +ve, Urease
+ve except Y. pestis
• Aerobic/facultative anaerobic or Microaerophilic.
• Most have animals as their natural hosts, but they can produce
serious disease in humans.
• Grow on ordinary media, however, growth is slower than other
• Wide temperature range (5-42 C)
• Best growth temp. 25-30C
• Yersinia has the ability to not only survive, but also to actively
proliferate at temperatures as low as 1–4°C (e.g., on cut salads and
other food products in a refrigerator).
11 named species in genus 3 are
human and animal pathogens
Y. pestis, Y. pseudotuberculosis, Y.
Y. pestis- plague
Y. pseudotuberculosis - human
diarrhoeal diseases (rarely infect
Y. enterocolitica is the cause of 1 – 3%
of diarrhea cases caused by bacteria
Y. ruckeri causes perioral
haemorrhagic imflammation in some
species of fish.
Genus: Yersinia (van Loghem, 1944)
Y. enterocolitica subsp.
enterocoliticaYersinia enterocolitica subsp. palearctica
Y. pseudotuberculosis subsp.
pestisY. pseudotuberculosis subsp. pseudotuberculosis
5. Serotyping and biotyping methods used for identifying pathogenic
Of the ten serotypes of Y. pseudotuberculosis, serotypes I , II and III contain
the majority of pathogenic isolates.
There are five biotypes and more than 50 serotypes of Y. enterocolitica.
Somatic antigens 2, 3, 5, 8 and 9 are present in isolates from clinical
infections caused by this species.
Serotype 0:9 is of particular importance because it shares common
antigens with BruceIla species and it may induce false-positive reactions in
brucella agglutination tests.
Yersinia pseudotuberculosis and Y. enterocolitica are found in the intestinal
tract of a wide range of wild mammals, birds and domestic animals.
All these animals may be reservoirs of infection.
Many avian species may act as amplifier hosts and may also transfer the
Both organisms can grow in a wide temperature range (5 to 42°C) and
survive for long periods in cool wet conditions.
In endemic areas, wild rodents are important reservoirs of Y. pestis.
Fleas, especially Xenopsylla cheopis, the Oriental rat flea, transmit the
infection to man and other animals.
Pathogenic yersiniae are facultative intracellular organisms which possess
plasmid and chromosomal encoded virulence factors, many of which are
required for survival and multiplication in macrophages.
Yersinia pseudotuberculosis and Y. enterocolitica are less virulent than Y. pestis
and rarely produce generalized infections.
The pathogenic mechanisms in enteric disease caused by Y. enterocolitica and Y.
pseudotuberculosis are incompletely understood.
It is probable that both organisms gain entry to the mucosa through M cells of
Adhesion to and subsequent invasion through these cells are facilitated by
factors such as invasin and adhesion-invasion proteins which have an affinity for
integrins on cell surfaces.
Once in the mucosa, the bacteria are engulfed by macrophages in which they
survive and are transported to the mesenteric lymph nodes (Brubaker, 1991).
Replication in the nodes follows with the development of necrotic lesions and
Survival of Y. pseudotuberculosis and Y. enterocoiitica is enhanced by
antiphagocytic proteins secreted by the organisms which interfere with the
normal functioning of neutrophils in the host.
Yersinia pestis is more invasive than Y. pseudotubercuIosis and Y. enterocolitica
and possesses additional virulence factors include an antiphagocytic protein
capsule (Fraction 1) and a pIasminogen activator which aids systemic spread.
Endotoxin, with properties similar to the endotoxin produced by other members
of the Enterobacteriaceae, also contributes to the pathogenesis of disease.
9. Clinical Infections
Yersinia pseudotuberculosis causes enteric infections, in a wide
variety of wild and domestic animals which are often subclinical.
The septicaemic form of disease, known as pseudotuberculosis,
can occur in laboratory rodents and aviary birds.
Sporadic abortions caused by Y. pseudotuberculosis have been
reported in cattle, sheep and goats.
Wild and domestic animals may act as reservoirs of Yersinia
enterocolitica which is primarily a human enteric pathogen.
The pig is the natural reservoir for Y. enterocolitica serotype 03
biotype 4, which is an important pathogen in humans.
Rare cases of enteric disease, precipitated by stress, may be
encountered in pigs, farmed deer, goats and lambs.
Yersinia enterocolitica has been implicated in sporadic ovine
Yersinia pestis, the cause of human bubonic plague (black death),
can infect both dogs and cats in endemic areas.
Cats, which are particularly susceptible, may be a source of
infection for owners and attending veterinarians.
10. Pathogenesis and Clinical Findings
Yersinia enterocolitica and Y. pseudotuberculosis
• An inoculum of 108 – 109 yersiniae must enter the alimentary tract to
• During IP of 5 – 10 days, yersiniae multiply in the gut mucosa
particularly the ileum
• Leads to inflammation and ulceration and leukocytes appear in feces
• The process may extend to mesenteric lymph nodes and rarely to
• Early symptoms include fever, abdominal pain, and diarrhoea
• Diarrhoea ranges from watery to bloody and may be due to an
enterotoxin or to invasion of the mucosa
• At times abdominal pain is severe and located in the right lower
quadrant suggesting appendicitis (Pseudoappendicitis)
• 1 to 2 weeks after onset some patients develop arthalgia, arthritis and
erythema nodosum suggesting immunologic reaction to the infection
• Very rarely, it produces pneumonia, meningitis or sepsis; in most
cases, it is self-limited
11. Enteric yersiniosis
Enteritis caused by Y. pseudoruberculosis is relatively common in
young farmed deer in New Zealand and Australia.
Outbreaks of the disease have been reported also in buffaloes in
Enteric disease has been reported in sheep, goats and cattle under
one year of age.
Subclinical infection in many species is common and clinical disease
may be precipitated in the winter months by stress factors such as
poor nutrition, weaning, transportation and cold wet conditions.
There may be prolonged survival of Y. pseudotuberculosis on pasture
in cold wet weather, facilitating faecal-oral transmission.
Enteritis in young deer and lambs is characterized by profuse watery
diarrhoea, sometimes blood-stained, which may be rapidly fatal if
The luminal contents of the small and large intestine are watery and
mucosal hyperaemia is evident at postmortem examination.
Severely affected animals may show mucosal ulceration.
The mesenteric lymph nodes are often enlarged and oedematous and
scattered pale necrotic foci may be present in the liver.
A clinically similar but less severe enterocolitis caused by Y.
enterocolitica has been described in young ruminants.
12. Septicaemic yersiniosis
Septicaemia, caused by Y. pseudotuberculosis occurs in birds
kept in cages or aviaries.
It is presumed that infection is acquired through contact with
the faeces of wild birds or rodents, or through the feeding of
contaminated leafy plants.
In aviaries, overcrowding may predispose to the development
Infected birds may die suddenly.
Some may display ruffling of feathers and listlessness shortly
Pin-point white necrotic foci are present in the Iiver at
Confirmation is based on the isolation and identification of Y.
pseudotuberculosis from the liver and other internal organs.
Treatment is seldom feasible due to the acute nature of the
Control should be aimed at preventing faecal contamination of
food and water by wild birds and rodents.
13. Pseudotuberculosis in Laboratory animals
Infection with Y. pseudotuberculosis in colonies
of G. pigs or rodents is usually introduced
through faecal contamination of food by wild
Diarrhoea and gradual weight loss leading to
emaciation and death are the signs most often
observed in affected animals.
Some animals may die suddenly from
At postmortem examination, numerous white
necrotic lesions are present in the liver.
Affected mesenteric lymph nodes are enlarged
and may show caseous necrosis.
Treatment is usually not desirable because some
animals in the colony may become carriers and
the organism is zoonotic.
Depopulation, disinfection and restocking are
the preferred control measures.
Exclusion of wild rodents is an essential step in
preventing infection with Y. pseudotuberculosis.
The species and age group affected, especially
during cold wet spells of weather, may suggest
HistoIogical examination of intestinal lesions may
reveal clusters of organisms in microabscesses
within the mucosa.
Confirmation requires isolation and identification
of Y. pseudutuberculosis or Y. entercolitica.
Samples from tissues can be plated directly onto
blood and MacConkey agars and incubated
aerobically at 37°C for up to 72 hours.
Faecal samples may be plated directly onto special
A cold enrichment procedure may facilitate
recovery of yersiniae from faeces especially if they
are present in low numbers.
A 5% suspension of faeces in PBS, held at 4°C for 3-4
weeks, is subcultured weekly onto MacConkey
Serotyping may be necessary to establish whether
the isolates belong to known pathogenic
15. Differentiation of Yersinia Species
Y. pestis – + – – + + +
+ + – + – – –
Y. enterocolitica – + – + + + +
16. Some diagnostic signs differentiating the bacteria of
plague from those of pseudotuberculosis in rodents
Yersinia pestis Yersinia pseudotuberculosis
1.Fresh strains do not usually
1. Fresh strains usually ferment
2. Do not ferment adonite 2. Ferment adonite with the
formation of acid
3. Do not ferment urea 3. Ferment urea
4. On desoxycholic citrate agar
they grow with the formation
of red colonies
4. On desoxycholic citrate agar
they grow with the formation
of yellow colonies
5. Are lysed by the plague
phage to the titre
5. Do not undergo lysis
17. Treatment and control
Fluid replacement therapy together with broad spectrum
antimicrobial treatment should be initiated promptly in young
A formalin-killed Y. pseudotuberculosis vaccine composed of
serotypes I, II and III, administered in two doses three weeks
apart has been shown to decrease the occurrence of clinical
disease in young deer.
StressfuI conditions should, where practicable, be minimized.
18. Yersinia pestis
• Gram negative rod with striking bipolar staining with special
• Grows as facultative anaerobe on many bacteriologic media
• Growth is more rapid in media containing blood or tissue fluids
and fastest at 30 C
• In cultures of blood agar at 37 C, colonies may be very small at
• A virulent inoculum, derived from infected tissue, produces
gray and viscous colonies but after passage in the laboratory
the colonies become irregular and rough
• Has little biochemical activity, is somewhat variable
19. Antigenic structure
• All yersiniae possess lipopolysaccharides that have endotoxic
activity when released
• Produce many antigens and toxins that act as virulence factors
• The envelope contains a protein (fraction I) produced mainly at
37 C and confers antiphagocytic properties
• Virulent, wild-type Y. pestis carries V-W antigens, which are
encoded by genes on plasmids
• A 72-kb plasmid is essential for virulence
• Avirulent strains lack the plasmid
• Some stable avirulent strains have served as live vaccines
• Produces coagulase at 28 C (normal temperature of the flea)
but not at 35 C (transmission via fleas is low or absent in very
• Among several exotoxins produced, one is lethal for mice in
amounts of 1 µg - this homogenous protein (MW 74,000)
produces beta-adrenergic blockage and is cardiotoxic in animals
20. Pathogenesis and Pathology
When a flea feeds on a rodent infected with Y. pestis, the ingested
organisms multiply in the gut of the flea and helped by coagulase,
block its proventriculus so that no food can pass through
Subsequently the blocked and hungry flea bites ferociuosly and the
aspirated blood contaminated with Y. pestis from the flea, is
regurgitated into the bite wound
Inoculated organisms may be phagocytosed by polymorphonuclear
cells and monocytes
The organisms are killed by the polymorphonuclear cells but can
multiply in the monocytes because bacteria are multiplying at 37 C,
they produce antiphagocytic proteins (antiphagocytic protein capsule
fraction 1) and subsequently are able to resist phagocytosis
The pathogens rapidly reach the lymphatics and an intense
hemorrhagic inflammation develops in the enlarged lymph nodes,
may undergo necrosis and become fluctuant
Often reach bloodstream and become widely disseminated
Hemorrhagic and necrotic lesions may develop in all organs
Meningitis, pneumonia and serosanguineous pleuropericarditis are
Primary pneumonic plague results from inhalation of infective drops
(from a coughing patient), with hemorrhagic consolidation, sepsis
21. Yersinia pestis: Clinical Findings
• After an IP of 2 – 7 days,
there is high fever and
commonly with greatly
enlarged, tender nodes
(buboes) in the groin or
• Vomiting and diarrhoea may
develop with early sepsis
• Later, DIC leads to
hypotension, altered mental
status, renal and cardiac
• Terminally, signs of
pneumonia and meningitis
• Y. pestis multiplies
intravascularly and can be
seen in blood smears
The disease is transmitted by the bites of fleas
(e.g., Xenopsylla cheopis, the rat flea) which
have previously sucked blood from an
infected animal. The ingested bacilli
proliferate in the intestinal tract of the flea
and eventually block the lumen of the
proventriculus. The hungry flea, upon biting
another rodent, regurgitates into the wound a
mixture of plague bacilli and aspirated blood.
22. Clinical Diseases: Feline Plague
Cats usually acquire infection with Y. pestis by ingestion of infected rodents.
Three clinical forms of the disease are recognized: bubonic, septicaemic and
The most common form of the disease is characterized by enlarged lymph
nodes (buboes) associated with lymphatic drainage from the site of
Affected superficial lymph nodes may rupture, discharging serosanguineous
fluid or pus.
Clinical signs include fever, depression and anorexia.
Septicaemia may occur without lymphadenopathy and is potentially fatal.
Pneumonic lesions may result from haematogenous spread.
Cats with pneumonic lesions are a potential source of human infection
through aerosol generation and should be euthanized.
Human infection can also be acquired through cat scratches and bites and
possibly through the bites of fleas from infected cats.
Care should be taken when handling infected animals
Rapid recognition and lab confirmation of disease are essential
in order to institute lifesaving therapy
Plague should be suspected in febrile patients who have been
exposed to rodents in known endemic areas
Lymphadenopathy and severe depression in cats in endemic
areas may suggest feline plague.
Specimens from suspect cases should be sent to specialized
Suitable specimens include pus, blood and lymph node
Giemsa-stained smears may reveal large numbers of bipolar-
Direct fluorescent antibody tests are carried out in reference
A passive haemagglutination test, using Fraction 1 A antigen,
can be used on paired serum samples taken two weeks apart
from suspect cats - A substantial increase in the antibody level is
usually indicative of active infection.
24. Yersinia pestis: Diagnostic Lab Tests
• Blood for culture
• Aspirates of enlarged lymph nodes for smear
• Acute and convalescent sera for antibody levels
• Sputum for culture
• CSF for smear and culture
• Examined after staining with Giemsa’s stain and
with specific immunofluorescent stains
• Definite identification is best done by
• With Wayson’s stain, may show striking bipolar
• On BA, MCA plates and in infusion broth
• Growth on solid media may be slow but blood
culture are often +ve in 24 hrs
• Tentatively identified by biochemical reactions
• In previously unvaccinated, a convalescent
serum antibody titre of 1:16 or greater is
presumptive evidence of infection
• A titre rise in two sequential specimens
confirms the serologic diagnosis
The Wayson stain is a
blue, ethyl alcohol-phenol
microscopic staining procedure.
It was originally a modified
methylene blue stain used for
diagnosing bubonic plague.
With this stain, Yersinia
pestis appears purple with a
appearance, which is due to
the presence of a central vacuole.
25. Treatment and control
Cats with suspected plague should be kept in isolation and
immediately treated for fleas to prevent those handling the
animal becoming exposed to flea bites.
The bubonic form of the disease may respond to parenterally
administered tetracyclines or chloramphenicol.
Multidrug resistance, mediated by a transferable plasmid, has
been reported recently in Y. pestis (Galimand et a!., 1997).
Unless promptly treated, have a mortality rate of about 50%;
pneumonic plague nearly 100%
Drug of choice is streptomycin
Tetracycline is an alternative drug and is sometimes given in
combination with streptomycin
In endemic areas, dogs and cats should be routinely treated for
Rodent control measures should be implemented after flea
control procedures are in place.
Yersinia Pestis: Epidemiology and Control
• Plague is an infection of wild rodents (field mice, gerbils, moles,
skunks, and other animals) that occurs in many parts of the world
• Chief enzootic areas are India, Southeast Asia (Vietnam), Africa, North
and South America
• The Western states of US and Mexico always contain reservoirs of
• Epizootics with high mortality rates occur intermitently; at such times,
the infection can spread to domestic rodents (rats) and other animals
(cats) and humans can be infected by flea bites or by contact
• The commonest vector is the rat flea Xenopsylla cheopis but other
fleas may also transmit the infection
• Control requires surveys of infected animals, vectors and human
• All patients with suspected plague should be isolated particularly if
pulmonary involvement has not been ruled out
• All specimens must be treated with extreme caution
• Contacts of patients with suspected plague pneumonia should receive
tetracycline as chemoprophylaxis
• A formalin-killed vaccine is available for travelers to hyperendemic
areas and for persons at special high risk
• The CDC ranks the plague as a
Category A disease
– “Agents in Category A have the
greatest potential for adverse
public health impact with mass
casualties, and most require broad-
based public health preparedness
efforts (e.g., improved surveillance
and laboratory diagnosis and
stockpiling of specific medications).
– Category A agents also have a
moderate to high potential for
large-scale dissemination or a
heightened general public
awareness that could cause mass
public fear and civil disruption.”
28. Yersinia pestis as a Weapon
• It is relatively easy to obtain
and mass produce.
• It can be delivered in aerosol
• Pneumonic plague causes a
rapid onset of illness with a
high fatality rate
• Pneumonic plague has a high
potential for secondary
spread of cases during an
• 100-500 bacteria are enough
to cause pneumonic plague
• Plague is fragile and dies
after about 1 hr
• Manufacturing an effective
weapon using Y. pestis would
require advanced knowledge
29. Additional Dangers of Yersinia pestis as a Weapon
• There is no currently available pre-exposure prophylaxis or
vaccine for plague
• Biological attack with plague might employ antimicrobial-
resistant strains that circumvent clinical efforts to deal with the
– In 1995 a patient in Madagascar was found who had a Y.
pestis with a transferable multidrug resistance plasmid
– Additionally, there are reports that the bioweapons
operations of the former Soviet Union engineered
multidrug resistant and fluoroquinolone resistant Y. pestis
30. Effectiveness of Y. pestis as a Weapon
• While antibiotic treatment of bubonic plague is usually effective,
pneumonic plague is difficult to treat and often results in death
regardless of treatment
• Most experts agree that “intentional dissemination of plague would
most probably occur via an aerosol of Y pestis, a mechanism that has
been shown to produce [pneumonic] disease in nonhuman
primates…The size of the outbreak would depend on the quantity of
biological agent used, characteristics of the strain, environmental
conditions, and methods of aerosilization…people would die quickly
following the onset of symptoms.” - JAMA May 3, 2000 Vol 283, No. 17
• In 1970, the WHO estimated that “if 50 kg of Y pestis were released as
an aerosol over a city of 5 million, plague could occur in as many as
150,000 persons, 36,000 of whom would be expected to die.” And this
does not take into account the people who would die from secondary
contraction of the disease.
80,000 to 100,000 hospitalized
500,000 secondary cases
Up to 100,000 deaths total
• According to the CDC, “The fatality rate of patients with pneumonic
plague when treatment is delayed more than 24 hours after symptom
onset is extremely high.”
32. Yersinia enterocolitica and Y. pseudotuberculosis
• Non-lactose fermenting Gram negative rods that are urease-
positive and oxidase negative
• Grow best at 25 C and are motile at 25 C but non-motile at 37 C
• Found in intestinal tract of a variety of animals in which they
cause disease and are transmissible to humans in whom they
can produce a variety of clinical syndromes
• Y. enterocolitica exists in >50 serotypes
• Most isolates from human disease belong to serotypes O3, O8
• There are striking geographic differences in the distribution of
Y. enterocolitica serotypes
• Y. pseudotuberculosis exists in at least 06 serotypes, but
serotypes O1 accounts for most human infections
33. Yersinia enterocolitica and Y. pseudotuberculosis
• Y. enterocolitica can produce a heat-stable enterotoxin but the
role of toxin in diarrhoea associated infection is not well
• Y. enterocolitica has been isolated from rodents and domestic
animals and water contaminated by them
• Transmission to humans occurs by contamination of food, drink
• Y. pseudotuberculosis occurs in domestic and farm animals and
birds which excrete the organisms in feces
• Human infection probably results from ingestion of materials
contaminated with animal feces
• Person to person transmission with either of these organisms
is probably rare
35. Diagnostic Lab Tests:
Yersinia enterocolitica and Y. pseudotuberculosis
• Stool, blood, or material obtained at surgical exploration
• Number in stool may be small and can be increased by cold
• A small amount of feces or a rectal swab is placed in buffered
saline, pH 7.6, and kept at 4 C for 2-4 weeks; many fecal
organisms do not survive but Y. enterocolitica will multiply
• Subcultures made at intervals on MCA may yield yersiniae
36. Yersinia enterocolitica and Y. pseudotuberculosis
• In paired serum specimens taken 2 or more weeks apart, a rise in
agglutinating antibodies can be shown
• However, cross reactions between yersiniae and other organisms
such as vibrios, salmonellae, brucellae may confuse the results
Yersinia enterocolitica and Y. pseudotuberculosis
• Most yersinia infections with diarrhoea are self-limited
• Y. enterocolitica is generally susceptible to aminoglycosides,
chloramphenicol, tetracycline, trimethoprim-
sulphamethoxazole, piperacillin, 3rd generation cephalosporins
• Typically resistant to ampicillin and 1st generation
• Proved yersinia sepsis or meningitis has a high mortality rate
but deaths occur mainly in immunocompromised patients
• Yersinia sepsis can be successfully treated with 3rd generation
cephalosporins possibly in combination with aminoglycosides
or a fluoroquinolone possibly with another antimicrobial
38. Prevention and Control:
Yersinia enterocolitica and Y. pseudotuberculosis
• Contact with farm and domestic animals, their feces or
materials contaminated by them probably accounts for most
• Meat and dairy products have occasionally been indicated as
sources of infection, and group outbreaks have been traced to
contaminated food or drink
• Conventional sanitary precautions are probably helpful
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